Activity In Cell-free Extracts Research Articles

Antiproliferative and Antitelomerase Effects of Silymarin on Human Colorectal and Hepatocellular Carcinoma Cells.

Silymarin, a widely-used hepatoprotective agent, has shown antitumor properties in both in vitro and animal studies. Currently, there is limited knowledge regarding silymarin's antitelomerase effects on human colorectal cancer and hepatocyte carcinoma cells. In this study, we investigated the antiproliferative and antitelomerase effects of silymarin on four human colorectal cancer and HepG2 hepatocyte carcinoma cell lines. The cell viability and telomerase activity were assessed using MTT and the telomerase repeat amplification protocol assay, respectively. We also investigated the effects of silymarin on the expression of human telomerase reverse transcriptase and its promoter methylation in HepG2 cells by real-time RT-PCR and methylation-specific PCR, respectively. Silymarin treatment inhibited cell proliferation and telomerase activity in all cancer cells. After 24 h of treatment, silymarin exhibited IC50 values ranging from 19 - 56.3 µg/mL against these cancer cells. A 30-min treatment with silymarin at the IC50 concentration effectively inhibited telomerase activity in cell-free extracts of both colorectal cancer and hepatocyte carcinoma cells. Treatment of HepG2 cells with 10 and 30 µg/mL of silymarin for 48 h resulted in a decrease in human telomerase reverse transcriptase expression to 75 and 35% of the level observed in the untreated control (p < 0.01), respectively. Treatment with silymarin (10, 30, and 60 µg/mL) for 48 h did not affect human telomerase reverse transcriptase promoter methylation in HepG2 cells. In conclusion, our findings suggest that silymarin inhibits cancer cell growth by directly inhibiting telomerase activity and downregulating its human telomerase reverse transcriptase catalytic subunit. However, silymarin did not affect human telomerase reverse transcriptase promoter methylation at the concentrations of 10 - 60 µg/mL used in this study.

Effects of resveratrol on the urease-positive urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis

Resveratrol (3, 5, 4’-trihydroxy-Trans-stilbene) is a polyphenolic plant metabolite with antibacterial, antioxidant, and anti-inflammatory activities. Staphylococcus saprophyticus and Proteus mirabilis were inhibited by pure resveratrol with minimum inhibitory concentrations of 250 to 500 µg ml-1. Urease activity in cell-free extracts and whole cells was partially inhibited by resveratrol but a concentration of 5 mmol l-1 was required to reduce it by more than 60%. Inhibition was competitive for S. saprophyticus but noncompetitive for P. mirabilis. Addition of resveratrol to the growth medium did not affect the specific activity of urease or prevent the increase in pH that occurs as a result of urease activity. Resveratrol reduced biofilm formation by S. saprophyticus at concentrations >125 µg ml-1 and partially inhibited it by P. mirabilis in rich media containing urea. While resveratrol has some potential for the treatment of urinary tract infections, it may need to be modified or combined with other urease inhibitors.

Bile salt hydrolase and cholesterol assimilation potential of lactobacilli fromNigerian fermented foods and human sources

Bile salt hydrolases (BSH) results in microbes releasing enzymes conferring physiological functions for both the producing organisms and the host and subsequently lower serum cholesterol. In recent years, these attributes have generated interest towards screening of lactic acid bacteria (LAB) for functional food production. In this present study, lactobacilli (n=34) (13 L. casei, 6 L. paracasei, 11 L. brevis, 3 L. pentosus and 1 L. plantarum) from varied sources viz. Nigerian fermented foods and human specimens were screened qualitatively and quantitatively for BSH activity using the direct plate assay and cell free extracts (CFE) of overnight cultures. To establish a correlation between BSH and cholesterol reduction, 12 isolates displaying high precipitation over bile salt agar plates, and high BSH enzyme activity in CFE were selected for in vitro cholesterol reduction assay. Results showed that 58% of the isolates from humans, and 36% from fermented food origin showed larger zones of precipitation. The highest BSH activity was exhibited by Lactobacillus casei GR4 (76mM) after 10 min of incubation, an isolate from fermented food. Isolates from human specimens expressed higher enzyme activity compared to those from fermented foods with significant increase (P&lt;0.05) after 30 min incubation. Cholesterol level was variable as L. pentosus 8ST5 and 8ST7 had 2 and 4% cholesterol levels in the medium; while L. casei and paracasei group (Lactobacillus casei group, LCG) 3MB4, 8BM6, BK4 and L. brevis GR29 had 7%/ml each proving their application in food formulation, reduction of hypercholesterolemia, and as prophylaxis for controlling serum cholesterol levels.

