Dihydrofolate Reductase Research Papers

Unusual Developmental Pattern of Expression of Enzymes Involved in DNA Biosynthesis in Trichinella spiralis and Trichinella pseudospiralis

by Patrycja Wińska and

2010, NATO Science for Peace and Security Series A: Chemistry and Biology

Fig. 1 Schematic overview of solid-phase cytometry. Steps include: membrane filtration, fluores- cent labelling, scanning, data analysis by a computer and microscopic validation Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry

Fig. 2 Overview of the software parameters used by the computer to discriminate among micro- organisms and autofluorescent particles. The four main discriminants are size, specific intensity, color ratio and signal pattern of the detected fluorescent spot

Physiological probes directly bind to particular molecules in the cell or are markers of metabolic activities (such as enzyme activity and membrane poten- tial). These probes are non-specific, i.e. their action is independent of the type and the identity of the cell. Depending on whether a stain is membrane permeant or impermeant it will label both viable and dead cells or dead cells only, respec- tively. ChemChrome V6 (formerly ChemChrome B or V3) contains carboxyfluo- rescein diacetate (Fig. 4), a nonfluorescent compound that is taken up by metabolically active cells and cleaved by intracellular esterases to yield an intensely green fluorescent product (carboxyfluorescein). When used in conjunc- tion with an appropriate labelling buffer (ChemSol B12), the fluorescent car- boxyfluorescein is only retained in cells with an intact cytoplasmatic membrane (Catala et al. 1999; Parthuisot et al. 2000). As the dye will rapidly leak from dead cells because of their damaged membranes, ChemChrome V6 functions as an activity and cell integrity probe that measures both enzymatic activity and cell- membrane integrity.

Fig. 4 Cleavage of ChemChrome V6 by esterases to yield the green fluorescent carboxyfluo- racrain 8-glucuronidase activity resulting from the use of a proprietary cocktail of inducers and stabilizers.

Fig. 5 Schematic presentation of tyramide signal amplification (TSA) Detection and Quantification of Bacteria and Fungi Using Solid-Phase Cytometry

Fig. 1 Hydrostratigraphic section through wells NN 1, 4, 7, 8, 10, 12. (1) Loam with gravel, pebble, basalt detritus, waterless. (2) Basalt detritus, pebble filled with loam. (3) Monolithe basalt. (4) Fractured basalt Vulnerability Assessment and Mapping of Akounq Groundwater Body, Armenia

Fig. 2 AFSSA screening strategy to detect the five major STEC serogroup involved in human cases (EFSA 2007)

Fig. 1 Dendrogram based on cluster analysis of digitized (GTG).-PCR fingerprints of type, refer- ence and Bryndza cheese strains. The dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using correlation levels expressed as percentage values of the Pearson correlation coefficient. Numbered clusters (1-9) correspond to identified species of Enterococcus sp. and Lactococcus sp.

Fig. 4 (continued) (b) PFGE, (GTG)5-PCR, ERIC-PCR fingerprints of 82 E. faecium isolates. The dendrogram was constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) using correlation levels expressed as percentage values of the Pearson correla- tion coefficient (For further legends see Fig. 4a)

The “threshold effect” (concentration above which foods become unusable) of aflatoxin B1 is 20 ug L" of the total content of aflatoxins. AFB, is formed when the temperature is 40-50°. Entering the organism through fodder it is metabolized to aflatoxin M, that is given off into the milk. The threshold of AFM, content in milk is 5 pg L™'. For the maze used to feed milk cows this level is 20 ug L", for calves used to produce meat and pigs it is 300 ug L™ and for breeding stock — 100 ug L”. The allowable quantities of aflatoxin B, in cotton seeds is 300 wg L™'. An important condition for the toxic and carcinogenic effects of AFB, to be active is its conversion into a number of metabolites (Murphy et al. 2006). The aflatoxins possess the following biological activity. They are the strongest carcinogenic substances in nature. They are also strongly toxic. They suppress the activity of the immune system. They possess an important biological specificity. They have a cumulative effect — manifested by evacuation through the liver, urine and faeces, a peak moment occurs, that is a carcinoma is formed in it, which is due to the accumulation of a certain quantity of aflatoxin. It has been proven that there is a connection between the virus causing hepatitis B and the occurrence of liver cancer when aflatoxins are taken in. These mycotoxins posses tetragenic effect (cause malformations) of the fetus when the mother has taken contaminated food that penetrates the fetus through the umbilical cord. Thus they cause irreversible deformations such as imbecility, defects in the heart valves, only one ear, no fingers or one hand shorter than the other (Vlahov and Ivanon 1996). Of all the known mycotoxins aflatoxin B1 (AFB1) is the major and best studied one because it possesses exceptional toxicity and is widespread in market foods and

These mycotoxins are produced by fungi pathogenic for maze like Fusarium verticiliodes and Fusarium proliferetum and to a very small extent Alternaria in tomatoes. At least 15 varieties of fumosinins have been identified. They are readily dissolved in water and are distinguished from the other mycotoxins by their lack of aromatic structure or a unique chromophore, which does not allow easy analytic determination. Foods containing maze are the main hazard of contamination with fumosinins in the food industry (Vlahov and Ivanon 1996). Most of the foods on the market contain fumosinins to 500 ng g™ or less thanks to the control exercised on the maze and the other components. Fumosinin B, is the most toxic representa- tive of the group.

Fig. 3_ Normalized excitation and fluorescence spectra of membrane Magnetic nanoparticles as immobilization materials have the following advantages: (1) more specific surface area obtained for the binding of larger amounts of biomolecules; (2) lower mass transfer resistance; (3) selective separation of the immobilized biomolecules from a reaction mixture on application of a magnetic field. Among these materials, Fe,O,, magnetic nanoparticles are the most commonly studied. Fe,O,, magnetic nanoparticles have good biocompatibility, strong superpara- magnetism, low toxicity, and an easy preparation process, and their use in biosensors has already shown attractive prospects (Sheng-Fu Wang and Tan Yu-Mei 2007). Horseradish peroxidase (HRP) is an important peroxidase that contains heme,

as a quantifiable o ptical signal. Optical biosensors offer the advantages of noninvasive nondestructive, continuous, and simultaneous multianalyte detection (Taitt et al 2005). Optical systems do not interfere with metabolism, and thus in vivo measure- ments are possib e. In this chapter, some applications of the following optical sensing principles in foodborne pathogen detection are discussed: (1) SPR, (2 resonance mirror (RM), (3) fiber-optic biosensor, (4) array biosensor, (5) Ramar spectroscopy, and (6) light-addressable potentiometric sensor (LAPS) (Knopk et al. 2007c).

Fig. 6 Standard curve for determination of Aflatoxin B1 using biosensor AvaSpec — Ox} Fig. 5 Stability of microbial lipoxygenase immobilized on granulated polymer carrier in time In a measurement cell 20 mL of 0.1 Tris-buffer with pH 7.2 the respective sensor was incubated for 3 min with aflatoxin B1 in concentration range of 0.0 5—0.5 uM oreviously diluted in dimethyl sulphoxide. After 3 min of incubation at 37°C 0.1 mL linoleic acid was added to the samples so that the final concentration of the sample was 4 uM. Based on the measurements, standard curves were constructed for the optical enzyme electrode (Fig. 6).

Fig. 8 Effect of pH on the activity of soluble and immobilized peroxidase

Fig. 9 Effect of temperature on soluble and immobilized peroxide

Table 1 Numerical scores assigned to water level below land surface (b.1.s.) and to soils according their thickness and lithology

Table 2 Classification of water-table aquifer by vulnerability degree

Table 3 Evaluation of water-table aquifer vulnerability by bacteriological contamination Period of vital functions of bacteria under slow contamination is taken 200 days, under mass infection (natural or man-caused disaster) or continuous contaminant spill conditions it is taken 400 days. Six categories of ground water vulnerability to bacteriological contamination have been distinguished (Table 3): Quantitative assessment of confined aquifer vulnerability that is first from land surface is carried on the base of two parameters: total thickness of overlain soils that have slow infiltration rates (m,) and by levels of water table (H.) and estab-

Table 4 Water-table aquifer intrinsic vulnerability assessment by numerical scores

Table 5 Water-table aquifer natural vulnerability assessment from bacteriological contamination

Table 6 Confined aquifer natural vulnerability assessment

performed in order to increase the cell numbers of the pathogen to a detectable level and to allow recovery of injured cel three parameters: (i) the temperature, (ii) s. The efficiency of this step rely on the length of incubation and (iii) the the broth composition. While growth of STEC is usually carried out at 37°C, some authors also incubate the broth at 4 °C (Vimont et al. 2006). The length of incubation is also variable, and may vary depending on the protocol and the matrice enriched, ranging from 6 to 24 h. Possé et al. (2008a) performed an enrichment method including a pre-enric hment phase at 42°C and an enrich- ment at 37°C. The two most employed enrichment media for E. coli O157:H7 and other STEC serogroups are modified trypton soy broth (mTSB) and E. coli broth (EC). These media may be supplemented with various selective agents such as bile salts, potassium tellurite and is the most commonly used (Vimont et al. antibiotics among which novobiocin 2006). However, the use of antibiot- ics may interfere with STEC growth and concentration of these additive has to be carefully chosen (Foster et al. 2003; Vimont et al. 2006). The International Standard Organization (ISO) reference method for isolation of

ther sorbitol-non-fermenting bacterial species tested. This agar was tested on attle rectal swabs and has increased the rate of STEC O157 isolation. Cefixime ellurite Sorbitol MacConkey Agar (CT-SMAC) is now the media of choice in the SO standard protocol (ISO 16654) for E. coli O157 although this medium has been eported to fail to isolate two sorbitol-fermenting STEC O157:H- on HUS patients Karch et al. 1996).

