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1.
Vet World ; 17(5): 1168-1176, 2024 May.
Article in English | MEDLINE | ID: mdl-38911073

ABSTRACT

Background and Aim: Klebsiella pneumoniae is one of the most common causes of clinical and asymptomatic mastitis in dairy cattle, as well as in milk and dairy products that affect milk quality. Mastitis caused by K. pneumoniae is even more serious due to its poor response to antibiotic therapy. The aim of this study was to detect and identify the presence of K. pneumoniae in milk and dairy products produced in Libya. Materials and Methods: A total of 234 samples were randomly collected from various locations in Libya. Samples were examined for the presence of K. pneumoniae using conventional cultural techniques, including cultivation in violet red bile agar plus 4-methylumbelliferyl-ß-D-glucuronide broth and CHROM agar, followed by polymerase chain reaction identification and partial sequencing of 16S rRNA. Results: Of the 234 samples of milk and dairy products collected, 16 (6.8%) isolates revealed mucoid colonies on agar media that were phenotypically suggested to be K. pneumoniae. Identification of isolates was confirmed using molecular techniques (16S rRNA). Among the examined samples, K. pneumoniae was recovered from camel's milk, raw cow's milk, raw fermented milk, Maasora cheese, Ricotta cheese, soft cheese, full cream milk powder, milk powder infant formula, cereal baby food, and growing-up formula. Antibiotic susceptibility testing was performed on 12 of the 16 K. pneumoniae isolates, and the results showed that K. pneumoniae isolates were resistant to more than eight antibiotics; interestingly, two isolates showed metallo-beta-lactamase (MBL) production. Conclusion: K. pneumoniae is considered a risk to human health because many of these products do not comply with the microbiological criteria of international and/or Libyan standards. This study emphasized the relationship between K. pneumoniae and raw milk, cheese, milk powder, and infant milk retailed in Libya. There is a need to take the necessary measures to ensure effective hygiene practices during production in dairy factories, handling, and distribution on the market, in particular at a small local production scale.

2.
Open Vet J ; 13(1): 108-113, 2023 01.
Article in English | MEDLINE | ID: mdl-36777432

ABSTRACT

Background: Cronobacter sspecies are the most significant foodborne pathogen in infant milk formula (IMF). These pathogens have been incriminated in severe forms of neonatal meningitis, sepsis, and necrotizing enterocolitis with a high mortality rate. Aim: This study was performed to elucidate the effect of heat stress on Cronobacter spp. (C. sakazakii and C. pulveris) in reconstituted IMF (RIMF). Methods: The reconstituted formula was inoculated with five C. sakazakii isolates and four C. pulveris isolates separately. The nine isolates of Cronobacter spp. were heated in RIMF at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C. The D- and z-values were determined by using linear regression analysis. Results: The D-values of all isolates of C. sakazakii (CS1, CS3, CS4, CS5, and CS6) at 48°C, 52°C, 56°C, 60°C, 64°C, and 66°C were in the ranges 7.29-23.47, 2.77-15.50, 0.62-1.04, 0.62-1.02, 0.62-1.00, 0.62-1.00 minutes, respectively; while, the z-values extended from 2.50°C to 4.28°C. The D- values of C. pulveris isolates (CP1, CP2, CP3, CP4) were in the ranges 7.60-22.32, 1.42-8.45, 0.62-1.08, 0.62-0.78, 0.62-0.78, 0.62-0.79 minutes at 48°C, 52°C, 56°C, 60°C, 64°C, 66°C, respectively and the calculated z-values ranged from 3.33°C to 4.89°C. Conclusion: This study may contribute to improving the understanding of the behavior of C. sakazakii and C. pulveris isolates in RIMF at various heat stress temperatures and may participate in the effective control of these pathogens in infant food production.