Evaluating metabolic potential of Thauera sp. M9 for the transformation of 4-chloroaniline (4-CA)

Abstract Thauera sp. M9, was isolated after enrichment of microbial diversity in nitrogen free mineral salt medium (NFMSM) supplemented with 300 mg/l of 4-chloroaniline (4-CA). The isolate M9 was able to achieve complete degradation of 300 mg/l 4-CA supplement to NFMSM, after 30 h incubation. In addition to 4-CA, isolate M9 was able to achieve complete degradation of 300 mg/l 3-chloroaniline (3-CA), 21% degradation of 300 mg/l 2-chloroaniline (2-CA), and complete transformation of 50 mg/l of 3, 4-dichloroaniline (3, 4-DCA), was observed only in the presence of 0.02% (w/v) yeast extract (YE) supplement to NFMSM. The presence of catechol 2, 3-dioxygenase (C23O) activity in cell free extract and detection of 4-chlorocatechol (4-CC); 2-hydroxy muconic semialdehyde (2-HMS) in biologically transformed samples by mass spectroscopy (MS) analysis indicated to meta-cleavage of the aromatic ring. A plug flow reactor (PFR) was developed using polyurethane foam (PUF) and refractory brick pieces (RBP) as immobilization matrix. The treatment of simulated effluent supplemented with 300 mg/l 4-CA through PFR, resulted in 88% transformation of 4-CA and 70% lowering of chemical oxygen demand (COD). These observations suggest that Thauera sp. M9 could be a potential inoculum for developing bioremediation protocols for waste rich in chloroanilines (CAs).

Removal of Hexavalent Chromium by Aspergillus niger Through Reduction and Accumulation

Industrial activities discharge a large amount of wastes containing hexavalent chromium [Cr(VI)] into the environment, which poses a threat to human health. Microorganisms can be used as efficient tools for Cr(VI) remediation. In this study, the Cr(VI) removal capacity of Aspergillus niger was evaluated. A. niger could tolerate and reduce Cr(VI) by nearly 100% at concentrations ranging from 10 to 50 mg/L. Overall, almost 97% of the Cr(VI) removal was caused by extracellular reduction whereas 3% was caused by accumulation. Extracellular reduction was mediated by non-enzymatic cell secretions, whereas extracellular accumulated Cr formed precipitates on the hyphal surfaces and was partially absorbed on the cell wall. Cr(VI) also entered the cell and was reduced by the strong chromate reductase activity in cell-free extracts and then accumulated within the cell. These data suggest that A. niger, which has the capacity to remove Cr(VI) by reduction and accumulation, can be a useful tool for Cr(VI) remediation.

IN VITRO ANTI-RADICAL ACTIVITY OF BIFIDOBACTERIUM BIFIDUM AND LACTOBACILLUS REUTERI CELL-FREE EXTRACTS

Probiotic microorganisms are known to be able to exhibit antioxidant activity. Most of beneficial probiotics effects are due to the action of their derivatives (structural components and metabolites). The aim of the work was to investigate the antiradical activity of cell-free extracts containing bacterial derivatives of Bifidobacterium bifidum 1 and Lactobacillus reuteri DSM 17938 probiotic strains, by their hydroxyl radical scavenging activity in the model system of their generation. Cell-free extracts were obtained from disintegrators and cultures of probiotic bacteria cultured in the disintegrators of their own cells. A metabiotic HYLAK FORTE was chosen as the reference drug. At a concentration 20% vol. in the incubation medium the hydroxyl radical scavenging by L. reuteri cell-free extracts was at a level of 75–90 %, and the hydroxyl radical scavenging by B. bifidum cell-free extracts was at a level of 50–60%. The metabiotic provided the same level of the hydroxyl radical scavenging activity when its content in the incubation medium was lower in 1.5 – 2.5 times. Extracts obtained from disintegrators showed a more pronounced antiradical activity than extracts from probiotic cultures. Based on the weight of the dry residue, concentrations for 50% hydroxyl radical scavenging (ІС50) by extracts and metabiotic were calculated. According to this indicator, L. reuteri extract obtained from disintegrate was the most effective (IC50 = 1.57 mg/ml). Other investigated derivative-containing products were arranged in decreasing order of antiradical activity as follows: B. bifidum extract from disintegrate (IC50 = 1, 64 mg/ml) &gt; B. bifidum extract from culture (IC50 = 1, 75 mg/ml) &gt; L. reuteri extract from culture (IC50 = 1, 86 mg/ml) &gt; HYLAK FORTE (IC50 = 11, 03 mg/ml). Thus, all the studied extracts showed antiradical activity with respect to the most reactive hydroxyl radical. The obtained results encourage further study of the antioxidant properties of L. reuteri and B. bifidum cell-free extracts in order to elucidate the mechanisms of their biological activity and justify the appropriateness of their therapeutic use.