Table 3 Commercialized immunological methods for the detection and isolation of E. coli O157:H7 in food. (Vernozy-Rozand et al., 2002)

indicator for viability of E. coli O157:H7 cells under typical growth conditions before and after thermal treatment. They found that 7fbE, stx/, hly and 16S RNA amplicons were detected during all growth phase. But only the RT-PCR signal of 16S rRNA and /fbE was stable. After heating at 60°C for 20 min, all the mRNA species were degraded, except the rRNA. The authors suggested that the 7fbE gene is the most appropriate target for the detection of viable E. coli O157:H7. However,

Table 1 Occurrence of enterococci in the gastro-intestinal tract ++: usual; + : frequent; (+): occasional; — : not mentioned (According to Klein (2003))

++: usual; + : frequent; (+): occasional; — : not mentioned; ND: not investigated (Adapted from Klein 2003) Table 2. Enterococci in food

Table 3. European cheese products which involved enterococci

Table 3 (continued) The enterococci contribute to the ripening and aroma development of cheese due to their proteolytic and esterolytic activities, as well as their diacetyl production by citrate metabolism (Centeno et al. 1999). The presence and growth of enterococci in cheeses and sausages results in organoleptically unique products, which contrib- ute to the local cuisine and the region’s heritage. Due to interlinked European and worldwide markets, these cheeses are widely distributed and are internationally considered as delicacies (Franz et al. 1999). ee q49,% 7 is 7 - x q aie 7 ‘es: n

Mili, LUALLY OLIULEE La IVIL ALI 1UUU SESE A eye: S. aureus is a bacterium which has gained notoriety over recent years due tc its ability to evolve new virulent and drug resistant strains (Chambers 2001) Strains of this species carry in their genomes genes encoding virulence factors suck as enterotoxins, toxic shock syndrome toxin, exfoliative toxins and immune evasior factors. These genes are often carried on mobile genetic elements including bacte- riophages. In recent years, increasingly virulent strains of S. aureus have emergec in the community, causing severe skin and soft tissue infections and a lethal forn of hemolytic pneumonia in children (Gillet et al. 2002). These new strains are asso: ciated with a bi-component toxin called Panton-Valentine leukocidin (PVL). The increasing levels of antibiotic resistance and the emergence of epidemic strains 0’ bacterial pathogens over the last decade highlight the adaptability of bacteria anc the remarkable speed of bacterial evolution (Enright et al. 2002).

Table 3. Comparison of genotyping systems applied to Staphylococcus aureus (modified according to Witte et al. 2006) The main typing systems used for epidemiological purposes in S. aureus are summarized in Table 3. The methods are compared in respect to their reproduc- bility and discriminatory power. Typing by means of PCR methods is rapid and nexpensive and provides results which are sufficient for quick analysis concerning he relatedness of nosocomial isolates. Spa sequence typing has the advantage of 1igh reproducibility; the sequence data are portable and can be stored in databases. Vloreover, spa typing has a good discriminatory power and can, therefore, replace ymal macrorestriction analysis.

—: 290% strains negative; +: 290% strains positive Table 1 Bacillus anthracis, B. cereus, B. thuringiensis characteristics

Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods associated to food poisoning only in recent years (van Netten et al. 1990). B. cereus, B. thuringiensis and B. anthracis are important species for human healt as they carry genes encoding virulence factors affecting pathogenesis (Sergeev et al. 2006). As some B. thuringiensis strains have been reported to possess genes known to be involved in B. cereus pathogenesis (Hansen and Hendriksen 2001) a differentiation of B. cereus ad B. thuringiensis detected in food products could be a real contri- bution to the collection of epidemiological data. B. cereus, B. thuringiensis and B. anthracis have been subjected to several genomic studies to understand the taxo- nomic relationships. Data reported by Helgason et al. (2000a) reveal that B. cereus, B. thuringiensis and B. anthracis are indistinguishable using MEE or sequence analyses of nine genes, according with data obtained using DNA-DNA hybridization analysis by Kaneko et al. (1978) and Priest et al. (1994). Genetic evidence indicates that B. cereus, B. thuringiensis and B. anthracis are one s Cherif et al. (2003a) grouped B. anthracis in a separa pecies (Helgason et al. 2000b). te branch from B. cereus and B. thuringiensis on the basis of the phylogenetic relationship between the species of the B. cereus group using the long ITS containing t DNA. Data on ITS sequences confirm the difficulty in distinguishing B. cereus and B. thuringiensis apart from

ea ena Te one teen ere IR eee ee a a a ee ERS ae ee other Bacillus species (Table 3). The emetic type toxin is a 1.2 kDa molecular weight cereulide (ionosphoric water-soluble peptide) resistant to a treatment at 120°C for | h (heat-stable toxin) (Agata et al. 2002). This toxin may persist in foods after vegetative cells death. It causes an acute attack of vomiting after ingestion of contaminated food. For diar- rheal syndrome food containing more than 10* cells/spore per g has been considered risky, whereas for the emetic syndrome the number of B. cereus varies from 10° to 10° per g. Cereulide production begins at the end of the log growth phase. Outbreaks caused by B. cereus and/or the related species are often confused with out- breaks caused by other etiological agents. The symptoms of the diarrheal syndrome due to the presence of B. cereus are similar to the C. perfringens syndrome symp- toms. Symptoms due to B. cereus emetic toxins are similar to Staphylococcus intoxication symptoms. For this reason it is necessary an accurate laboratory investigation. Moreover B. cereus and B. thuringiensis are very closely related species belonging to the B. cereus group which main difference is the presence on B. thuringiensis plasmids of genes coding for the insecticidal toxins (Chen and Tsen 2002; Rasko et al. 2005). Plasmids loss and horizontal spreading of plasmids cause many difficulties in distinguishing B. thuringiensis from B. cereus affecting taxonomy and pathogenicity (Ward and Ellard 1983; Wilcks et al. 1998). Gonzales et al. (1982) used the high-frequency transfer of plasmids between strains of B. thuringiensis to study the genetic relarionship between plasmid and toxin produc- tion. Schnepf et al. (1998) worked on pesticidal proteins. Molecular methods have been developed to detect microbial toxins in foods. Radhika et al. (2002) utilized a probe to detect the phospholipase C. Virulence factors (enterotoxins, phospholi- pases and exotoxins) important for pathogenesis, were explored by the microarray- based detection by Sergeev et al. (2006).

Table 4 Characteristics used for differentiating Bacillus cereus group species +: 90-100% positive; — : 90-100% positive; +/—: 50-50% positive

strain level (Table 5). These molecular techniques can be used either on isolates from agar plate medium or on microbial DNA directly extracted from food. The need to obtain fast methods in food analyses stimulate research to optimize mo ecular protocols. Pulsed field gel electrophoresis (PFGE) and multilocus enzymes elecrophoresis are methods useful to analyze the genetic diversity of B. cereus and B. thuringiensis isolates (Helgason et al. 2000b). Fricker et al. (2007) proposed Real-Time PCR assays for the detection of emetic Bacillus cereus strains in foods. The advantage obtained in using Real-time PCR is the higly specificity, sensitivi fication (Mackay 2004). The assay targeted a specific part of the ce (ces) gene allowing the detection of the microorganism responsi ty and the quanti- reulide synthetase ble for the emetic food-poisoning outbreaks. The developed method was able to detect 10° CFUg"' after an enrichment time of 4-6 h, whereas the detection limit was 10'-103 CFUg"! directly form food (Fricker et al. 2007). Also a PCR method using colony hybrid- ization has been developed to detect B. cereus in foods by Radhika et al. (2002).

Table 6 Microorganisms used to check primers BCFWI1 and BCREVNEW

Table 7 Bacillus strains used for PCR optimization in RE-PCR protocol

Table 9 Identification results by the RE method (Hinf 1) on PCR product obtained by BCFW1 and BCRW1

Table 10 Strain distribution in clusters Primer REP 1R-Dt of 15 bp and the primer REP 2-Dt of 22 bp were used (Gevers et al. 2001). As reported in Fig. 2, ten clusters (A to L) were obtained by the rep-PCR fingerprints analysis using 80% similarity (Fig. 4). rep-PCR was more effective in clustering Bacillus strains. The technique clusters strains from the same origin as reported in cluster C, D, F, I and L. In cluster E are grouped strains from pesticides and in cluster C strains from patients (Table 11) (Manzano et al. 2009). According with Abriouel et al. (2007) “the utilization of conserved repetitive sequences dispersed in the whole genome is useful in contributing to the differen- tiation of genomic groups and check their association with foods”.

to an increasingly denaturing environment partially melts till the completely dissociation into single strands at the reaching of extreme denaturing conditions. The position in the gel w here a DNA fragment subjected to denaturing gradient, melts and stops as migrating is dependent on the nucleotide sequence in the melted region. Different sequences stops at different positions (levels). DGGE/TTGE is a good method of finding po ymorphisms when DNA samples amplified from different individuals are run on denaturing gradient gels. This method has been applied in many protocols for identi et al. 2001; Ercolini 2004 BCRW1 were used for am fication or differentiation of strains from food (Vasquez ). Primers BCFW1 with a 40 bp GC clamp added, and amplicons from B. thuring plification of 69 amplicons from B. cereus strains and 15 iensis. Amplicons were used in a sequence-specific sepa- ration obtained by the TTGE technique. B. cereus amplicons were splitted in seven levels as reported in Table B. thuringiensis were 12. splitted in two levels: the first for seven strains from

Table 12 Levels obtained by PCR-TTGE analyses applied to B. cereus strains present in this microbial group (Sayama and Bork 2001; Rasko et al. 2005). These regions can be the result of changes due to the insertion or deletion of mobile elements. Also utilizing molecular methods it is difficult to identify a Bacillus strain as the genetic exchange between Bacillus strains, in the absence of genetic barriers, rules out this kind of classification at the species and subspecies level.