Subject(s)
Cronobacter sakazakii , Animals , Milk , Food Microbiology , Infant Formula
3.
Cureus ; 15(12): e50981, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38259413

ABSTRACT

BACKGROUND: Helicobacter pylori infection is prevalent among Saudi adults and has been linked to gastric cancer and other tumor-like conditions. We aimed to explore the pathological characteristics of endoscopic gastric biopsies among symptomatic adult Saudi patients and their relation to H. pylori infection. RESULTS: Among 151 gastric biopsies, gastritis was detected in 97 (64.2%) cases, chronic active gastritis in 26 patients (17.2%), duodenitis in 20 (13.2%) patients, and total metaplasia in 14 (9.3%) patients. H. pylori was detected in 83 cases (55%), with a recurrence or reinfection rate of 9.8%. The patients with H. pylori infection were considerably young (median age: 34 (IQR: 15) vs. 35.5 (IQR: 11), p = 0.024) and had a low frequency of epigastric pain (78.3% vs. 91.2%, p = 0.031), reflux/regurgitation (7.2% vs. 20.6%, p = 0.016), and dysphagia (4.85% vs. 14.7%, p = 0.037). However, they exhibited a higher incidence of chronic active gastritis (96.2% vs. 3.8%, p < 0.001) and intestinal metaplasia (85.7% vs. 14.3%, p = 0.015). Young age (OR = 1.09, 95% CI = 1.02-1.16, p = 0.011) and H. pylori infection (OR = 30.85, 95% CI = 3.26-291.60, p = 0.003) were identified as a positive predictor of intestinal metaplasia while heartburn (OR = 0.08, 95% CI = 0.01-0.58, p = 0.012) was a negative predictor. CONCLUSION: H. pylori infection is prevalent among Saudi adults experiencing upper gastrointestinal symptoms and is associated with intestinal metaplasia. Infection rate and intestinal metaplasia were higher in patients with milder symptoms. Therefore, screening for H. pylori is highly recommended for Saudi individuals with upper gastrointestinal symptoms. Old age and H. pylori infection were identified as positive predictors of intestinal metaplasia, emphasizing the importance of early detection and management of H. pylori infection in the Saudi population.

4.
Vet World ; 15(5): 1185-1190, 2022 May.
Article in English | MEDLINE | ID: mdl-35765472

ABSTRACT

Background and Aim: Foodborne illnesses are a serious challenge to human health and the economic sector. For example, salmonellosis remains a burden in developed and developing nations. Rapid and reliable molecular methods to identify Salmonella strains are essential for minimizing human infection. This study aimed to identify Salmonella spp. in raw milk and dairy products using conventional and molecular techniques and to test the antibiotic susceptibility of the isolated strains. Materials and Methods: One hundred and thirty-one milk and dairy product samples were randomly collected from different localities in Libya. Samples were examined for the presence of Salmonella by conventional culture techniques, including cultivation in Rappaport-Vassiliadis broth and streaking on xylose lysine deoxycholate agar. Identification also used polymerase chain reaction and partial sequencing of 16S rDNA. Twenty-four antibiotics were used for the examination of antimicrobial resistance of Salmonella spp. isolates with the agar disk diffusion method (Kirby-Bauer technique). Multi-antibiotic resistance index and antibiotic resistance index (ARI)for Salmonella enterica isolates were calculated. Results: Twenty-one of 131 samples (16%) were positive for Salmonella spp. recovered from 9 (16%), 2 (11%), 4 (22.2%), and 6 (46%) samples of raw cow milk, fermented raw milk, and fresh locally made soft cheeses, Maasora and Ricotta), respectively. Samples of ice cream, milk powder, and infant formula showed no Salmonella spp. contamination. Only 9 of 21 (42.8%) isolates were confirmed as S. enterica by partial sequence 16S rDNA analysis. All isolates were resistant to amoxycillin, bacitracin, penicillin G, lincomycin, vancomycin, clindamycin, and cloxacillin with an ARI of 0.042. In contrast, all tested strains were sensitive to levofloxacin, doxycycline, and ciprofloxacin. In addition, all of the tested isolates (100%) were resistant to more than one antibiotic. Conclusion: This study demonstrated the applicability of molecular techniques, compared with conventional methods, as preferable for the identification of Salmonella in milk and dairy products and thus reduction of milk-borne transmission to the consumers. From the view of public health, isolation and identification of Salmonella multidrug-resistant strains from raw cow`s milk and locally prepared dairy products sold in the Libyan markets indicate the need to improve the handling and processing of milk and dairy products to minimize the prevalence of Salmonella, one of the most important foodborne microorganisms that cause food poisoning.

