Biochemistry

Menstrual toxic shock syndrome (TSS) is a serious illness that afflicts women of premenopausal age worldwide and arises from vaginal infection by Staphylococcus aureus and concurrent production of toxic shock syndrome toxin-1 (TSST-1). Studies have illustrated the capacity of lactobacilli to reduce S. aureus virulence, including the capacity to suppress TSST-1. We hypothesized that an aberrant microbiota characteristic of pathogenic bacteria would induce the increased production of TSST-1 and that this might represent a risk factor for the development of TSS. A S. aureus TSST-1 reporter strain was grown in the presence of vaginal swab contents collected from women with a clinically healthy vaginal status, women with an intermediate status, and those diagnosed with bacterial vaginosis (BV). Bacterial supernatant challenge assays were also performed to test the effects of aerobic vaginitis (AV)-associated pathogens toward TSST-1 production. While clinical samples from healthy and BV women suppressed toxin production, in vitro studies demonstrated that Streptococcus agalactiae and Enterococcus spp. significantly induced TSST-1 production, while some Lactobacillus spp. suppressed it. The findings suggest that women colonized by S. aureus and with AV, but not BV, may be more susceptible to menstrual TSS and would most benefit from prophylactic treatment.

 In this paper, we explain the basic structure and properties of both single- and double-stranded DNA in vivo (in living organisms). We also review the first in vitro (test tube) experiment that solved a mathematical problem, The Directed Hamiltonian Path Problem, by manipulating DNA strands. Lastly, we give a list of bio-operations that have so far been used in DNA computation.

This paper proposes a DNA algorithm for solving an NP-complete problem (The Shortest Common Superstring Problem) by manipulation of biomolecules, and presents partial results of the experiment that implements our algorithm. We also discuss practical constraints that have to be taken into account when implementing the algorithm, propose a coding system as a solution to these practical restrictions, and discuss the control experiments performed for establishing the parameters controlling the speci city of the assay.

Insertions and deletions of small circular DNA strands into long linear DNA strands are phenomena that happen frequently in nature and thus constitute an attractive paradigm for biomolecular computing. This paper presents a new model for DNA-based computation that involves circular as well as linear molecules, and that uses the operations of insertion and deletion. After introducing the formal model we investigate its properties and prove in particular that the circular insertion/deletion systems are capable of universal computation. We also give the results of an experimental laboratory implementation of our model. This shows that rewriting systems of the circular insertion/deletion type are viable alternatives in DNA computation.

Apoptosis, or programmed cell death, is a vital cellular process often impaired in diseases such as cancer. Aspartic acid-directed proteases known as caspases cleave a broad spectrum of cellular proteins and are central constituents of the apoptotic machinery. Caspases are regulated by a variety of mechanisms including protein phosphorylation. One intriguing mechanism by which protein kinases can modulate caspase pathways is by blocking substrate cleavage through phosphorylation of residues adjacent to caspase cleavage sites. To explore this mechanism in detail, we recently undertook a systematic investigation using a combination of bioinformatics, peptide arrays, and peptide cleavage assays to identify proteins with overlapping protein kinase and caspase recognition motifs (Duncan et al., Sci Signal 4:ra30, 2011). These studies implicated protein kinase CK2 as a global regulator of apoptotic pathways. In this article, we extend the analysis of proteins with overlapping CK2 and caspase consensus motifs to examine the convergence of CK2 with specific caspases and to identify CK2/caspase substrates known to be phosphorylated or cleaved in cells. Given its constitutive activity and elevated expression in cancer, these observations suggest that the ability of CK2 to modulate caspase pathways may contribute to a role in promoting cancer cell survival and raise interesting prospects for therapeutic targeting of CK2.

C-reactive protein (CRP) and surfactant protein A (SP-A) are phosphatidylcholine (PC) binding proteins that function in the innate host defense system. We examined the effects of CRP and SP-A on the surface activity of bovine lipid extract surfactant (BLES), a clinically applied modified natural surfactant. CRP inhibited BLES adsorption to form a surface-active film and the film's ability to lower surface tension (γ) to low values near 0 mN/m during surface area reduction. The inhibitory effects of CRP were reversed by phosphorylcholine, a water-soluble CRP ligand. SP-A enhanced BLES adsorption and its ability to lower γ to low values. Small amounts of SP-A blocked the inhibitory effects of CRP. Electron microscopy showed CRP has little effect on the lipid structure of BLES. SP-A altered BLES multilamellar vesicular structure by generating large, loose bilayer structures that were separated by a fuzzy amorphous material, likely SP-A. These studies indicate that although SP-A and...