Table 1 Behaviour of L. monocytogenes in dry cured ham treated with ozonated water * Mean values with different letters are statistically different (P < 0.001) Table 2, Behaviour of L. monocytogenes in pig skin treated with ozonated water

« Mean values with different letters are statistically different (P < 0.001) mut the same decrease was observed in the control, where the samples were treated vith water without added ozone. The samples obtained with water without added yzone showed a lower concentration of L. monocytogenes than the untreated sam- les. Indeed, viable L. monocytogenes was found in ozonized water obtained from he treated samples. Significant differences (P < 0.001) were observed when com- yaring the treated (with or without added ozone) and the untreated samples. Conversely, there are no significant differences between the samples treated with ind without added ozone. Therefore, the decrease might be due to the mechanical iction (washing) of water on the samples. The lack activity of the ozonized water night be due to the water temperature (15°C) and to a low ozone concentration in water (0.2 ppm). The ozone generator that we used did not allow to cool water lown to 4°C or to increase the ozone concentration. The bactericidal activity of yzonized water could be improved if water at 4°C was added (Kaothien et al. 2001) ind if its concentration were equal to or higher than 20 ppm (Wade et al. 2003). Indeed, the matrix (DCH or PS) played a role in the ineffectiveness of the treat- nent. The presence of organic compounds in the matrix may degrade the ozone and “onsequently its oxidative activity (Moore et al. 2000). For this reason, Beltran

Table 3. Behaviour of L. monocytogenes in dry cured ham treated with different concentrations of H,O, and kept in air

Table 4 Behaviour of Listeria monocytogenes in pig skin treated with different concentrations of H,O, and kept in air

Table 5 Behaviour of L. monocytogenes in dry cured ham treated with different concentrations of H,O, and kept under vacuum

3% (Table 5) hydrogen peroxide solution respectively in PS kept in air and pack- aged under vacuum.

Table 7 Behaviour of Listeria monocytogenes in DCH and PS treated with different mixes of essential oil Legend: DCH: dry cured ham; PS: Pig skin; A: solution A B: Solution B; C: solution (mix A + B + water) X: average; SD: standard deviation.

Table 8 Behaviour of Leuconostoc carnosum CH versus Listeria monocytogenes in Data: UFC/cm?

Other sources of infection include contaminated surgical instruments anc donor organs. Health care workers, dentists, and others who have frequent contac with infected blood or blood products are at highest risk. HBV transmissior from infected healthcare personnel to patients is relatively uncommon, but ha: occurred during invasive surgical, obstetrical, or dental procedures. Renal dialysi: mentioned by Alter et al. (1983), and long-term household or intimate non-sexua contact with an HBsAg-positive individual also exposes people to the risk o: acquiring HBV unless necessary precautions have been taken beforehand (HBV vaccination, using separate dialysis equipment etc.). Binallyvy came “trendv”’ annlicatione ench ac tattqninga ear/hodyv niercino o

Table 1 Advantages and disadvantages of the main techniques used by laboratories in serological and virological dengue diagnostic assays

Table 2. Examples of commercial kits for DENV diagnostics

Table 3 Comparison of the chemiluminescent OFIS method to the chemiluminescent and colorimetric MAC-ELISA assays. (Atias et al. 2009) ising chemiluminescent OFIS (Atias et al. 2009). Since IgM is a much large nolecule than IgG, it is difficult to work with in chemiluminescent OFIS, as the ussay is based on bottom to top covalent binding and top to bottom detection but 1ot adsorption. However, the main serological routine test in dengue endemic -ountries is based on anti-DENV IgM detection, so it is important to focus on this <ind of diagnostic.

Table 1 (continued) “The term ‘microparasite’ was used in the sense of Anderson and May (1998): small-sized parasites able to replicate usually at very high rates within the host In the context of the present work, microparasites are exclusively eukaryotic microorganisms (protists and fungi). Most data come from Woolhouse et a (2006) and Fagherazzi-Pagel (2009).

‘Data from AFSSA (2002), Millar et al. (2002), Dawson (2005) and Smith et al. (2007) Table 3. Reported cases of foodborne cryptosporidiosis (non-exhaustive list)*

Table 4 Emerging and reemerging foodborne helminthes*

* Most data come from Woolhouse et al. (2006) and Fagherazzi-Pagel (2009)

Table 1 Specific activities of selected enzymes involved in thymidylate de novo biosynthesis in T. spiralis and T: pseudospiralis muscle larvae, T. spiralis adult forms, C. elegans adult forms, and L1, L3 and dauer larvae. For both Trichinella species time between infection and isolation is indicated

Table 1 Major parasitic helminths of humans

Table 2 Helminth* products modulating host immune response

On day 18, blood was taken from saline-, OVA- and OVA plus Systemic (Sy) HP-NAP-treated animals. Blood smears were stained to calculate the proportions and the mean (+ SE) absolute counts of lymphocytes, neutrophils, and eosinophils. Levels of total serum IgE were assessed by a specific ELISA assay. ** P < 0.01, *** P < 0.03 OVA plus Sy HP-NAP versus OVA alone. Table 3. HP-NAP delivery reduces serum IgE levels and eosinophil count in the blood of OVA eencitizved mire

Table 4 HP-NAP delivery reduces the Th2 cytokine release in the bronchoalveolar lavage of OVA sensitized mice On day 18, bronchoalveolar lavage samples were collected from OVA- and OVA plus Systemic (Sy) or mucosal (Mu) HP-NAP-treated OVA-sensitized mice. Cytokines were assayed in the supernatants. Mean values (+ SE) are reported. * P < 0.05, ** P < 0.04 OVA plus Sy or Mu HP-NAP versus OVA alone mice.

Table 2 Statistical processing of standard curves The obtained standard curves were statistically processed and are shown in Table 2. above. The nerformed investiscations nrove that they can he ncsed for meagurine real food.

Table 3 Investigation on the activity of the free and immobilized enzyme

Table 4 Kinetic parameters of soluble and immobilized peroxidase Acknowledgment This review is supported for publication from NATO Scientific Committee and our research investigation was financial supported by project 322, National Fund “Scientific Investigations” of Republic of Bulgaria.

Fig. 5 Antibiotic resistance behaviour of 308 enterococci isolated from Bryndza. Ampicillin (AMP), streptomycin (STR), gentamicin (GEN), vancomycin (VAN), teicoplanin (TEI), rifampi- cin (RIF), erythromycin (ERY), ciprofloxacin (CIP), and nitrofurantoin (NIT)

Fig. 6 Incidence of sensitive, intermediary and resistant E. faecium strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), cip- rofloxacin (CIP), and nitrofurantoin (NIT) Figures 6-8 illustrate antibiotic resistance patterns of E. faecium, E. faecali: ind E. durans isolates from Bryndza cheese. No one of the E. faecium, E. duran: ind E. faecalis iso ates were resistant to ampicillin, streptomycin, gentamicin, van -omycin, and teicoplanin. Thirty six percent of the E. faecium isolates and 22% o he E. faecalis iso ‘esistance to rifam ates were resistant to erythromycin. E. faecium showed simila picin (31%) as E. faecalis (29%). Both E. faecium and E. faecali strains exhibited the same resistance to ciprofloxacin (2%). E. durans isolate showed very low level of resistance to rifampicin, erythromycin, ciprofloxacin, an 1itrofurantoin (1-4 I%). E. faecium, E. faecalis and E. durans were also found to b he predominant s pecies recovered from naturally ripened European cheeses an

Fig. 7 Incidence of sensitive, intermediary and resistant E. faecalis strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), cip- rofloxacin (CIP), and nitrofurantoin (NIT)

Fig. 8 Incidence of sensitive, intermediary and resistant E. durans strains (%) isolated from Bryndza (I — January, VI — June and X — October) to rifampicin (RIF), erythromycin (ERY), ciprofloxacin (CIP), and nitrofurantoin (NIT)

Fig. 1 Amplicons obtained using different inocula of Bacillus cereus in the coffee concentrate samples (Manzano et al. 2003b). Lanes 1-7, samples artificially contaminated; lane 1, 1 x 10° cells mL7!; lane 2, 1 x 10° cells mL7!; lane 3, 1 x 10* cells mL7!; lane 4, 1 x 10° cells mL~!; lane 5, 1 x 10° cells mL~'; lane 6, 10 cells mL~'; lane 7, 1-10 cells mL“; lane 8, B. cereus DSMZ 2301 (positive control); lane 9, negative control; lane 10, (MWM) molecular weight marker (100 bp; Sigma, Germany)

Fig. 2 PCR products digested by Hinf I endonuclease. Lane 1: B. thuringiensis DSMZ 2046; Lane 2: B. thuringiensis from coffee; Lane 3: Bacillus thuringiensis DSMZ 350; Lane 4: Bacillus cereus DSMZ 4282; Lane 5: B. cereus from ricotta ; Lane 6: B. cereus DSMZ 2301; Lane 7: uncut PCR product B. cereus DSMZ 2301 ; Lane 8: Molecular Weight Marker, (Sigma, Milan, Italy)

Fig. 4 rep-PCR dendrogram of Bacillus cereus and B. thuringiensis strains determined by the Unweighted Pair Group Method using Arithmetic Average (UPGMA), correlation coefficient of 80% Molecular Methods to Detect Bacillus cereus and Bacillus thuringiensis in Foods

Fig. 1 Slices of dry cured ham and pig skin

Fig. 2. Box and ionizer system 40 DCH and 40 PS samples were inoculated. 10 ml of water (control samples) or 10 ml of three different solutions of hydrogen peroxide (20%, 10%, and 3%) were added to the samples (ten each). The samples were split in two groups. One group was kept in air, the second group was packaged under vacuum in PET. After 10 min

Fig. 3_ Behaviour of Listeria monocytogenes in dry cured ham packaged in MAP at 4°C. Legend: SD — sodium diacetate; LD — sodium diacetate/sodium lactate; L: sodium lactate; Aw: control sample

Fig. 4 Behaviour of Listeria monocytogenes in dry cured ham packaged under vacuum at 4°C. Legend: SD — sodium diacetate; LD — sodium diacetate/sodium lactate; L: sodium lactate; Aw: control sample

Fig. 5 Behaviour of L. monocytogenes in pig skin treated with ionized air. Legend: NT: not treated; T: treated