СКРИНІНГ ЦВІЛЕВИХ ГРИБІВ НА ЗДАТНІСТЬ ДО СИНТЕЗУ КРЕАТИНІНДЕІМІНАЗИ

Determination of creatinine concentration in biological fluids is becoming increasingly relevant as a clinical test. Various chemical and physico-chemical methods have been proposed to determine creatinine concentration in biological samples, including various modifications of the well-known Jaffe color reaction, although it has several drawbacks, namely, a low specificity and sensitivity to positive and negative interference from related substances. In addition, the available methods are time consuming, require a complicated sample preparation procedure, expensive reagents, highly qualified personnel and, most importantly, do not allow real-time analysis. Therefore, increasing attention has been paid to enzymatic methods for the determination of creatinine, particularly, using microbial creatinine deiminase. In this regard, the search for new microorganisms – effective producers of specific creatinine deiminases – is an actual task for microbial enzymology and analytical biotechnology. We have analyzed creatinine deiminase activity for 10 strains of different molds: Botrytis aclada , Botrytis allii 100(5), Monilinia fructicola , Sporobolomyces salmonicolor , Totula sp., Stachybotris chartarum , Pichiapini , Aspergillus oryzea , Trichoderma lignorum , Aspergillus glaucus , Trichothecium roseum , Aureobasidium pullulans . Among the strains tested, the enzyme activity was detected in the following species: M. fructicola, A. pullulans, B. allii, T. roseum, and C. g lobosum , although only two fungi, B. allii and C. globosum , were able to use creatinine as the sole nitrogen source. Creatinine deiminase activity in cell free extracts of B. allii and C. globosum , grown in glucose medium supplemented with creatinine (as the sole carbon and nitrogen source, respectively) was 2.5 and 1.5 times higher, related to that for the strains grown in glucose-sodium nitrate medium. Therefore, the fungi B. allii and C. globosum can be used for isolation of creatinine deiminase to be used in construction of biosensors selective to creatinine.

Enzymes involved in the anaerobic degradation of phenol by the sulfate-reducing bacterium Desulfatiglans anilini

BackgroundThe sulfate-reducing bacterium Desulfatiglans anilini can grow with phenol as sole source of carbon and energy under strictly anaerobic, sulfate-reducing conditions. In the nitrate-reducing bacterium Thauera aromatica, the enzymes involved in phenol degradation have been well elucidated, whereas the anaerobic phenol degradation pathway by D. anilini was not studied in detail yet.ResultsThe pathway of anaerobic phenol degradation by the sulfate-reducing bacterium Desulfatiglans anilini was studied by identification of genes coding for phenylphosphate synthase (encoded by pps genes) and phenylphosphate carboxylase (encoded by ppc genes) in the genome of D. anilini, by analysis of the transcription and translation of pps-ppc genes, and by measurement of phenylphosphate synthase activity in cell-free extracts of phenol-grown cells. The majority of genes involved in phenol degradation were found to be organized in one gene cluster. The gene cluster contained genes ppsα (phenylphosphate synthase alpha subunit), ppsβ (phenylphosphate synthase beta subunit), ppcβ (phenylphosphate carboxylase beta subunit), as well as 4-hydroxybenzoyl-CoA ligase and 4-hydroxylbenzoyl-CoA reductase-encoding genes. The genes ppsγ (phenylphosphate synthase gamma subunit), ppcα (phenylphosphate carboxylase alpha subunit) and ppcδ (phenylphosphate carboxylase delta subunit) were located elsewhere in the genome of D. anilini, and no obvious homologue of ppcγ (phenylphosphate carboxylase gamma subunit) was found in the genome. Induction of genes pps and ppc during growth on phenol was confirmed by reverse transcription polymerase chain reaction. Total proteome analysis revealed that the abundance of enzymes encoded by the gene cluster under study was much higher in phenol-grown cells than that in benzoate-grown cells. In in-vitro enzyme assays with cell-free extracts of phenol-grown cells, phenylphosphate was formed from phenol in the presence of ATP, Mg2+, Mn2+, K+ as co-factors.ConclusionsThe genes coding for enzymes involved in the anaerobic phenol degradation pathway were identified in the sulfate-reducing bacterium D. anilini. The results indicate that the first steps of anaerobic phenol degradation in D. anilini are phosphorylation of phenol to phenylphosphate by phenylphosphate synthase and carboxylation of phenylphosphate by phenylphosphate carboxylase.