5.
ACS Synth Biol ; 10(2): 228-235, 2021 02 19.
Article in English | MEDLINE | ID: mdl-33535752

ABSTRACT

Conjugated alkenes such as dienes and polyenes have a range of applications as pharmaceutical agents and valuable building blocks in the polymer industry. Development of a renewable route to these compounds provides an alternative to fossil fuel derived production. The enzyme family of the UbiD decarboxylases offers substantial scope for alkene production, readily converting poly unsaturated acids. However, biochemical pathways producing the required substrates are poorly characterized, and UbiD-application has hitherto been limited to biological styrene production. Herein, we present a proof-of-principle study for microbial production of polyenes using a bioinspired strategy employing a polyketide synthase (PKS) in combination with a UbiD-enzyme. Deconstructing a bacterial iterative type II PKS enabled repurposing the broad-spectrum antibiotic andrimid biosynthesis pathway to access the metabolic intermediate 2,4,6-octatrienoic acid, a valuable chemical for material and pharmaceutical industry. Combination with the fungal ferulic acid decarboxylase (Fdc1) led to a biocatalytic cascade-type reaction for the production of hepta-1,3,5-triene in vivo. Our approach provides a novel route to generate unsaturated hydrocarbons and related chemicals and provides a blue-print for future development and application.


Subject(s)
Anti-Bacterial Agents/metabolism , Aspergillus niger/enzymology , Carboxy-Lyases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Fungal Proteins/metabolism , Metabolic Engineering/methods , Biocatalysis , Escherichia coli/genetics , Fatty Acids, Unsaturated/metabolism , Polyenes/metabolism , Polyketide Synthases/metabolism , Pyrroles/metabolism
6.
Open Vet J ; 10(3): 276-288, 2020 10.
Article in English | MEDLINE | ID: mdl-33282699

ABSTRACT

Background: Whole muscle meat, meat products, and seafood contain different nutrients in adequate quantity providing a better environment for presence and replication of different microorganisms. There are underreported and inaccurate estimations of foodborne diseases due to the lack of effective surveillance systems in Libya. Aim: To determine the extent of microbiological contamination of whole muscle meat, meat products, and seafood. Methods: A total number of 731 samples of retail meat were collected from different stores in four cities in Libya. Samples were analyzed for aerobic plate count and subjected to microbiological enumeration and isolation techniques, followed by molecular identification by PCR and partial sequencing of 16S rDNA. Results: The results showed contamination of samples with enteric and spoilage bacteria. Fifteen genera of spoilage bacteria yielded 149 isolates which were detected and identified by PCR and partial sequencing of 16S rDNA as: Proteus spp., Provedencia spp., Raouttella ornithinolytical, Citrobacter spp., Enterobacter spp., Morganella morgi, Shewanella algea, Rhodobacter capsulatus, Listonella pelagia, Kluyvera spp., Pectobacterium spp., Brenneria spp., Klebsiella spp., Acintobacter radioresistens, and Pantoea spp. While for pathogenic bacteria, 143 isolates distributed among nine genera were identified by PCR and partial sequencing of 16S rDNA as: Bacillus spp., Escherichia spp., Shigella spp., Enterococci spp., Cronobacter spp., Staphylococci spp., Salmonella spp., Aeromonas spp., and Vibrio spp.. Many isolated bacteria are zoonotic bacteria with high importance for public health. Conclusion: Excessive handling and processing of meat and meat products seems to be one of the poorest microbiological qualities. These findings ought to be helpful in risk assessments and quality assurance of meat in order to improve food safety.