The captive bubble tensiometer was employed to study interactions of phospholipid (PL) mixtures of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) at 50 microg/ml with physiological levels of the surfactant protein (SP) A SP-B, and SP-C alone and in combination at 37 degrees C. All surfactant proteins enhanced lipid adsorption to equilibrium surface tension (gamma), with SP-C being most effective. Kinetics were consistent with the presence of two adsorption phases. Under the conditions employed, SP-A did not affect the rate of film formation in the presence of SP-B or SP-C. Little difference in gamma(min) was observed between the acidic POPG and the neutral POPC systems with SP-B or SP-C with and without SP-A. However, gamma(max) was lower with the acidic POPG system during dynamic, but not during quasi-static, cycling. Considerably lower compression ratios were r...

Reactive oxygen species produced by activated leukocytes in the alveolar epithelial lining fluid have been implicated in the inactivation of pulmonary surfactant and the impairment of lung function. Oxidation of bovine lipid extract surfactant (BLES), a therapeutic surfactant, with hypochlorous acid (H-BLES) or the Fenton reaction (F-BLES) led to temporary increases in conjugated dienes and formation of malondialdehyde and 4-hydroxy-2-nonenal. Electrospray ionization mass spectrometry revealed the appearance of lipid hydroperoxides, peroxides, lysophospholipids, and free fatty acids. Captive bubble tensiometer studies of H-BLES demonstrated prolonged adsorption times, film instability at low surface tensions during film compression, and reduced respreadability during film expansion. F-BLES exhibited prolonged adsorption times, a marked effect on increasing compressibility during compression, and a lesser effect on reducing respreadability on expansion. Addition of native bovine or rat surfactant-associated protein A (SP-A) reversed the effects of oxidation on surfactant biophysical properties. Studies using mutant recombinant rat SPAs indicated that an intact carbohydrate recognition domain and disulfide-dependent oligomeric assembly are critical for these effects, but the collagen-like region is not required. We conclude that SPA can reverse the detrimental effects of surfactant oxidation on the biophysical properties of surfactant, by a mechanism that is dependent on interchain disulfide bond formation and the Cterminal domains of the protein. Pulmonary surfactant is essential for normal lung function. By lowering surface tension (␥) 1 during inspiration, surfactant

Monolayers of a functional pulmonary surfactant (PS) can reach very low surface tensions well below their equilibrium value. The mechanism by which PS monolayers reach such low surface tensions and maintain film stability remains unknown. As shown previously by fluorescence microscopy, phospholipid phase transition and separation seem to be important for the normal biophysical properties of PS. This work studied phospholipid phase transitions and separations in monolayers of bovine lipid extract surfactant using atomic force microscopy. Atomic force microscopy showed phospholipid phase separation on film compression and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. The tilted-condensed phase consisted of domains not only on the micrometer scale, as detected previously by fluorescence microscopy, but also on the nanometer scale, which is below the resolution limits of conventional optical methods. The nanodomains were embedded uniformly within the liquidexpanded phase. On compression, the microdomains broke up into nanodomains, thereby appearing to contribute to tiltedcondensed and liquid-expanded phase remixing. Addition of surfactant protein A altered primarily the nanodomains and promoted the formation of multilayers. We conclude that the nanodomains play a predominant role in affecting the biophysical properties of PS monolayers and the monolayer-to-multilayer transition.

How does pulmonary surfactant reduce surface tension to very low values? Background: Although initially proposed by von Neergaard in 1929 (14), direct evidence for surface-active material at the air-fluid interface of the lung was first reported by Richard Pattle and John Clements in the 1950s (5, 10). Pattle deduced that microbubbles formed from lung washings could reduce surface tension (␥) to near 0 mN/m. Using his modified Langmuir-Wilhelmy balance, Clements demonstrated that pulmonary extracts generated surface films that could be compressed to ␥s below 10 mN/m. These were (and remain) extraordinarily low values. Von Neergaard's studies went virtually unnoticed, but Avery and Mead employed a Clements-type balance to demonstrate that infants succumbing to the respiratory distress syndrome lacked a lung substance present in infants dying of other causes (2). Not surprisingly, this discovery focused attention on the clinical significance of surfactant, resulting in the enormous scientific interest that surfactant research still enjoys today (7, 8, 12). However, as emphasized by the article of Yan et al. (16) in this issue of the Journal of Applied Physiology, despite being recognized for over 80 years and investigated for over 50, the mechanism by which pulmonary surfactant reduces ␥ at the air-liquid interface to Ͻ1 mN/m is still not understood. The classical model for surfactant function: Pattle proposed that surfactant was a lipoprotein, and it was soon discovered that surfactant contained phospholipids (PL), particularly phosphatidylcholine (PC), and that dipalmitoylphosphatidylcholine (DPPC) was the major constituent (12). DPPC bilayers (e.g., liposomes) at atmospheric pressure undergo a gel to liquidcrystalline phase transition at 41°C. Below this temperature, (T c), hydrated DPPC molecules are essentially motionless, although they may rotate occasionally. Above T c , the fatty acyl chains become mobile (i.e., melt) and individual DPPC molecules can diffuse within the bilayer leaflet. Lateral compression of DPPC molecules at the air-water interface (i.e., monolayers) on a Langmuir-Wilhelmy balance leads to analogous but not identical phase transitions. Such monolayers can be compressed from the very dilute gaseous phase (Ͼ120 Å 2 /molecule) to the interacting liquid-expanded (LE) phase (ϳ100 Å 2 /molecule) and then to the tightly packed tilted-condensed (TC) phase (ϳ40 Å 2 /molecule). DPPC isotherms (␥ vs. surface area at constant temperature) exhibit LE/TC phase coexistence plateaus. DPPC monolayers can be readily compressed to ␥s ϭ 0 mN/m at either room temperature or 37°C and can remain at these low surface tensions for considerable periods of time. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) monolayers cannot normally be compressed to low ␥s at these temperatures, presumably because they are far above their bilayer T c of Ϫ6.5°C. (Note that T c cannot be defined for a monolayer unless lateral compression is specified. This is because PL isotherms vary with temperature due to thermal agitation. See Refs. 11 and 12 for additional details.) The classical model for surfactant function was formulated to explain how pulmonary surfactant, which normally contains only 30-40% DPPC/total PL, can achieve ␥s near zero during