Fig. 6 Behaviour of L. monocytogenes in dry cured ham treated with ionized air. Legend: NT: not treated; T: treated

Fig. 7 (a) Dry cured ham treated with H,O, (20% v/v) (b) Dry cured ham treated with H,O, (10% v/v) (c) Dry cured ham treated with H,O, (3% v/v)

Fig. 1 Geographical distribution of HEV isolates according to genotypes (Gt). HEV Gt | and 2: epidemic strains causing human infection. HEV Gt 3 and 4: zoonotic strains isolated from humans and a variety of animals, particularly pigs. In some countries, different genotypes co-circulate in distinct ecological niches: Gt 1 and 4 in China, India, and Vietnam; Gt | and 2 in several African countries, including Namibia, Chad, and Sudan; Gt 3 and 4 in Japan; Gt | and 3 in Cambodia; Gt 2 and 3 in Mexico (www.eht-forum.org) death rate during pregnancy approaches 15-25% with possibilities of foetal death, abortion, premature delivery, or death of a live-born baby soon after birth (Smith 2001; Chibber et al. 2004). HEV is transmitted primarily by the faecal-oral route and waterborne epidemics are characteristics of hepatitis E. HEV, known for being the cause of major outbreaks of waterborne hepatitis in Asia and Africa, is an emerging pathogen in industrialized countries (Bihl and Negro 2008). Clinical cases of HEV infection in developed countries have generally been associated with travel to tropical or subtropical countries. There has also been an increasing number of reports from Europe and the United States of sporadic hepatitis attributable to HEV but not associated with travel, leading to suggestions that HEV may be endemic at low levels in developed countries. HEV has been reported in Netherlands (Widdowson et al. 2003), Spain (Peralta et al. 2009), France (Legrand-Abravanel et al. 2009), Greece (Stefanidis et al. 2004), Sweden (Olsen et al. 2006), UK (Ijaz et al. 2009), Italy (Di Bartolo et al. 2008), United States (Atiq et al. 2009), Japan (Tanaka et al. 2001) more cases are likely to be reported (Fig. 1). It has been recognised that HEV and HEV related viruses circulate in domestic animals native to industrial countries and should be considered as a potential

HEV is the major etiologic agent of enterically transmitted non-A, non-B hepatitis worldwide. It is a spherical, non-enveloped, single (plus) stranded RNA virus that is approximately 32-34 nm in diameter (Fig. 2). HEV belongs to a genus of HEV-like viruses (unassigned genus). Hepatitis E virus (HEV) is a positive- stranded RNA virus with a 7.2 kb genome that is capped and polyadenylated. The virus is currently unclassified: the organisation of the genome resembles that of the Caliciviridae but sequence analyses suggest it is more closely related to the Togaviridae (Yang and Vyas 1996).

Fig. 3. Hepatitis E virus, complete genome (http://www.ncbi.nlm.nih.gov/nuccore/9626440?repo rt=graph&from=1&to=7105) The incubation period following exposure to HEV ranges from 3 to 8 weeks, with a mean of 40 days. The period of communicability is unknown. There are no chronic infections reported. However, recent reports present biochemical, histological and genetic evidence of chronic HEV infection in transplant patients (reviewed by Chandra et al. 2008). 4° oe 4 . . 44 er ate: a

Fig. 4 Genetic relatedness between HEV strains from human and non-human sources and from various countries. Minimal spanning trees of 81 sequences of 148 nucleotides HEV RNA showing genetic distances between genotype three HEV strains from humans and animals. (a) Strains labelled by geographical origin. (b) Strains labelled by biological origin. The 13 recent Dutch cases are marked with ID numbers inside the coloured circles (adapted from Borgen et al. BMC Infectious Diseases 2008 8:61 doi:10.1186/1471-2334-8-61)

Fig. 1 Descriptive scheme of the experimental setups for dengue virus detection. (A) Photon counting unit. (Al) Hamamatsu HC135-01 PMT Sensor Module. (A2) PMT fixation ring. (A3) Manual shutter (71430, Oriel). (A4) Fiber holder that prevents the movement of the fiber inside the photon counting unit. (A5) Fiber optic. (A6) Connection wire of PMT to computer. (A7) Electricity cable. (B) The outside handle of manual shutter that enables light access to the PMT. (C) Immobilization unit. (C1) Fiber optic. (C2) 100ul pipette tip. (C3) Conical tube cup. (C4) Point of fixation of fiber. (C5) Optical fiber core. (C6) Biorecognition elements according to MAC-ELISA chemiluminescent OFIS (Atias et al. 2009). (C7) Test samples. (E) Connection to computer

Fig. 1 Development of Cryptosporidium muris in the SCID mouse. Stomach section from a SCID mouse euthanatized 46 days post-infection. Gastric glan are filled with numerous parasites at different developmental stages. Hematoxylin & Eosin staining. Bar = 10 um

Fig. 1 (a) Thymidylate synthase and histone H.3-like mRNA, and 18S rRNA, bands in T. spira- lis muscle larvae (ML), adult forms (A) and newborn larvae (NBL). (b) Thymidylate synthase mRNA level in T.spiralis muscle larvae (ML), adult forms (A) and newborn larvae (NBL) related to 18S rRNA level. (c) Histone H.3-like mRNA level in T:spiralis muscle larvae (ML), adult forms (A) and newborn larvae (NBL) related to 18S rRNA level. Densitometric results in B and C are presented as mean result of three separate experiments + SD for ML and (A), and as mean result of two separate experiments for NBL + % difference from each of the two results.

Fig. 2. Immunoblot analysis of cross-reactivity of monoclonal antibody generated against rat hepatoma thymidylate synthase. T: spiralis muscle larvae crude extract (lane 1; 100 tg protein), purified recombinant (lane 2; 1 jg protein) or purified muscle larvae endogenous (lane 3; 1 ug protein) thymidylate synthase, rat purified regenerating liver endogenous (lane 4; | Lg protein) or rat recombinant (lane 5; 1 ug protein) thymidylate synthase and L5178Y crude extract (lane 6; 100 ig protein) were separated by SDS polyacrylamide (12%) gel electrophoresis and proteins were transferred into PVDF membrane. Thymidylate synthase was detected by treatment with mono- clonal antibody and goat anti-mouse IgG (H + L) — HRP conjugate (BioRad) as a secondary antibody

Fig. 1 Effect of HP-NAP treatment on plasma levels of total and TsE/S-specific IgE. In control animals (open squares) TsE/S-specific IgE levels were high up to day 42, whereas in HP-NAP- treated animals (solid squares) IgE levels progressively decreased after day 14. Results are pre- sented as means + SD in 2 groups of 45 animals. **P < .0005 and ***P < .0001

Fig. 2. Effect of HP-NAP treatment on blood Th2 and Th1 parameters. Eosinophils and total IgE, IL-4, IL-5, IFN-y, and IL-12 levels were measured in the blood of control animals (open squares) and compared with values in animals treated on days 10 and 28 with HP-NAP (solid squares). Results are presented as means + SD

Fig. 3 Experimental protocol of the study. HP-NAP has been delivered systemically (i.p.) (a) or via mucosal route (b). Groups of C57BL/6j mice were treated with saline, with OVA alone, with OVA plus i.p. HP-NAP or with OVA plus mucosal HP-NAP. In both systemic and mucosal proto- cols, mice were treated with OVA according to a procedure consisting of a first phase of sensitiza- tion with OVA i.p. (100 j1g/mouse) and a second phase of induction of the allergic response with aerosolized OVA (2% in PBS) for 5 min on day 8, and finally exposed to aerosolized antigen (1% in PBS) for 20 min daily on days 15-18. Control animals, designed as saline, were injected with PBS alone and then exposed to aerosolized PBS. In the systemic protocol (a) mice were treated with ip. HP-NAP (10 j1g/mouse) on day 1, whereas in the mucosal protocol (b) mice received intra-nasal HP-NAP (10 ug/mouse) on days 7 and 8

Fig. 4 Intraperitoneal administration of HP-NAP inhibited the development of airway eosinophilia in OVA-sensitized animals. (a) On day 1, mice were sensitized with OVA alone or with OVA plus sys- temic HP-NAP (Sy HP-NAP), and then exposed to aerosolized OVA. Control animals were injected with PBS alone and then exposed to aerosolized PBS. Cytocentrifuge preparations from BAL of the three groups of animals on day 18 were stained to calculate the proportions of eosinophils, mac- rophages, neutrophils and lymphocytes. Absolute counts (+ SE) for each cell type were calculated from the number of total cells in the BAL. *** P < 0.01 versus OVA-treated mice. (b) Frozen sections of lungs from OVA-sensitized (eft), and Sy HP-NAP-treated OVA-sensitized animals (right). Eosinophil infiltration in the lung is virtually absent in OVA-sensitized mice treated with Sy HP-NAP Immunodulation and Helminths: Towards New Strategies for Treatment

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Expanding the Genetic Code of Escherichia coli

by lei wang

2001, Science

A unique transfer RNA (tRNA)/aminoacyl-tRNA synthetase pair has been generated that expands the number of genetically encoded amino acids in Escherichia coli. When introduced into E. coli, this pair leads to the in vivo incorporation of... more

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Plasmodium falciparum:Detection of Polymorphisms in theDihydrofolate ReductaseandDihydropteroate SynthetaseGenes by PCR and Restriction Digestion

by David Warhurst

1998, Experimental Parasitology

treatment of clinical malaria. It is becoming increasingly falciparum: Detection of polymorphisms in the Dihydrofolate reducimportant as an alternative to chloroquine, to which resistase and dihydropteroate synthetase genes by PCR and... more

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Synthesis, dihydrofolate reductase inhibition, antitumor testing, and molecular modeling study of some new 4 (3H)-quinazolinone analogs

by Laila Abouzeid

2006, Bioorganic & medicinal …

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Evolution and Classification of P-loop Kinases and Related Proteins

by L Aravind

2003, Journal of molecular biology

Sequences and structures of all P-loop-fold proteins were compared with the aim of reconstructing the principal events in the evolution of P-loop-containing kinases. It is shown that kinases and some related proteins comprise a... more