Subject(s)
Bacteria/isolation & purification , Food Microbiology , Meat Products/microbiology , Meat/microbiology , Seafood/microbiology , Libya
7.
J Environ Public Health ; 2020: 5379619, 2020.
Article in English | MEDLINE | ID: mdl-32405302

ABSTRACT

This study aimed to investigate the poisoning symptoms occurring in pesticide-exposed farmers. This study was conducted in a red onion farming center area in Wanasari Subdistrict, Brebes, Indonesia, from May to June 2018. This study was designed as the descriptive study. The significance value of p < 0.005 showed that the variable was correlated with the health problems, including excessive fatigue (p value = 0.041), excessive saliva (p value = 0.006), hard breathing (p value = 0.021), frequent urination (p value = 0.047), blurred vision (p value = 0.059), dizziness (p value = 0.032), and finger pain (p value = 0.007). The significance value (p < 0.005) of 0.000 showed that the use of personal protective equipment was correlated with the health problems. Based on the odds ratio value of 1.137, 95% confidence interval = 0.042-0.444 meant that the personal protective equipment was a risk factor of health problems. The results showed that 89.2% of the farmers who used the personal protective equipment were categorized in the healthy group.


Subject(s)
Agricultural Workers' Diseases/epidemiology , Farmers/statistics & numerical data , Occupational Exposure/adverse effects , Personal Protective Equipment/statistics & numerical data , Pesticides/poisoning , Adult , Agricultural Workers' Diseases/pathology , Agricultural Workers' Diseases/physiopathology , Agricultural Workers' Diseases/prevention & control , Humans , Indonesia/epidemiology , Male , Occupational Exposure/prevention & control , Occupational Exposure/statistics & numerical data , Risk Factors
8.
Transplant Proc ; 52(1): 175-178, 2020.
Article in English | MEDLINE | ID: mdl-31924404

ABSTRACT

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are newly introduced hypoglycemic drugs that work by inhibiting glucose reabsorption at proximal renal tubules. The use of SGLT2 inhibitors in nontransplant diabetic patients with or without cardiovascular disease has well-established efficacy and safety. The risks of renal graft dysfunction and urinary tract infections might be the limiting factors for their use in renal transplant patients. Data regarding the safety and long-term efficacy of SGLT2 inhibitors use in diabetic renal transplant patients is scanty. The aim of the study is to report our experience with use of SGLT2 inhibitors in 8 diabetic renal transplant patients supported by literature review. Eight diabetic renal transplant patients were recruited from Tawam hospital during the period between June 2016 and January 2019. Demographic, clinical, and laboratory data were collected and analyzed. Adding SGLT2 resulted in significant decrease in hemoglobin A1c and body mass index after 12 months of treatment. There was significant negative correlation between the duration of treatment with SGLT2 and hemoglobin A1c. Diabetic renal transplant patients with stable kidney function had better glycemic control with use of SGLT2 inhibitors. There was no deterioration of kidney function and risk of recurrent urinary tract infection was low.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Hypoglycemic Agents/therapeutic use , Kidney Transplantation , Sodium-Glucose Transporter 2 Inhibitors/therapeutic use , Aged , Female , Humans , Male , Middle Aged , Retrospective Studies , Sodium-Glucose Transporter 2
9.
Ann Chir Plast Esthet ; 65(3): 263-268, 2020 Jun.
Article in English | MEDLINE | ID: mdl-31607500

ABSTRACT

We report a case of pedicled omental flap use together with osteosynthesis treatment of a chronic L4-L5 spondylodiscitis due to a large sacral eschar. The 43-years-old patient was paraplegic and had depleted regional flaps solutions due to multiple previous surgeries. The procedure was carried out in supine position then in prone position whereby the dissected flap was recovered through the spine. The surgery was performed by a multidisciplinary team. First, we used an anterior approach for spine osteosynthesis with a metal implant and flap harvest. Then, in a prone position, we completed the vertebral reconstruction by an L3 athrodesis to the pelvis. The flap was recovered through the spine defect, on the side of the implant. It was a right sided pedicled. Complete wound healing was 120 days. The omental flap proved to be a reliable solution in the absence of recipient vessels for free flap transfer and depleted regional flap solutions. It also spared the latissimus dorsi muscle required for a wheelchair user as in our case. The omental flap is still performed in spine surgery especially in oncologic context to prevent wound dehiscence and for spondylodiscitis coverage. The anterior approach allows for both spine osteosynthesis and flap dissection.


