Papers by Hector Rodriguez
p -Coumaric acid decarboxylase from Lactobacillus plantarum : Structural insights into the active site and decarboxylation catalytic mechanism
Proteins: Structure, Function, and Bioinformatics, 2010
p-Coumaric acid decarboxylases (PDCs) catalyze the nonoxidative decarboxylation of hydroxycinnami... more p-Coumaric acid decarboxylases (PDCs) catalyze the nonoxidative decarboxylation of hydroxycinnamic acids to generate the corresponding vinyl derivatives. Despite the biotechnological relevance of PDCs in food industry, their catalytic mechanism remains largely unknown. Here, we report insights into the structural basis of catalysis for the homodimeric PDC from Lactobacillus plantarum (LpPDC). The global fold of LpPDC is based on a flattened beta-barrel surrounding an internal cavity. Crystallographic and functional analyses of single-point mutants of residues located within this cavity have permitted identifying a potential substrate-binding pocket and also to provide structural evidences for rearrangements of surface loops so that they can modulate the accessibility to the active site. Finally, combination of the structural and functional data with in silico results enables us to propose a two-step catalytic mechanism for decarboxylation of p-coumaric acid by PDCs where Glu71 is involved in proton transfer, and Tyr18 and Tyr20 are involved in the proper substrate orientation and in the release of the CO(2) product.
Stability and folding properties of a model β-sheet protein, escherichia coli cspa
Protein Science, 1998
Protein Science (1998). 7470479. Cambridge University Press. Printed in the USA Copyright 0 1998 ... more Protein Science (1998). 7470479. Cambridge University Press. Printed in the USA Copyright 0 1998 The Protein Society ... Stability and folding properties of a model P-sheet ... KATHERINE L. REID, HECTOR M. RODRIGUEZ, BRIAN J. HILLIER, AND LYDIA M. GREGORET ...
Examination of the folding of E. coli CspA through tryptophan substitutions
Protein Science, 2001
Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an... more Escherichia coli cold shock protein, CspA, folds very rapidly (time constant, tau = 4 msec) by an apparent two-state mechanism. However, recent time-resolved infrared (IR) temperature-jump experiments indicate that the folding trajectory of CspA may be more complicated. The sole tryptophan of wild-type CspA (Trp11), which is used to monitor the folding process by fluorescence spectroscopy, is located in an unusual aromatic cluster on the surface of CspA within the nucleic acid binding site. To gain a more global picture of the folding kinetics of CspA and to determine if there are any previously undetected intermediates, we have introduced a second tryptophan at three different surface locations in the protein. The three mutations did not significantly alter the tertiary structure of CspA, although two of the substitutions were found to be slightly stabilizing. Two-state folding, as detected by stopped-flow fluorescence spectroscopy, is preserved in all three mutants. These results indicate that the fast folding of CspA is driven by a concerted mechanism.
Allosteric inhibition of lysyl oxidase–like-2 impedes the development of a pathologic microenvironment
Nature Medicine, 2010
We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation ... more We have identified a new role for the matrix enzyme lysyl oxidase-like-2 (LOXL2) in the creation and maintenance of the pathologic microenvironment of cancer and fibrotic disease. Our analysis of biopsies from human tumors and fibrotic lung and liver tissues revealed an increase in LOXL2 in disease-associated stroma and limited expression in healthy tissues. Targeting LOXL2 with an inhibitory monoclonal antibody (AB0023) was efficacious in both primary and metastatic xenograft models of cancer, as well as in liver and lung fibrosis models. Inhibition of LOXL2 resulted in a marked reduction in activated fibroblasts, desmoplasia and endothelial cells, decreased production of growth factors and cytokines and decreased transforming growth factor-beta (TGF-beta) pathway signaling. AB0023 outperformed the small-molecule lysyl oxidase inhibitor beta-aminoproprionitrile. The efficacy and safety of LOXL2-specific AB0023 represents a new therapeutic approach with broad applicability in oncologic and fibrotic diseases.