Sequences and structures of all P-loop-fold proteins were compared with the aim of reconstructing the principal events in the evolution of P-loop-containing kinases. It is shown that kinases and some related proteins comprise a monophyletic assemblage within the P-loop NTPase fold. An evolutionary classification of these proteins was developed using standard phylogenetic methods, analysis of shared sequence and structural signatures, and similarity-based clustering. This analysis resulted in the identification of approximately 40 distinct protein families within the P-loop kinase class. Most of these enzymes phosphorylate nucleosides and nucleotides, as well as sugars, coenzyme precursors, adenosine 5′-phosphosulfate and polynucleotides. In addition, the class includes sulfotransferases, amide bond ligases, pyrimidine and dihydrofolate reductases, and several other families of enzymes that have acquired new catalytic capabilities distinct from the ancestral kinase reaction. Our reconstruction of the early history of the P-loop NTPase fold includes the initial split into the common ancestor of the kinase and the GTPase classes, and the common ancestor of ATPases. This was followed by the divergence of the kinases, which primarily phosphorylated nucleoside monophosphates (NMP), but could have had broader specificity. We provide evidence for the presence of at least two to four distinct P-loop kinases, including distinct forms specific for dNMP and rNMP, and related enzymes in the last universal common ancestor of all extant life forms. Subsequent evolution of kinases seems to have been dominated by the emergence of new bacterial and, to a lesser extent, archaeal families. Some of these enzymes retained their kinase activity but evolved new substrate specificities, whereas others acquired new activities, such as sulfate transfer and reduction. Eukaryotes appear to have acquired most of their kinases via horizontal gene transfer from Bacteria, partly from the mitochondrial and chloroplast endosymbionts and partly at later stages of evolution. A distinct superfamily of kinases, which we designated DxTN after its sequence signature, appears to have evolved in selfish replicons, such as bacteriophages, and was subsequently widely recruited by eukaryotes for multiple functions related to nucleic acid processing and general metabolism. In the course of this analysis, several previously undetected groups of predicted kinases were identified, including widespread archaeo-eukaryotic and archaeal families. The results could serve as a framework for systematic experimental characterization of new biochemical and biological functions of kinases.

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Genetic characterization of CHO production host DG44 and derivative recombinant cell lines

by Jacques S Beckmann

2006, Biochemical and Biophysical Research Communications

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and... more

Fig. 1. Flow cytometry of recombinant CHO DG44 cell lines expressing GFP. The analysis was performed at the end of the three-week period of clonal expansion prior to the removal of selective pressure. For each histogram, 10,000 cells were analyzed. Since the fluorescence intensity of the cells was very high, it was not possible to display the GFP-negative and GFP-positive cells on the same graph.

Fig. 2. Relationship between integrated transgene copy number and specific fluorescence for recombinant DG44-derived cell lines expressing GFP. The specific fluorescence corresponded to the RFUs divided by the number of cells multiplied by 100 and was determined at the end of clonal expansion prior to the removal of selective pressure. The GFP copy number was determined by Southern blot using the GFP gene as a probe.

Fig. 3. Flow cytometry of recombinant DG44-derived cell lines expressing GFP. The cells were analyzed prior to the removal of selective pressure (passage P1) and then after 15-18 passages (P15—P18) in the absence of puromycin. The GFP-negative cells in each population were marked CHO. The cell line number is indicated at the upper left of each graph.

Fig. 4. Karyotype of CHO DG44 cells. Metaphase chromosome spreads of CHO DG44 were stained with Giemsa (G-banding). The normal hamster chromosomes are numbered in the top row, followed by the Z group chromosomes, the derivative chromosomes (der), and the marker chromosomes (mar).

Fig. 5. Variation in the structure of chromosome 4 within CHO DG44 cells. The normal chromosome 4 (A), the abnormal add(4) (B), and add add(4) (C) chromosomes with additional genetic material at the end of the long arm are shown.

Fig. 6. Cytogenetic analysis of recombinant DG44-derived cell lines. The FISH (first column), inverse DAPI FISH (second column), and G-banding karyotype (third column) of different recombinant cell lines 1 (A), 3 (B), 10 (C), and 14 (D, a metaphase from a cell with add(8p)) are shown. For the FISH and inverted DAPI FISH the arrows indicate the position of the integrated plasmid. For the karyotypes, the rearranged chromosomes are identified with red arrows and integration sites with black arrows.

Characterization of recombinant DG44-derived cell lines Table 1

* The chromosome with integration as determined by FISH. No signal detected (—). © As determined by karyotype analysis. None and balanced correspond to no apparent change in chromosomal material and unbalanced corresponds to gains and losses of chromosomal segments. © Chromosomal translocation (t), loss of chromosome (—), gain of chromosome (+), deletion (del), additional genetic material (add), isochromosome (iso) (one arm is present in two copies, separated from each other by a centromere), short arm (p), and long arm (q). 4 As determined by flow cytometry during 8 weeks of cultivation in the absence of puromycin. Not determined (ND). Cytogenetics, FISH, and stability of expression of DG44-derived cell lines

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ACTIVIDAD INHIBITORIA SOBRE LA ENZIMA DIHIDROFOLATO REDUCTASA DE EXTRACTOS DE ESPONJAS MARINAS DEL GOLFO DE URABÁ INHIBITORY ACTIVITY OF SOME MARINE SPONGE EXTRACTS FROM URABÁ GULF ON DIHYDROFOLATE REDUCTASE ENZYME

by Diego Zabala

La enzima dihidrofolato reductasa está implicada en la producción de la base pririmidínica timidina, componente esencial de la estructura del ADN. Por tanto, cualquier sustancia que la inhiba tiene como efecto la inhibición de la síntesis... more

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MOLECULAR EPIDEMIOLOGY OF CRYPTOSPORIDIOSIS OUTBREAKS AND TRANSMISSION IN BRITISH COLUMBIA, CANADA

by Han Sulaiman

1999

Isolates from 25 (13 sporadic and 12 outbreak) cryptosporidiosis cases, 24 of which were from British Columbia, Canada, were characterized using nested polymerase chain reaction amplification of the polymorphic internal transcribed spacer... more

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An affinity selection–mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries

by John Athanasopoulos and

2004, International Journal of Mass Spectrometry

The NeoGenesis Automated Ligand Identification System (ALIS), an affinity selection-mass spectrometry (AS-MS) process consisting of a rapid size-exclusion chromatography stage integrated with reverse-phase chromatography, electrospray... more

Sinceits firstreportin 1991 [12], the direct analysis of non- covalent biomolecule-ligand complexes, particularly using electrospray ionisation-MS (ESI-MS), has become a very ac- tive and fruitful area of study [13] and has been successfully applied to screening compound pools for small molecule lig- ands [14,15]. This technique enjoys the advantage of hav- ing all reaction components free in solution; however, as a generic library screening method it suffers the limitation that resulting receptor-ligand complexes must remain stable un- der the conditions of ESI-MS. Furthermore, the method is not Combining a soluble protein and a mass-encoded small molecule library in a physiologically relevant buffer leads to the formation of a complex of the protein with any suitable ‘ibrary member. The complex is separated from non-binding ‘ibrary members by a rapid, low temperature (<30s at 4°C) SEC step. A rapid SEC separation insures that even weakly bound Hoan (Kq <10 1M) with moderate dissociation rates (Kore < 0.1 s") are captured for identification as possible lead structures. The SEC band containing the complex is immedi- ately transferred to a reverse-phase chromatography column. The column is maintained at 60°C and pH <2 to promote dissociation of ligands from the complex. The dissociated igands are eluted into a high-resolution mass spectrometer for analysis, and automated software algorithms [29] search the mass spectral data to identify the ligands by virtue of their molecular weight. The A LIS method allows interrogation of a protein surface without modification of its structure, does not require immobilization of the protein or the small molecule 9001 against which the target is screened, and consumes only sub-milligram amounts of a purified, soluble protein to screen Fig. 1. Diagram of the Automated Ligand Identification System (ALIS) affinity selection-mass spectrometry method.

Fig. 2. Schematic of the instrument configuration used in ALIS. The libraries investigated in this study were designed and synthesized such that each memberis constructed from build-

Fig. 3. UV detector responses from the SEC eluant path showing the DHFF protein-ligand complex eluting at 24 seconds and unbound library compo nents eluting later. Upper trace: detector 1, before protein peak transfer tc RPC-MS; lower trace: detector 2 showing components not transferred tc RPC-MS.

Fig. 4. Synthetic scheme for mass-encoded library NGL127A 443 containing isobaric positional isomers 1 and 19

Fig. 5. Amine building blocks used in the synthesis of library NGL127A 443. AS-MS analysis were conducted by incubating 2500- member libraries at 2.5mM cumulative compound concen- tration with 5M E. coli DHFR [38] in a final volume of 2 pL pH 7.5 phosphate buffer containing 2.5% DMSO and 100mM NaCl. As such, 2 pmol of each library component (at 1.0 ~.M/component) and 10 pmol (0.18 jg) protein were ganic layer was dried over sodium sulfate and concen- trated to give a clear oil. The intermediate was dissolved in DCM/2- propanol (3 mL:1 mL) to which was added (R)-1-(4- methoxyphenyl)ethylamine (50 L, 0.40 mol), Y b(III) cata- lyst solution (100 wL of a 120 mg solution in 1.5mL THF), and DIEA (50 pL, 0.28mmol). The mixture was heated to 45-50°C for 24h then concentrated and purified by flash chromatography (SiOz, 1-5% MeOH/DCM) to give a clear oil, which was freeze-dried to a hygroscopic powder (81 mg, 56%). HPLC and HPLC-MS analysis indicated >95% pu- rity. 'H NMR (300 MHz, CDCl) 6 7.17 (d, 1H, J =8.2 Hz), 7.15-7.10 (m, 3H), 6.81-6.70 (m, 4H), 6.34 (s, 1H), 4.20 (dd, 1H, J =3.9, 10.9Hz), 4.13 (dd, 1H, 5.9, 10.9Hz), 3.94 (m, 1H), 3.80 (apparent t, 4H, 5.1 Hz), 3.73 (s, 3H), * B (m, 1H), 3.19-3.16 (m, 5H), 2.68-2.58 (m, 2H), 1.41 (d 7

“ig. 6. (A) Extracted ion chromatogram (XIC) of m/z 515.2 (M + H)* fro in ALIS experiment with DHFR and NGL127A 443. (B) XIC of m/z 515.2 rom control experiment (no library). (C) Mass spectrum of the region neal n/z 515.2 underlying the XIC peak in (A). (D) LC-MS/MS spectrum of the arly-eluting isomer 1. (E) LC-MS/MS spectrum of the late-eluting isomer 19. (F) ALIS-MS/MS spectrum of 1 from an ALIS experiment with DHFR ind the NGL127A 443 sub-library.