ENZYMES ACTIVITY OF GLUTATHIONE ANTIOXIDANT SYSTEM OF CHLOROBIUM LIMICOLA IMV K-8 BACTERIA UNDER THE INFLUENCE OF COPPER (II) SULFATE

Aim. Investigation of changes of glutathione antioxidant system enzymes activity of Chlorobium limicola ІМV K-8 bacteria under the influence of copper (II) sulfate. Methods. In this investigation there were used C. limicola ІМV K-8 bacteria which were cultivated in Green sulfur bacteria medium. Glutathione peroxidase activity was determined by the rate of oxidation of reduced glutathione before and after incubation with tert-butyl hydroperoxide by color reaction with 5,5-dithiobis-2-nitrobenzoic acid. Glutathione S-transferase activity was determined by the rate of formation of conjugate of reduced glutathione with 1-chloro-2,4-dinitrobenzene. Results. Glutathione-S-transferase, glutathione peroxidase and glutathione reductase activity of cell free extracts of C. limicola ІМV K-8 were investigated under the influence of copper (II) sulfate in concentrations that caused decrease of biomass accumulation from 10 to 70%. It was shown that under the influence of all investigated copper (II) sulfate concentrations glutathione-S-transferase and glutathione peroxidase activity increased in comparison with control. Activity of these enzymes changed in dependence on copper (II) sulfate concentration in incubation medium and duration of cultivation. Also it was determined that glutathione reductase activity is absent in investigated bacteria. Conclusion. The increase of activity of glutathione peroxidase by 2–12 times and glutathioneS-transferase by 1.5–2 times, in comparison with control, in cell free extracts of C. limicola ІМV К-8 under the influence of copper (II) sulfate indicates on accumulation of hydrophobic and hydrophilic organic peroxides inside the cells. It also shows that, possibly, this system is one of the important ways of cell defense of green photosynthetic bacteria, in particular C. limicola, against reactive oxygen species.

Plant Glyoxylate/Succinic Semialdehyde Reductases: Comparative Biochemical Properties, Function during Chilling Stress, and Subcellular Localization.

Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (cytosolic GLYR1 and plastidial/mitochondrial GLYR2) are considered to be of particular importance under abiotic stress conditions. Here, the apple (Malus × domestica Borkh.) and rice (Oryza sativa L.) GLYR1s and GLYR2s were characterized and their kinetic properties were compared to those of previously characterized GLYRs from Arabidopsis thaliana [L.] Heynh. The purified recombinant GLYRs had an affinity for glyoxylate and succinic semialdehyde, respectively, in the low micromolar and millimolar ranges, and were inhibited by NADP+. Comparison of the GLYR activity in cell-free extracts from wild-type Arabidopsis and a glyr1 knockout mutant revealed that approximately 85 and 15% of the cellular GLYR activity is cytosolic and plastidial/mitochondrial, respectively. Recovery of GLYR activity in purified mitochondria from the Arabidopsis glyr1 mutant, free from cytosolic GLYR1 or plastidial GLYR2 contamination, provided additional support for the targeting of GLYR2 to mitochondria, as well as plastids. The growth of plantlets or roots of various Arabidopsis lines with altered GLYR activity responded differentially to succinic semialdehyde or glyoxylate under chilling conditions. Taken together, these findings highlight the potential regulation of highly conserved plant GLYRs by NADPH/NADP+ ratios in planta, and their roles in the reduction of toxic aldehydes in plants subjected to chilling stress.