Subject(s)
Free Tissue Flaps , Omentum/transplantation , Pressure Ulcer/surgery , Sacrum , Adult , Discitis/complications , Humans , Lumbar Vertebrae , Lumbosacral Region , Male , Pressure Ulcer/etiology , Plastic Surgery Procedures/methods
10.
Pak J Med Sci ; 35(4): 1122-1127, 2019.
Article in English | MEDLINE | ID: mdl-31372154

ABSTRACT

OBJECTIVES: To explore the correlation between maternal and cord blood prolactin, the correlation between cord prolactin and birth weight, and to compare cord blood prolactin in new-borns of women with normal pregnancy and women with pregnancy complications namely; gestational hypertension, gestational diabetes and preterm labour. METHODS: This study was performed from September to December 2018. Thirty-two women, delivered at Baghdad teaching hospital, and their newborns (32) were included. Maternal blood (5 ml) was taken before labour and cord blood (5 ml) was collected after placenta expulsion. Maternal and cord blood prolactin were analysed using fluorescence immunoassay. RESULTS: Cord blood prolactin was higher in babies born to hypertensive women (405.28±77.52 ng/ml) than normal pregnancy women (244.80±60.80 ng/ml), P=0.000. Also, cord prolactin in gestational hypertension group was significantly higher than diabetic (P=0.006) and preterm labour (P=0.000) groups. No significant difference was noticed in cord blood prolactin in newborns of diabetic and normal pregnancy women (299.28±37.01, 244.80±60.80 ng/ml respectively, P=0.053). Preterm babies had lower cord prolactin (204.57±22.90 ng/ml) than normal pregnancy babies (244.80±60.80 ng/ml), however the difference was non-significant, P=0.118. Positive correlation was found between cord and maternal prolactin (P=0.000) and between cord prolactin and birth weight (P=0.018). CONCLUSION: Cord blood prolactin is high in newborns of hypertensive women, low in preterm neonates. Diabetes has no effect on cord prolactin level.

11.
Chembiochem ; 20(6): 785-792, 2019 03 15.
Article in English | MEDLINE | ID: mdl-30431225

ABSTRACT

The monoterpenoid lactone derivative (+)-dihydrocarvide ((+)-DHCD) can be polymerised to form shape-memory polymers. Synthetic biology routes from simple, inexpensive carbon sources are an attractive, alternative route over chemical synthesis from (R)-carvone. We have demonstrated a proof-of-principle in vivo approach for the complete biosynthesis of (+)-DHCD from glucose in Escherichia coli (6.6 mg L-1 ). The pathway is based on the Mentha spicata route to (R)-carvone, with the addition of an 'ene'-reductase and Baeyer-Villiger cyclohexanone monooxygenase. Co-expression with a limonene synthesis pathway enzyme enables complete biocatalytic production within one microbial chassis. (+)-DHCD was successfully produced by screening multiple homologues of the pathway genes, combined with expression optimisation by selective promoter and/or ribosomal binding-site screening. This study demonstrates the potential application of synthetic biology approaches in the development of truly sustainable and renewable bioplastic monomers.


Subject(s)
Lactones/metabolism , Monoterpenes/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli/metabolism , Glucose/chemistry , Lactones/chemistry , Metabolic Engineering/methods , Monoterpenes/chemistry , Proof of Concept Study , Stereoisomerism , Synthetic Biology/methods
12.
Biochemistry ; 57(13): 1997-2008, 2018 04 03.
Article in English | MEDLINE | ID: mdl-29533655

ABSTRACT

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMOPhi1) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMOPhi1. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMOPhi1 for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.


Subject(s)
Bacterial Proteins/chemistry , Lactones/chemistry , Mixed Function Oxygenases/chemistry , Monoterpenes/chemistry , Pseudomonas/enzymology , Rhodococcus/enzymology , Catalysis
13.
Nat Commun ; 6: 7907, 2015 Aug 12.
Article in English | MEDLINE | ID: mdl-26264192

ABSTRACT

The coenzyme B12-dependent photoreceptor protein, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids in response to light. On binding of coenzyme B12 the monomeric apoprotein forms tetramers in the dark, which bind operator DNA thus blocking transcription. Under illumination the CarH tetramer dissociates, weakening its affinity for DNA and allowing transcription. The mechanism by which this occurs is unknown. Here we describe the photochemistry in CarH that ultimately triggers tetramer dissociation; it proceeds via a cob(III)alamin intermediate, which then forms a stable adduct with the protein. This pathway is without precedent and our data suggest it is independent of the radical chemistry common to both coenzyme B12 enzymology and its known photochemistry. It provides a mechanistic foundation for the emerging field of B12 photobiology and will serve to inform the development of a new class of optogenetic tool for the control of gene expression.