Journal of Agricultural and Food Chemistry, 2008
It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 T were able t... more It was previously reported that cell cultures from Lactobacillus plantarum CECT 748 T were able to decarboxylate phenolic acids, such as p-coumaric, m-coumaric, caffeic, ferulic, gallic, and protocatechuic acid. The p-coumaric acid decarboxylase (PDC) from this strain has been overexpressed and purified. This PDC differs at its C-terminal end when compared to the previously reported PDC from L. plantarum LPCHL2. Because the C-terminal region of PDC is involved in enzymatic activity, especially in substrate activity, it was decided to biochemically characterize the PDC from L. plantarum CECT 748 T . Contrarily to L. plantarum LPCHL2 PDC, the recombinant PDC from L. plantarum CECT 748 T is a heat-labile enzyme, showing optimal activity at 22°C. This PDC is able to decarboxylate exclusively the hydroxycinnamic acids p-coumaric, caffeic, and ferulic acids. Kinetic analysis showed that the enzyme has a 14-fold higher K M value for p-coumaric and caffeic acids than for ferulic acid. PDC catalyzes the formation of the corresponding 4-vinyl derivatives (vinylphenol and vinylguaiacol) from p-coumaric and ferulic acids, respectively, which are valuable food additives that have been approved as flavoring agents. The biochemical characteristics showed by L. plantarum PDC should be taken into account for its potential use in the food-processing industry.
International Journal of Food Microbiology, 2008
In foods, tannins are considered nutritionally undesirable. Spectrophotometric methods have been ... more In foods, tannins are considered nutritionally undesirable. Spectrophotometric methods have been used to detect tannin degradation by L. plantarum strains isolated from food substrates. Enzymatic degradation of tannic acid by L. plantarum CECT 748 T was examined in liquid cultures and in cell-free extracts by HPLC. Significative reduction of tannic acid was not observed during incubation in the presence of L. plantarum cells after 7 days incubation. However, tannic acid was effectively degraded by cell-free extracts of L. plantarum during 16 h incubation. We have partially characterized L. plantarum tannase activity by measuring its esterase activity on methyl gallate. Tannase activity was optimal at pH 5.0 and 30°C, and showed nearly 75% of the maximal activity at 50°C. The biochemical characteristics showed by L. plantarum tannase are considered favourable for tannin biodegradation in the food-processing industry.
International Journal of Food Microbiology, 2007
Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wi... more Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wine lactobacilli such as Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis. We establish the specificity of 16S rDNA sequencing in the differentiation of these species and in the rejection of the Lactobacillus vermiforme species name. Moreover, we succeeded in differentiating these heterofermentative species by means of recA gene sequence comparison. Short homologous regions were amplified by PCR with degenerate consensus primers, sequenced, and 280 bp were analysed and considered for the inference of phylogenetic trees. The phylogram obtained was coherent and clearly separated the three species. The recA gene sequence was a reliable and useful method that allowed a good discrimination among closely related species. The validity of the recA gene sequence, restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR), and random amplified polymorphic DNA (RAPD) to study the L. hilgardii intraspecies heterogeneity was tested in five strains isolated from stuck wine fermentations at the same winery in the same vintage. The results indicated that L. hilgardii is a heterogeneous species. Since L. hilgardii is a malolactic species that can influence the final quality of the wine, the presence of oenological relevant genes, such as those involved in ethyl carbamate or biogenic amine production, was investigated.
International Journal of Food Microbiology, 2009
Phenolic compounds are important constituents of food products of plant origin. These compounds a... more Phenolic compounds are important constituents of food products of plant origin. These compounds are directly related to sensory characteristics of foods such as flavour, astringency, and colour. In addition, the presence of phenolic compounds on the diet is beneficial to health due to their chemopreventive activities against carcinogenesis and mutagenesis, mainly due to their antioxidant activities. Lactic acid bacteria (LAB) are autochthonous microbiota of raw vegetables. To get desirable properties on fermented plant-derived food products, LAB has to be adapted to the characteristics of the plant raw materials where phenolic compounds are abundant. Lactobacillus plantarum is the commercial starter most frequently used in the fermentation of food products of plant origin. However, scarce information is still available on the influence of phenolic compounds on the growth and viability of L. plantarum and other LAB species. Moreover, metabolic pathways of biosynthesis or degradation of phenolic compounds in LAB have not been completely described. Results obtained in L. plantarum showed that L. plantarum was able to degrade some food phenolic compounds giving compounds influencing food aroma as well as compounds presenting increased antioxidant activity. Recently, several L. plantarum proteins involved in the metabolism of phenolic compounds have been genetically and biochemically characterized. The aim of this review is to give a complete and updated overview of the current knowledge among LAB and food phenolics interaction, which could facilitate the possible application of selected bacteria or their enzymes in the elaboration of food products with improved characteristics.