Fig. 8. Effect of added DHFR ligands on the recovery of (S)-1 from ALIS competition experiments.

A portion of the membership of the ALIS screening library, composed of NGL127A 443 (library 3 in this table) and four other libraries, which yielded DHER ligand 1 (entry 11)

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Overcoming methotrexate resistance in breast cancer tumour cells by the use of a new cell-penetrating peptide

by Emelia Eiriksdottir

2006, Biochemical Pharmacology

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 4 1 6 -4 2 5 Drug delivery Methotrexate MDA-MB-231 cells Drug resistance Drug delivery Abbreviations: Ac, acetylated APA, 4-amino-4-deoxy-N 10methyl pteroic acid BOP,... more

b i o c h e m i c a l p h a r m a c o l o g y 7 1 ( 2 0 0 6 ) 4 1 6 -4 2 5 Drug delivery Methotrexate MDA-MB-231 cells Drug resistance Drug delivery Abbreviations: Ac, acetylated APA, 4-amino-4-deoxy-N 10methyl pteroic acid BOP, benzotriazol-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate CPP, cell-penetrating peptide DHFR, dehydrate folate reductase LDH, lactate dehydrogenase MALDI, matrix assisted laser desorption ionization MS, mass spectrometry MTX, methotrexate RP-HPLC, reversed phase high performance liquid chromatography a b s t r a c t Resistance to chemotherapy limits the effectiveness of anti-cancer drug treatment. Here, we present a new approach to overcome the setback of drug resistance by designing a conjugate of a cell-penetrating peptide and the cytostatic agent methotrexate (MTX). Two different peptides, YTA2 and YTA4, were designed and their intracellular delivery efficiency was characterized by fluorescence microscopy and quantified by fluorometry. MTX was conjugated to the transport peptides and the ability of the peptide-MTX conjugates to inhibit dihydrofolate reductase, the target enzyme of MTX, was found to be 15 and 20 times less potent than MTX. In addition, in vitro studies were performed in a drug resistant cell model using the 100-fold MTX resistant breast cancer cells MDA-MB-231. At a concentration of 1 mM, the peptide-MTX conjugates were shown to overcome MTX resistance and kill the cells more efficiently than MTX alone. Estimated EC 50 's were determined for MTX, MTX-YTA2 and YTA2 to be 18.5, 3.8 and 20 mM, respectively. In summary, cell-penetrating peptide conjugation of MTX is a new way of increasing delivery, and thereby, the potency of already well-characterized therapeutic molecules into drug resistant tumour cells. (M. Lindgren). a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o c h e m p h a r m 0006-2952/$ -see front matter #

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Synthesis, dihydrofolate reductase inhibition, antitumor testing, and molecular modeling study of some new 4(3H)-quinazolinone analogs

by Mahmoud Nagi and

2006, Bioorganic & Medicinal Chemistry

In order to produce potent new leads for anticancer drugs, a new series of quinazoline analogs was designed to resemble methotrexate (MTX, 1) structure features and fitted with functional groups believed to enhance inhibition of mammalian... more

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Specific chemotherapy of Chagas disease: controversies and advances

by Roberto Docampo

2003, Trends in Parasitology

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Investigation of inter-individual variability of the one-carbon folate pathway: a bioinformatic and genetic review

by Munir Pirmohamed

2009, The Pharmacogenomics Journal

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The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis

by David Boehr

2006, Science

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Pearson versus Spearman Kendall's Tau Correlation Analysis on StructureActivity Relationships of Biologic Active Compounds

by Sorana Bolboaca

A sample of sixty-seven pyrimidine derivatives with inhibitory activity on E.

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Drug resistance and genetic diversity of Plasmodium falciparum parasites from Suriname

by D. Panchoe

2005, The American journal of tropical medicine and hygiene

Plasmodium falciparum in Suriname was studied for the presence of drug resistance and genetic variation in blood samples of 86 patients with symptomatic malaria. Drug resistance was predicted by determining point mutations in the... more

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Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: structural basis of antifolate resistance

by Tirayut Vilaivan

2000, Bioorganic & Medicinal Chemistry

AbstractÐThe nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was... more

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Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis

by Herbert Pinedo

2002, Biochemical Pharmacology

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Antibiotic Resistance in Food-Borne Bacterial Contaminants in Vietnam

by Linh Tran

2007, Applied and Environmental Microbiology

This study was conducted to examine the rate of contamination and the molecular characteristics of enteric bacteria isolated from a selection of food sources in Vietnam. One hundred eighty raw food samples were tested; 60.8% of meat... more

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Design, synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens

by Seema Bag

2010, Bioorganic & Medicinal Chemistry

The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii... more

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Urban malaria in Dakar, Senegal: chemosusceptibility and genetic diversity of Plasmodium falciparum isolates

by Sidy Ka

2006, The American journal of tropical medicine and hygiene

The chemosusceptibility and genetic polymorphism of Plasmodium falciparum populations from 48 patients hospitalized for malaria at the Hospital Principal in Dakar, Senegal were investigated during the 2002 malaria transmission season.... more

FiGuRE 2. Frequency of merozoite surface protein 1 (msp1) and msp2 allelic families in Plasmodium falciparum isolates from the study sample. The distribution was determined for 24 alleles for K1, 8 for Mad20, and 1 for RO33 for msp1, and 18 alleles for 3D7 and 6 FC27 for msp2.

Ficure 1. Multiplicity of infection with Plasmodium falciparun deduced by genotyping of merozoite surface protein 1 and merozoite surface protein 2 in 37 isolates from the study sample.

* dhfr = dihydrofolate reductase; dhps = dihydropteroate synthetase; crt = chloroquine resistance transporter. Frequency of Plasmodium falciparum (codons 51, 59, 108), dhps (codons 436, 437, 540), and crt (codon 76) mutations among 48 blood samples from Dakar*

* Values are mean 50% inhibitory concentration (ICs) ie., the drug concentration cor- responding to 50% of the uptake of 3"-hypoxanthine by the parasites in drug-free control wells. + Values are proportion of resistant isolates according to cut-off values. In vitro activity of chloroquine, quinine, mefloquine, halofantrine. cycloguanil, artesunate, and atovaquone assessed on 23 Plasmo- dium falciparum isolates collected in Dakar, Senegal in 2002

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Incidence, Distribution, and Spread of Tetracycline Resistance Determinants and Integron-Associated Antibiotic Resistance Genes among Motile Aeromonads from a Fish Farming Environment

by I. Dalsgaard

2001, Applied and Environmental Microbiology

A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I.... more

A collection of 313 motile aeromonads isolated at Danish rainbow trout farms was analyzed to identify some of the genes involved in high levels of antimicrobial resistance found in a previous field trial (A. S. Schmidt, M. S. Bruun, I. Dalsgaard, K. Pedersen, and J. L. Larsen, Appl. Environ. Microbiol. 66:4908-4915, 2000), the predominant resistance phenotype (37%) being a combined oxytetracycline (OTC) and sulphadiazine/trimethoprim resistance. Combined sulphonamide/trimethoprim resistance (135 isolates) appeared closely related to the presence of a class 1 integron (141 strains). Among the isolates containing integrons, four different combinations of integrated resistance gene cassettes occurred, in all cases including a dihydrofolate reductase gene and a downstream aminoglycoside resistance insert (87 isolates) and occasionally an additional chloramphenicol resistance gene cassette (31 isolates). In addition, 23 isolates had "empty" integrons without inserted gene cassettes. As far as OTC resistance was concerned, only 66 (30%) out of 216 resistant aeromonads could be assigned to resistance determinant class A (19 isolates), D (n ‫؍‬ 6), or E (n ‫؍‬ 39); three isolates contained two tetracycline resistance determinants (AD, AE, and DE). Forty OTC-resistant isolates containing large plasmids were selected as donors in a conjugation assay, 27 of which also contained a class 1 integron. Out of 17 successful R-plasmid transfers to Escherichia coli recipients, the respective integrons were cotransferred along with the tetracycline resistance determinants in 15 matings. Transconjugants were predominantly tetA positive (10 of 17) and contained class 1 integrons with two or more inserted antibiotic resistance genes. While there appeared to be a positive correlation between conjugative R-plasmids and tetA among the OTCresistant aeromonads, tetE and the unclassified OTC resistance genes as well as class 1 integrons were equally distributed among isolates with and without plasmids. These findings indicate the implication of other mechanisms of gene transfer besides plasmid transfer in the dissemination of antibiotic resistance among environmental motile aeromonads.