Subject(s)
Bacterial Proteins/metabolism , Cobamides/metabolism , Gene Expression Regulation, Bacterial/physiology , Thermus thermophilus/metabolism , Bacterial Proteins/genetics , Chromatography , Cobamides/chemistry , Computer Simulation , Light , Models, Chemical , Models, Molecular , Photochemical Processes , Protein Conformation , Spectrum Analysis/methods , Thermus thermophilus/genetics
14.
J R Soc Interface ; 12(103)2015 Feb 06.
Article in English | MEDLINE | ID: mdl-25505136

ABSTRACT

Environmental exposure to electromagnetic fields is potentially carcinogenic. The radical pair mechanism is considered the most feasible mechanism of interaction between weak magnetic fields encountered in our environment and biochemical systems. Radicals are abundant in biology, both as free radicals and reaction intermediates in enzyme mechanisms. The catalytic cycles of some flavin-dependent enzymes are either known or potentially involve radical pairs. Here, we have investigated the magnetic field sensitivity of a number of flavoenzymes with important cellular roles. We also investigated the magnetic field sensitivity of a model system involving stepwise reduction of a flavin analogue by a nicotinamide analogue-a reaction known to proceed via a radical pair. Under the experimental conditions used, magnetic field sensitivity was not observed in the reaction kinetics from stopped-flow measurements in any of the systems studied. Although widely implicated in radical pair chemistry, we conclude that thermally driven, flavoenzyme-catalysed reactions are unlikely to be influenced by exposure to external magnetic fields.


Subject(s)
Basidiomycota/enzymology , Carbohydrate Dehydrogenases/chemistry , Dinitrocresols/chemistry , Fungal Proteins/chemistry , Magnetic Fields , Models, Chemical , Kinetics , Oxidation-Reduction
15.
FEBS Lett ; 587(17): 2832-41, 2013 Sep 02.
Article in English | MEDLINE | ID: mdl-23831062

ABSTRACT

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a "cycle of knowledge" strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.


Subject(s)
Glycolysis , Models, Biological , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Computer Simulation , Isoenzymes/chemistry , Kinetics , Metabolic Networks and Pathways , Saccharomyces cerevisiae/metabolism , Systems Biology
16.
FEBS Lett ; 587(17): 2860-7, 2013 Sep 02.
Article in English | MEDLINE | ID: mdl-23856461

ABSTRACT

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction.


Subject(s)
Adenine Nucleotides/chemistry , Glycolysis , Models, Biological , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/enzymology , Adenine Nucleotides/physiology , Allosteric Regulation , Fermentation , Kinetics , Saccharomyces cerevisiae Proteins/antagonists & inhibitors , Subcellular Fractions/enzymology , Thermodynamics
17.
J Biol Chem ; 288(29): 21295-21306, 2013 Jul 19.
Article in English | MEDLINE | ID: mdl-23720742