Metabolism of food phenolic acids by Lactobacillus plantarum CECT 748T
Food Chemistry, 2008
Phenolic acids account for almost one third of the dietary phenols and are associated with organo... more Phenolic acids account for almost one third of the dietary phenols and are associated with organoleptic, nutritional and antioxidant properties of foods. This study was undertaken to assess the ability of Lactobacillus plantarum CECT 748T to metabolize 19 food phenolic acids. Among the hydroxycinnamic acids studied, only p-coumaric, caffeic, ferulic and m-coumaric acids were metabolized by L. plantarum. Cultures of
Food Chemistry, 2008
Lactobacillus plantarum is the main species responsible for the spontaneous fermentation of Spani... more Lactobacillus plantarum is the main species responsible for the spontaneous fermentation of Spanish-style green olives. Olives and virgin oil provide a rich source of phenolic compounds. This study was designed to evaluate inhibitory growth activities of nine olive phenolic compounds against four L. plantarum strains isolated from different sources, and to explore the L. plantarum metabolic activities against these phenolic compounds. None of the nine compounds assayed (oleuropein, hydroxytyrosol, tyrosol, as well as vanillic, p-hydroxybenzoic, sinapic, syringic, protocatechuic and cinnamic acids) inhibited L. plantarum growth at the concentration found in olive products. Oleuropein and tyrosol concentrations higher than 100 mM were needed to inhibit L. plantarum growth. On the other hand, sinapic and syringic acid showed the highest inhibitory activity since concentrations ranging from 12.5 to 50 mM inhibited L. plantarum growth in all the strains analyzed. Among the nine compounds assayed, only oleuropein and protocatechuic acid were metabolized by L. plantarum strains grown in the presence of these compounds. Oleuropein was metabolized mainly to hydroxytyrosol, while protocatechuic acid was decarboxylated to catechol. Metabolism of oleuropein was carried out by inducible enzymes since a cell-free extract from a culture grown in the absence of oleuropein was unable to metabolize it. Independent of their isolation source, the four L. plantarum strains analysed showed similar behaviour in relation to the inhibitory activity of phenolic compounds, as well as their ability to metabolize these compounds.
Food Chemistry, 2008
The ability of Lactobacillus plantarum CECT 748 T to degrade hydrolysable tannins was evaluated. ... more The ability of Lactobacillus plantarum CECT 748 T to degrade hydrolysable tannins was evaluated. Three commercial tannic acids were incubated in presence of cell-free extracts containing soluble proteins from L. plantarum. By HPLC analyses, almost a complete tannic acid degradation was observed in the three samples assayed. By using HPLC-DAD/ESI-MS, we partially determined the composition of tannic acid from Quercus infectoria galls. This tannic acid is a gallotannin mainly composed of monomers to tetramers of gallic acid. We studied the mechanism of its degradation by L. plantarum. The results obtained in this work indicated that L. plantarum degrades gallotannins by depolymerisation of high molecular weight tannins and a reduction of low molecular weight tannins. Gallic acid and pyrogallol were detected as final metabolic intermediates. Due to the potential health beneficial effects, the ability to degrade tannic acid is an interesting property in this food lactic acid bacteria.
Food Chemistry, 2010
The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis... more The potential to degrade 15 food phenolic acids was investigated for several Lactobacillus brevis strains isolated from different sources. All the strains analysed in this study showed a similar metabolism on phenolic acids. Among the cinnamic acids assayed, only p-coumaric, ferulic and caffeic acids were metabolized by the L. brevis strains. These acids were decarboxylated to produce their corresponding vinyl derivatives. Contrarily to the results previously reported on Lactobacillus plantarum, the L. brevis strains analysed in this study were unable to subsequently reduce or metabolize these vinyl derivatives. In L. brevis, vinyl phenol, vinyl catechol, and vinyl guaiacol were the final metabolic products from p-coumaric, caffeic or ferulic acids, respectively. From the benzoic acids analysed, and similarly to L. plantarum strains, only gallic and protocatechuic acids were modified by L. brevis strains. Both acids were decarboxylated to pyrogallol and catechol, respectively. Currently, the enzymes involved in the metabolism of phenolic acids in L. brevis remain uncharacterized.