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Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate

by Anna Eleuteri and

2008, Proteins: Structure, Function, and Bioinformatics

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In... more

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P. falciparum dihydrofolate reductase and dihydropteroate synthase mutations: epidemiology and role in clinical resistance to antifolates

by ogobara doumbo

1998, Drug Resistance Updates

Plasmodium falciparum resistance to the antifolates has arisen rapidly in Asia and South America, and threatens the usefulness of these drugs in Africa. In vitro resistance to the antifolates is determined by mutations in parasite... more

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Specific chemotherapy of Chagas disease: controversies and advances

by Julio Urbina

2003, Trends in Parasitology

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Molecular epidemiology of malaria in Yaounde, Cameroon I. Analysis of point mutations in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum

by Leonardo Basco

1998, The American journal of tropical medicine and hygiene

Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of proguanil, and pyrimethamine) is associated with a Ser- to Asn-108 point mutation in the dihydrofolate reductase-thymidylate synthase gene of... more

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Haloadaptation: insights from comparative modeling studies of halophilic archaeal DHFRs

by Panagiotis Kastritis

2007, International journal of …

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In silico and in vitro validation of serine hydroxymethyltransferase as a chemotherapeutic target of the antifolate drug pemetrexed

by Roberto Contestabile

2011, European Journal of Medicinal Chemistry

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Chloroquine resistance before and after its withdrawal in Kenya

by abdi abdirahman

2009, Malaria Journal

The spread of resistance to chloroquine (CQ) led to its withdrawal from use in most countries in sub-Saharan Africa in the 1990s. In Malawi, this withdrawal was followed by a rapid reduction in the frequency of resistance to the point... more

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The phylogeny of rodent malaria parasites: Simultaneous analysis across three genomes

by Neil Sarkar

2007, Infection, Genetics and Evolution

Species of Plasmodium that naturally infect wild rodents but can also be maintained in laboratory mice have long been used as model systems in which to study the biology of malaria parasites. Several of these rodent parasites are now... more

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Pyrimethamine-sulfadoxine efficacy and selection for mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase in Mali

by Ogobara Doumbo

1999, The American journal of tropical medicine and hygiene

To assess pyrimethamine-sulfadoxine (PS) efficacy in Mali, and the role of mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) in in vivo PS resistance, 190 patients with uncomplicated P.... more

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MOLECULAR EPIDEMIOLOGY OF MALARIA IN YAOUNDE, CAMEROON.VII. ANALYSIS OF RECRUDESCENCE AND REINFECTION IN PATIENTS WITH UNCOMPLICATED FALCIPARUM MALARIA

by Leonardo Basco

2000

In an endemic area where malaria transmission is intense and continuous, reappearance of asexual parasites may be ascribed to either recrudescence or reinfection. To distinguish between recrudescence and reinfection after oral treatment... more

* Type A refers to the presence of identical bands (same size and same number). Type B refers to the presence of multiple bands at day 0 and the same bands minus 1 or more bands post-treatment. Type C refers to the presence of additional bands not seen before treatment. + Number of cases presenting a new infection. + In Group 3, reinfection occurred between Day 52 and Day 78 (chloroquine), between Day 55 and Day 80 (sulfadoxine-pyrimethamine), between Day 38 and Day 99 (amodia- quine), and between Day 36 and Day 63 (pyronaridine). Recrudescence occurred on Day 35 (amodiaquine), Day 35 (pyronaridine), and Day 42 (pyronaridine). Recrudescence and reinfection occurring within 100 days after antimalarial drug therapy

* Recrudescence Type A, identical bands; Type B, selection; Type C, appearance of new bands. Reinfection 1 and 2 refer to two different new infections. Patient no. 1062 (Day 258) had no detectable asexual parasites but carried gametocytes. The presence of gametocytes is not an evidence of recrudescence; but unless microscopic examination is conducted, the polymerase chain reaction alone may lead to a wrong conclusion. + SP = sulfadoxine-pvrimethamine. Multiple episodes of recrudescence and reinfections occurring in the same patients

Comparison of the in vitro chloroquine response between pre-treatment and post-treatment isolates

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Structure-Based Design of Selective Inhibitors of Dihydrofolate Reductase: Synthesis and Antiparasitic Activity of 2,4-Diaminopteridine Analogues with a Bridged Diarylamine Side Chain

by N. Galitsky

1999, Journal of Medicinal Chemistry

As part of a larger search for potent as well as selective inhibitors of dihydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N-... more

As part of a larger search for potent as well as selective inhibitors of dihydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N- [(2,4-diaminopteridin-6-yl)methyl]dibenz [b,f]azepine (4a) and the corresponding dihydrodibenz[b,f]azepine, dihydroacridine, phenoxazine, phenothiazine, carbazole, and diphenylamine analogues were synthesized from 2,4-diamino-6-(bromomethyl)pteridine in 50-75% yield by reaction with the sodium salts of the amines in dry tetrahydrofuran at room temperature. The products were tested for the ability to inhibit DHFR from Pneumocystis carinii (pcDHFR), Toxoplasma gondii (tgDHFR), Mycobacterium avium (maDHFR), and rat liver (rlDHFR). The member of the series with the best combination of potency and species selectivity was 4a, with IC 50 values against the four enzymes of 0.21, 0.043, 0.012, and 4.4 µM, respectively. The dihydroacridine, phenothiazine, and carbazole analogues were also potent, but nonselective. Of the compounds tested, 4a was the only one to successfully combine the potency of trimetrexate with the selectivity of trimethoprim. Molecular docking simulations using published 3D structural coordinates for the crystalline ternary complexes of pcDHFR and hDHFR suggested a possible structural interpretation for the binding selectivity of 4a and the lack of selectivity of the other compounds. According to this model, 4a is selective because of a unique propensity of the seven-membered ring in the dibenz[b,f]azepine moiety to adopt a puckered orientation that allows it to fit more comfortably into the active site of the P. carinii enzyme than into the active site of the human enzyme. Compound 4a was also evaluated for the ability to be taken up into, and retard the growth of, P. carinii and T. gondii in culture. The IC 50 of 4a against P. carinii trophozoites after 7 days of continuous drug treatment was 1.9 µM as compared with previously observed IC 50 values of >340 µM for trimethoprim and 0.27 µM for trimetrexate. In an assay involving [ 3 H]uracil incorporation into the nuclear DNA of T. gondii tachyzoites as the surrogate endpoint for growth, the IC 50 of 4a after 5 h of drug exposure was 0.077 µM. The favorable combination of potency and enzyme selectivity shown by 4a suggests that this novel structure may be an interesting lead for structure-activity optimization.

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Transcriptional control of the cell cycle

by Irma Sanchez

1996, Current Opinion in Cell Biology

Cell division is a highly coordinated process. In the last decades, many plant cell cycle regulators have been identified. Strikingly, only a few transcriptional regulators are known, although a significant amount of the genome is... more

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Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript

by Ana Serra

2007, Nature

Alternative promoters within the same gene are a general phenomenon in gene expression 1,2 . Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the... more

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Design, synthesis, biological evaluation and X-ray crystal structure of novel classical 6,5,6-tricyclic benzo[4,5]thieno[2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors

by Vivian Cody

2011, Bioorganic & Medicinal Chemistry

Design, synthesis, biological evaluation and X-ray crystal structure of novel classical 6,5,6-tricyclic benzo thieno [2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors a b s t r a c t Classical... more

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A Heterodimeric Coiled-coil Peptide Pair Selected from a Designed Library-Library Ensemble

by Joelle Pelletier and

of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here... more

Figure 1. (a) Schematic representation of a parallel dimeric coiled coil. Side view: the helical backbones are rep- resented by cylinders, the side-chains by knobs. The path of the polypeptide chain is indicated by a line wrapped around the cylinders. For simplicity, the supercoiling of the helices is not shown. While residues at positions a and d make up the hydrophobic interface, residues at positions e and g pack against the hydrophobic core. They can par- ticipate in interhelical electrostatic interactions between residue i (g position) of one helix and residue i’ +5 of the other helix (e’ position, belonging to the next heptad), as indicated by the hatched bars. Top view: arrangement of the heptad positions. (b) Schematic representation of the protein-fragment complementation assay as described in the text. Each library is genetically fused to one of the two DHFR-fragments ([1] or [2]), and only an interaction between the two library peptides restores the enzyme activity which is crucial for cell survival under selective conditions. Introduction of a mutation at the DHFR interface (I114A) was used to increase selection stringency where indicated (Pelletier et al., 1998). (c) Overview of the library design depicted as a helical wheel plot from the N to the C terminus (inside to outside). The black residues correspond to the original residues from c-Jun (library A) and c-Fos (library B). Changes introduced by the design are in blue. The randomized positions are in red (*, equimolar mixture of Q, E, K, R; x, equimolar mixture of N, V) and are boxed with the same colors as used in (a). The selected residues from the predominant pair, WinZip-A1B1 (clone 1 in Table 3), are next to the randomized positions in the respective boxes.

egg a Ae Tee OMe ee: Se Ry Ae Re eee ee meet the selected heterodimer (a) and the two putative homo- dimers (b) from the statistically expected distribution in the absence of selection. Interactions are potentially attractive (E-K, K-E, E-R, R-E; hatched bars), neutral (Q- Q, Q-E, E-Q, Q-K, K-Q, Q-R, R-Q; grey bars) or repul- sive (E-E, K-K, K-R, R-K, R-R; black bars). Low strin- gency selection: clones (n=14, Table 1) were subdivided into those with an N-N pair in the core a position (1 = 8) and those with an N-V or V-V pair (n = 6). Medium stringency selection: only clones with an Asn pair in the core a position were considered (n = 23, clones 1-23 in Table 2). High stringency selec- tion: clones which survived the competition selection at least up to passage 10 (clones 1-10 in Table 3) were con- sidered. These were analyzed counting each sequence once (not weighted, n=10) or according to their fre- quency of appearance (weighted, n = 37). For example, the high stringency selection yielded in total 82 attrac- tive (opposite charges) interactions in the 37 sequences analyzed. In a random distribution (37 pairs with six e/ g interactions each), 25% or 55.5 e/g interactions would be expected to be attractive (opposite charges). Thus, the observed number of attractive pairs exceeds the stat- istically expected one by 48 %.