Rat pulmonary surfactant protein A is an oligomer of 18 polypeptide chains which are associated by triple helix formation in the collagen-like domain and interchain disulfide bridges at the NH 2 terminus. The roles of the intermolecular bond at Cys 6 and the collagen-like domain (Gly 8-Pro 80) in the interactions of SPA with phospholipids and alveolar type II cells were investigated using mutant forms of the protein. Wild type SPA (SP-A hyp), SPA with the substitution Cys 6 3 Ser to prevent disulfide formation (SP-A hyp,C6S), and SPA with the collagen-domain deleted (SP-A ⌬G8-P80) were synthesized in insect cells using recombinant baculoviruses. The SPAs were glycosylated and secreted from the invertebrate cells and the binding affinities of the wild type and mutant proteins for the mannose-Sepharose matrix used for purification were nearly identical. The SPA hyp and SPA ⌬G8-P80 were at least nonameric in solution based on gel exclusion chromatography, and demonstrated extensive sulfhydryl-dependent oligomerization under nonreducing conditions. The SPA hyp,C6S was also oligomeric in solution and formed disulfidedependent dimers, indicating the presence of at least one additional interchain disulfide bond. The SPA ⌬G8-P80 but not the SPA hyp,C6S aggregated lipid vesicles at 20°C and augmented the surface tension lowering effect of extracts of natural surfactant. The SPA ⌬G8-P80 competed poorly with native SPA for receptor occupancy on isolated alveolar type II cells and was a potent but nonspecific (concanavalin Alike) inhibitor of surfactant secretion. In contrast, the SPA hyp,C6S partially competed for receptor occupancy and weakly inhibited surfactant secretion in a specific manner. Neither the SPA ⌬G8-P80 nor the SPA hyp,C6S supported the association of phospholipid liposomes with type II cells. We conclude that: 1) the Cys 6 interchain disulfide bond of SPA is required for aggregation of liposomes and for potent inhibition of surfactant secretion. 2) The collagen-like region is required for competition with 125 I-SPA for receptor occupancy and specific inhibition of surfactant secretion in the presence of competing sugars. 3) Both the NH 2-terminal disulfide and the collagen-like region are required to enhance the association of phospholipid vesicles with type II cells.