Native State EX2 and EX1 Hydrogen Exchange of Escherichia coli CspA, a Small β-Sheet Protein †
Biochemistry, 2002
Escherichia coli CspA is a small all-beta-sheet protein that folds fast (tau = 4 ms) via an appar... more Escherichia coli CspA is a small all-beta-sheet protein that folds fast (tau = 4 ms) via an apparent two-state mechanism. Our previous studies have shown that a large aromatic cluster on the surface of the protein participates in the rate-limiting step of folding and thus may be part of the folding nucleus of this protein. To obtain a more detailed picture of molecular events at the peptide backbone during unfolding and folding of CspA, we used native state hydrogen exchange and nuclear magnetic resonance spectroscopy (NMR). The experiments with native CspA were performed over a range of pH values from low pH, where exchange is governed by a rapid equilibrium before chemical exchange (EX2 exchange), to high pH, where exchange is dictated by the rate of unfolding (EX1 exchange). Rates of folding and unfolding were determined for 11 residues. The distribution of rates of folding within the structure of CspA suggests that hairpin turns, including one near the aromatic cluster, may nucleate the folding of CspA.
Discovery of Omecamtiv Mecarbil the First, Selective, Small Molecule Activator of Cardiac Myosin
ACS Medicinal Chemistry Letters, 2010
We report the design, synthesis, and optimization of the first, selective activators of cardiac m... more We report the design, synthesis, and optimization of the first, selective activators of cardiac myosin. Starting with a poorly soluble, nitro-aromatic hit compound (1), potent, selective, and soluble myosin activators were designed culminating in the discovery of omecamtiv mecarbil (24). Compound 24 is currently in clinical trials for the treatment of systolic heart failure.
Patient Activation is Inconsistently Associated with Positive Health Behaviors Among Obese Safety Net Patients
Journal of Immigrant and Minority Health, 2015
We examine the association of patient activation and physical activity and fruit and vegetable co... more We examine the association of patient activation and physical activity and fruit and vegetable consumption among obese safety net patients. Adult obese patients (n = 198) of three safety net clinics completed a survey assessing patient activation, physical activity, fruit and vegetable consumption, care experiences, and health status. Multivariate logistic regression models incrementally assessed the adjusted relation of patient activation and physical activity and fruit and vegetable consumption. In adjusted analyses, higher activated patients had higher odds [Odds ratio (OR) 1.58, p < 0.01] of consuming fruits and vegetables daily than less activated patients. There was no significant association between patient activation and regular physical activity. Engaging in regular physical activity appears to be difficult, even for highly activated patients. In contrast, additional fruit and vegetable consumption is a relatively easier change. Patient activation was inconsistently associated with two positive health behaviors among obese safety net patients.
The Effect of Item Screeners on the Quality of Patient Survey Data: A Randomized Experiment of Ambulatory Care Experience Measures
Background: The use of item screeners is viewed as an essential feature of quality survey design ... more Background: The use of item screeners is viewed as an essential feature of quality survey design because only respondents who are 'qualified' to answer questions that apply to a subset of the sample are directed to answer. However, empirical evidence supporting this view is scant. Abstract: Objective: This study compares data quality resulting from the administration of ambulatory care experience
Population health management, Jan 28, 2015
The objectives were to assess the associations between fall-related injuries (FRIs) treated in th... more The objectives were to assess the associations between fall-related injuries (FRIs) treated in the emergency department (ED) among older adults in California and contextual county-level physical, social, and economic characteristics, and to assess how county-level economic conditions are associated with FRIs when controlling for other county-level factors. Data from 2008 California ED discharge, Medicare Impact File, and County Health Rankings were used. Random effects logistic regression models estimated contextual associations between county-level factors representing economic conditions, the built environment, community safety, access to care, and obesity with patient-level FRI treatment among 1,712,409 older adults, controlling for patient-level and hospital-level characteristics. Patient-level predictors of FRI treatment were consistent with previous studies not accounting for contextual associations. Larger and rural hospitals had higher odds of FRI treatment, while teaching a...
The Journal of ambulatory care management
Teamlets of physicians and medical assistants may help improve obesity management in primary care... more Teamlets of physicians and medical assistants may help improve obesity management in primary care settings. We aimed to understand the barriers and facilitators of implementing a teamlet approach to managing obesity in 3 safety net clinics. Key stakeholder interviews (n = 21) were conducted both during early implementation of practice change and 6 months later. Patient surveys (n = 393) examined obese patient activation and health status. Insufficient program resources and limited patient engagement due to external factors were implementation barriers despite fairly high patient activation. Staff members need time and resources to execute new responsibilities to support obesity management in safety net settings. Because of high turnover, multiple supporters may improve sustainability.
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Papers by Hector Rodriguez