ABSTRACT

Despite high similarity in sequence and catalytic properties, the l-lactate dehydrogenases (LDHs) in lactic acid bacteria (LAB) display differences in their regulation that may arise from their adaptation to different habitats. We combined experimental and computational approaches to investigate the effects of fructose 1,6-bisphosphate (FBP), phosphate (Pi), and ionic strength (NaCl concentration) on six LDHs from four LABs studied at pH 6 and pH 7. We found that 1) the extent of activation by FBP (Kact) differs. Lactobacillus plantarum LDH is not regulated by FBP, but the other LDHs are activated with increasing sensitivity in the following order: Enterococcus faecalis LDH2 ≤ Lactococcus lactis LDH2 < E. faecalis LDH1 < L. lactis LDH1 ≤ Streptococcus pyogenes LDH. This trend reflects the electrostatic properties in the allosteric binding site of the LDH enzymes. 2) For L. plantarum, S. pyogenes, and E. faecalis, the effects of Pi are distinguishable from the effect of changing ionic strength by adding NaCl. 3) Addition of Pi inhibits E. faecalis LDH2, whereas in the absence of FBP, Pi is an activator of S. pyogenes LDH, E. faecalis LDH1, and L. lactis LDH1 and LDH2 at pH 6. These effects can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modeled in protonation states corresponding to pH 6 and pH 7. Overall, the results show a subtle interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDHs of different LABs.


Subject(s)
Bacteria/enzymology , Lactate Dehydrogenases/metabolism , Lactic Acid/metabolism , Allosteric Regulation/drug effects , Bacteria/drug effects , Binding Sites , Biocatalysis/drug effects , Crystallography, X-Ray , Enzyme Activation/drug effects , Fructosediphosphates/pharmacology , Hydrogen-Ion Concentration/drug effects , Isoenzymes/metabolism , Kinetics , Lactate Dehydrogenases/chemistry , Lactate Dehydrogenases/isolation & purification , Models, Biological , Phosphates/pharmacology , Sodium Chloride/pharmacology , Static Electricity
18.
Methods Enzymol ; 500: 215-31, 2011.
Article in English | MEDLINE | ID: mdl-21943900

ABSTRACT

In order to produce a full quantitative description of yeast metabolism, a number of kinetic parameters of enzymes that are important for energy metabolism must be determined experimentally. We aim to determine the prospective in vivo kinetic properties of a range of yeast-purified isoenzymes that are important in energy metabolism, with respect to the concentration of their substrates and products. This endeavor forms part of our systems biology pipeline to facilitate the production of bottom-up models of metabolism. Within this workflow, we implement an infrastructure for medium- to high-throughput determination of the kinetic properties of purified isoenzymes in in vivo like conditions. This includes the use of the KineticsWizard software for data capture and analysis. The captured experimental data are analyzed by the software and subsequently stored in appropriate repositories (MeMo-RK and SABIO-RK). While we focus initially on glycolysis in Saccharomyces cerevisiae, our methodology is generic and can be widely applied to the study of other enzymes and pathways in yeast and other organisms.


Subject(s)
Energy Metabolism , Enzyme Assays/methods , Isoenzymes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Algorithms , Databases, Protein , Glycolysis , Information Management , Isoenzymes/isolation & purification , Kinetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/isolation & purification
20.
BMC Bioinformatics ; 11: 582, 2010 Nov 29.
Article in English | MEDLINE | ID: mdl-21114840

ABSTRACT

BACKGROUND: The behaviour of biological systems can be deduced from their mathematical models. However, multiple sources of data in diverse forms are required in the construction of a model in order to define its components and their biochemical reactions, and corresponding parameters. Automating the assembly and use of systems biology models is dependent upon data integration processes involving the interoperation of data and analytical resources. RESULTS: Taverna workflows have been developed for the automated assembly of quantitative parameterised metabolic networks in the Systems Biology Markup Language (SBML). A SBML model is built in a systematic fashion by the workflows which starts with the construction of a qualitative network using data from a MIRIAM-compliant genome-scale model of yeast metabolism. This is followed by parameterisation of the SBML model with experimental data from two repositories, the SABIO-RK enzyme kinetics database and a database of quantitative experimental results. The models are then calibrated and simulated in workflows that call out to COPASIWS, the web service interface to the COPASI software application for analysing biochemical networks. These systems biology workflows were evaluated for their ability to construct a parameterised model of yeast glycolysis. CONCLUSIONS: Distributed information about metabolic reactions that have been described to MIRIAM standards enables the automated assembly of quantitative systems biology models of metabolic networks based on user-defined criteria. Such data integration processes can be implemented as Taverna workflows to provide a rapid overview of the components and their relationships within a biochemical system.


Subject(s)
Metabolic Networks and Pathways , Systems Biology/methods , Databases, Factual , Models, Biological
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