Pattle, who provided some of the initial direct evidence for the presence of pulmonary surfactant in the lung, was also the first to show surfactant was susceptible to proteases such as trypsin. Pattle concluded surfactant was a lipoprotein. Ž. Our group has investigated the roles of the surfactant proteins SP-SPA , SP-B, and SP-C using a captive bubble Ž. tensiometer. These studies show that SP-C) SP-B) SPA in enhancing surfactant lipid adsorption film formation to the equilibrium surface tension of ; 22᎐25 mNrm from the 70 mNrm of saline at 37ЊC. In addition to enhancing adsorption, surfactant proteins can stabilize surfactant films so that lateral compression induced through surface area Ž. Ž. reduction results in the lowering of surface tension ␥ from ; 25 mNrm equilibrium to values near 0 mNrm. These low tensions, which are required to stabilize alveoli during expiration, are thought to arise through exclusion of fluid phospholipids from the surface monolayer, resulting in an enrichment in the gel phase component dipalmitoylphos-Ž. phatidylcholine DPPC. The results are consistent with DPPC enrichment occurring through two mechanisms, selective Ž. DPPC adsorption and preferential squeeze-out of fluid components such as unsaturated phosphatidylcholine PC and Ž. phosphatidylglycerol PG from the monolayer. Evidence for selective DPPC adsorption arises from experiments showing that the surface area reductions required to achieve ␥ near 0 mNrm with DPPCrPG samples containing SP-B or SPA plus SP-B films were less than those predicted for a pure squeeze-out mechanism. Surface activity improves during quasi-static or dynamic compression᎐expansion cycles, indicating the squeeze-out mechanism also occurs. Although SP-C was not as effective as SP-B in promoting selective DPPC adsorption, this protein is more effective in promoting the reinsertion of lipids forced out of the surface monolayer following overcompression at low ␥ values. Addition of SPA to samples containing SP-B but not SP-C limits the increase in ␥ during expansion. It is concluded max that the surfactant apoproteins possess distinct overlapping functions. SP-B is effective in selective DPPC insertion during monolayer formation and in PG squeeze-out during monolayer compression. SPA can promote adsorption during film formation, particularly in the presence of SP-B. SP-C appears to have a superior role to SP-B in formation of the surfactant reservoir and in reinsertion of collapse phase lipids.

Exposing bovine lipid extract surfactant (BLES), a clinical surfactant, to reactive oxygen species arising from hypochlorous acid or the Fenton reaction resulted in an increase in lipid (conjugated dienes, lipid aldehydes) and protein (carbonyls) oxidation products and a reduction in surface activity. Experiments where oxidized phospholipids (PL) were mixed with BLES demonstrated that this addition hampered BLES biophysical activity. However the effects were only moderately greater than with control PL. These results imply a critical role for protein oxidation. BLES oxidation by either method resulted in alterations in surfactant proteins SP-B and SP-C, as evidenced by altered Coomassie blue and silver staining. Western blot analyses showed depressed reactivity with specific antibodies. Oxidized SP-C showed decreased palmitoylation. Reconstitution experiments employing PL, SP-B, and SP-C isolated from control or oxidized BLES demonstrated that protein oxidation was more deleterious than lipid oxidation. Furthermore, addition of control SP-B can improve samples containing oxidized SP-C, but not vice versa. We conclude that surfactant oxidation arising from reactive oxygen species generated by air pollution or leukocytes interferes with surfactant function through oxidation of surfactant PL and proteins, but that protein oxidation, in particular SP-B modification, produces the major deleterious effects.

This review is focused on the evolution and function of alveolar proteins. The lung faces physical and environmental challenges, due to changing pressures/volumes and foreign pathogens, respectively. The pulmonary surfactant system is integral in protecting the lung from these challenges via two groups of surfactant proteins-the small molecular weight hydrophobic SPs, SP-B and-C, that regulate interfacial adsorption of the lipids, and the large hydrophilic SPs, SPA and-D, which are surfactant collectins capable of inhibiting foreign pathogens. Further aiding pulmonary host defence are non-surfactant collectins and antimicrobial peptides that are expressed across the biological kingdoms. Linking to the first symposium session, which emphasised molecular structure and biophysical function of surfactant lipids and proteins, this review begins with a discussion of the role of temperature and hydrostatic pressure in shaping the evolution of SP-C in mammals. Transitioning to the role of the alveolus in innate host defence we discuss the structure, function and regulation of antimicrobial peptides, the defensins and cathelicidins. We describe the recent discovery of novel avian collectins and provide evidence for their role in preventing influenza infection. This is followed by discussions of the roles of SPA and SP-D in mediating host defence at the alveolar surface and in mediating inflammation and the allergic response of the airways. Finally we discuss the use of animal models of lung disease including knockouts to develop an understanding of the role of these proteins in initiating and/or perpetuating disease with the aim of developing new therapeutic strategies.

Hydrolysis of surfactant phospholipids (PL) by secretory phospholipases A2(sPLA2) contributes to surfactant damage in inflammatory airway diseases such as acute lung injury/acute respiratory distress syndrome. We and others have reported that each sPLA2exhibits specificity in hydrolyzing different PLs in pulmonary surfactant and that the presence of hydrophilic surfactant protein A (SP-A) alters sPLA2-mediated hydrolysis. This report tests the hypothesis that hydrophobic SP-B also inhibits sPLA2-mediated surfactant hydrolysis. Three surfactant preparations were used containing varied amounts of SP-B and radiolabeled tracers of phosphatidylcholine (PC) or phosphatidylglycerol (PG): 1) washed ovine surfactant (OS) (pre- and postorganic extraction) compared with Survanta (protein poor), 2) Survanta supplemented with purified bovine SP-B (1–5%, wt/wt), and 3) a mixture of dipalmitoylphosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and 1-palmitoyl-2-oleoyl-pho...