Figure 3. Number of selected pairs having a certain difference of attractive (grey bars) or repulsive inter- actions (black bars), respectively, between the heterodi- mer and its constituting homodimers. For example, a value of —3 for the repulsive interactions (black bars) indicates that there are three fewer potentially repulsive interactions in the heterodimer than in the average of both homodimers. (a) Fourteen clones from the low stringency selection, (b) 23 clones from the medium stringency selection, (c) ten different clones from the high stringency selection (not weighted), (d) 37 clones from the high stringency selection (weighted).

Figure 4. Positional distribution of amino acids at each e and g position in sequences from library A (a) and library B (b), obtained from the highest stringency selection. The statistically expected random occurrence of each amino acid at each position was subtracted from the relative occurrence observed in the selection (Q is hatched, E is grey, K/R is black).

Figure 5. CD measurements of the synthesized peptides of WinZip-A1 peptide concentration at 5°C for the heterodimer WinZip-A1B1 (——), the homodimers WinZi and WinZip-B1 (-— - —), and the calculated average between WinZip-Al assuming no interaction (—----- ). (b) Temperature dependence of Q]y. for WinZip-A1B1 ( B1. (a) Far-UV CD-spectra of 150 uM total p-AL (+++ 0+: ) and WinZip-B1, which would be expected , light grey), WinZip-A1 (A, dark grey), WinZip-B1 (W, black), and the calculated average of both homodimers (— - —). (c) Depen- dence of t,, and At,, (¢, white) on pH, and (d) on salt. The t,, was calculated from thermal denaturation curves (same symbols are used as in panel (b)). The At,, was calculated as the d and the averaged t,, of both homodimers. It must be noted that thermal d tely reversible at 1 M salt concentration, and only 71% of the starting signal was regained. ifference between the t,, of the heterodimer enaturation of WinZip-Al was not comple-

Figure 6. Determination of the molecular mass of WinZip-A1B1 by sedimentation eq (a) Measured data (©) obtained at 10 uM pe centration at 25°C, as well as the fit for a (Gogo ), dimer ( ), or trimer (- — — -). (b) between measured data obtained at 10 uM pe uilibrium. ptide con- monomer Residuals ptide con- centration at 25°C and data fitted as monomer (top), dimer (middle), or trimer (bottom). (c) Residuals for a dimeric fit of the data set for WinZip-A1B1 o btained at 150 uM peptide concentration, 10°C, demonstrating that over this concentration and temperature range Zip-A1B1 is dimeric. , the Win-

Figure 8. Urea titration of the heterodimer WinZip- A1B1 (1, light grey), and the homodimers WinZip-A1 (A, dark grey) and WinZip-B1 (¥, black). Designed coiled coils usually are judged only for being stable in vitro and, in certain cases, for hetero- specificity, whereas naturally occurring coiled coils also must function reliably in a cellular environ- ment. Similar demands are imposed during our

Figure 7. Native gel electrophoresis of WinZip-A1, WinZip-B1 and WinZip-A1B1, performed as described in Materials and Methods.

Table 1. Sequence pairs selected in the lowest stringency selection (single-step selection using wild-type DHFR fragments)

2 Sequence of library A: VAQL(e1)E(g1) VKTL(e2)A(g2) (a3) YEL(e3)S(g3) VORL(e4)E(¢4) VAQL. » Sequence of library B: VDEL(1)A(g’1) VDQL(e’2)D(g’2) (a3) YAL(@’3)T(e’3) VAQL(¢’4)K(g’4) VEKL. Table 2. Sequence pairs selected in the medium stringency selection (single-step selection using DHFR-I114A

* The frequency and distribution of each clone in passages 12 and 10 is shown. Clone 6 also was found twice in P8; clones 2 anc 7 were each found once in P6; clone 11 was found twice in P4; clones 12 and 13 were each found once in P2. > Sequence of library A: VAQL(e1)E(g1) VKTL(e2)A(g2) (a3) YEL(€3)S(¢3) VORL(e4)E(g4) VAQL. © Sequence of library B: VDEL(e’1)A(@’1) VDQL(¢’2)D(g’2) (a3) YAL(e’3)T(@’3) VAQL(e’4)K(9’4) VEKL. Table 3. Sequence pairs selected in the highest stringency selection (competition selection

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Synthesis and biological evaluation of biguanide and dihydrotriazine derivatives as potential inhibitors of dihydrofolate reductase of opportunistic microorganisms

by Seema Bag

2010, Journal of Enzyme Inhibition and Medicinal Chemistry

Twenty-one biguanide and dihydrotriazine derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms: Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium... more

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Development of a Lead Inhibitor for the A16V+S108T Mutant of Dihydrofolate Reductase from the Cycloguanil-Resistant Strain (T9/94) of Plasmodium falciparum †

by Tirayut Vilaivan

2000, Journal of Medicinal Chemistry

The Ala16Val+Ser108Thr (A16V+S108T) mutant of the Plasmodium falciparum dihydrofolate reductase (DHFR) is a key mutant responsible for cycloguanil-resistant malaria due to steric interaction between Val-16 and one of the C-2 methyl groups... more

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Genomic Organization and Characterization of the white Locus of the Mediterranean Fruitfly, Ceratitis capitata

by Giuseppe Saccone

2001

An ‫-41ف‬kb region of genomic DNA encoding the wild-type white eye (w ϩ ) color gene from the medfly, Ceratitis capitata has been cloned and characterized at the molecular level. Comparison of the intron-exon organization of this locus... more

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A toolbox facilitating stable transfection of Eimeria species

by Karen Soppe

2008, Molecular and Biochemical Parasitology

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria... more

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Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: Implications for applied site-specific recombination

by Matthew O'Neill

2011, International Journal for Parasitology

The ability to manipulate the genome and induce site-specific recombination using either Flippase (FLP) or Cre recombinase has been useful in many systems including Plasmodium berghei for specific deletion events or to obtain conditional... more

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Molecular epidemiology of drug-resistant malaria in western Kenya highlands

by Yaw Afrane

2008, BMC Infectious Diseases

Since the late 1980s a series of malaria epidemics has occurred in western Kenya highlands. Among the possible factors that may contribute to the highland malaria epidemics, parasite resistance to antimalarials has not been well... more

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Allosteric Communication in Dihydrofolate Reductase: Signaling Network and Pathways for Closed to Occluded Transition and Back

by Ruxandra i. Dima

2007, Journal of Molecular Biology

Escherichia coli dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. During the catalytic cycle, DHFR undergoes conformational transitions between the closed (CS) and occluded (OS) states that,... more

Escherichia coli dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate. During the catalytic cycle, DHFR undergoes conformational transitions between the closed (CS) and occluded (OS) states that, respectively, describe whether the active site is closed or occluded by the Met20 loop. The CS→OS and the reverse transition may be viewed as allosteric transitions. Using a sequence-based approach, we identify a network of residues that represents the allostery wiring diagram. Many of the residues in the allostery wiring diagram, which are dispersed throughout the adenosine-binding domain as well as the loop domain, are not conserved. Several of the residues in the network have been previously shown by NMR experiments, mutational studies, and molecular dynamics simulations to be linked to equilibration conformational fluctuations of DHFR. To further probe the nature of events that occur during conformational fluctuations, we use a self-organized polymer model to monitor the kinetics of the CS→OS and the reverse transitions. During the CS→OS transition, coordinated changes in a number of residues in the loop domain enable the Met20 loop to slide along the α-helix in the adenosine-binding domain. Sliding is triggered by pulling of the Met20 loop by the βG-βH loop and the pushing action of the βG-βH loop. The residues that facilitate the Met20 loop motion are part of the network of residues that transmit allosteric signals during the CS→OS transition. Replacement of M16 and G121, whose C α atoms are about 4.3 Å in the CS, by a disulfide cross-link impedes that CS→OS transition. The order of events in the OS→CS transition is not the reverse of the forward transition. The contact Glu18-Ser49 in the OS persists until the sliding of the Met20 loop is nearly complete. The ensemble of structures in the transition state in both the allosteric transitions is heterogeneous. The most probable transition-state structure resembles the OS (CS) in the CS→OS (OS→CS) transition, which is in accord with the Hammond postulate. Structures resembling the OS (CS) are present as minor (∼ 1-3%) components in equilibrated CS (OS) structures.

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Molecular Analysis of Plasmodium Falciparum From Drug Treatment Failure Patients in Papua New Guinea

by John Reeder

2004

A study was conducted in Papua New Guinea to analyze Plasmodium falciparum drug resistance poly- morphisms in patients presenting with resistant malaria. One hundred ninety-nine P. falciparum-positive patients were recruited at two sites,... more

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Inhibitors of Multiple Mutants of Plasmodium f alciparum Dihydrofolate Reductase and Their Antimalarial Activities

by Tirayut Vilaivan

2004, Journal of Medicinal Chemistry

Novel analogues of pyrimethamine (Pyr) and cycloguanil (Cyc) have been synthesized and tested as inhibitors of Plasmodium falciparum dihydrofolate reductase carrying triple (N51I+C59R+S108N, C59R+S108N+I164L) and quadruple... more

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Lipophilic conjugates of methotrexate with short-chain alkylamino acids as DHFR inhibitors. Synthesis, biological evaluation, and molecular modeling

by Rosario Pignatello

2004, Bioorganic & Medicinal Chemistry

Pursuing previous researches on lipophilic conjugates of methotrexate, aimed at over-crossing a form of transport resistance shown by some tumor cell lines toward the drug, a new series of derivatives is described in which the drug a-and... more

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Novel triazine JPC-2067-B inhibits Toxoplasma gondii in vitro and in vivo

by stephen muench

2008, PLoS neglected tropical …

Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar... more

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Molecular basis for nonadditive mutational effects in Escherichia coli dihydrofolate reductase

by Scott Singleton

1995, Biochemistry

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Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine–pyrimethamine resistance in malaria-infected patients from Nigeria

by Omotayo O Ebong

2005, Acta Tropica

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of... more

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Dihydrofolate Reductase

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