Glycogen synthase from the parabasalian parasite Trichomonas vaginalis: An unusual member of the starch/glycogen synthase family

Trichomonas vaginalis, a parasitic protist, is the causative agent of the common sexually-transmitted infection trichomoniasis. The organism has long been known to synthesize substantial glycogen as a storage polysaccharide, presumably mobilizing this compound during periods of carbohydrate limitation, such as might be encountered during transmission between hosts. However, little is known regarding the enzymes of glycogen metabolism in T. vaginalis. We had previously described the identification and characterization of two forms of glycogen phosphorylase in the organism. Here, we measure UDP-glucose-dependent glycogen synthase activity in cell-free extracts of T. vaginalis. We then demonstrate that the TVAG_258220 open reading frame encodes a glycosyltransferase that is presumably responsible for this synthetic activity. We show that expression of TVAG_258220 in a yeast strain lacking endogenous glycogen synthase activity is sufficient to restore glycogen accumulation. Furthermore, when TVAG_258220 is expressed in bacteria, the resulting recombinant protein has glycogen synthase activity in vitro, transferring glucose from either UDP-glucose or ADP-glucose to glycogen and using both substrates with similar affinity. This protein is also able to transfer glucose from UDP-glucose or ADP-glucose to maltose and longer oligomers of glucose but not to glucose itself. However, with these substrates, there is no evidence of processivity and sugar transfer is limited to between one and three glucose residues. Taken together with our earlier work on glycogen phosphorylase, we are now well positioned to define both how T. vaginalis synthesizes and utilizes glycogen, and how these processes are regulated.

Purification and Characterization of Hexavalent Chromate Reductase Activity in Cell Free Extract of Bacillus subtilis Strain Isolated From Treated Tannery Effluent

A Bacillus subtilis strain previously isolated from treated tannery effluent was tolerant to a maximum of 1400 mg/L Cr(VI) and reduced 77% toxic Cr(VI) to less toxic Cr(III) within 24 hrs. at pH 8.5 and 35°C temperature under shaking condition (120 rpm). The chromate reductase enzyme was found to be membrane bound and it has been purified using ammonium sulphate fractionation, anionexchange chromatography and gel filtration chromatography on Sephadex G-75 by cell free extract of the bacterium. The enzyme was characterized based on optimal temperature, pH, metal ions and initial Cr(VI) concentration in the reaction mixture. Maximum chromate reductase activity was observed at pH 8.5 and 35ºC temperature. The enzyme was significantly active between pH 6.5-10.0 and was found to be stable between 30ºC-40ºC. The chromate reductase activity was slightly decreased with increasing “the concentration of Cr(VI). The enzymatic activity was completely inhibited by Hg(II) and As(III) while Ni(II), Cd(II), Mn(II), Zn(II), Pb (II) and Co (II) showed moderate effect on chromate reductase activity of Bacillus subtilis and the chromate reduction was enhanced by Cu(II) and Fe(III). Keywords: Bacillus subtilis, chromate reductase, Cr(VI), tannery.

OPTIMATION OF ANTIMICROBIAL SUBSTANCE PRODUCTION IN CELL FREE EXTRACT FROM THERMOPHYLIC BACTERIA FERMENTATION AFTER MERAPI ERRUPTION

The aims of this study was to investigate the effect of medium with different pH (6,7,9), salt concentration (0,5; 1; 2 %) and fermentation periode (24 and 48 hr) to the antimicrobial activity of cell free extract to three pathogens, Eschericia coli, Staphylococcus aureus, and Candida albican. This study consists of antimicrobial compounds production with different medium and continued by antimicrobial tested to fungi and bacterial pathogens Eschericia coli, Staphylococcus aureus, and Candida albican by Kirby Bauer method with paper disk. Different salt concentration, pH and fermentation periode affected the antimicrobial activity potency of cell free extract yielded by thermophylic bacteria. Treatment which yielded CFE with the best antimicrobial activity was treatment with 24 hr fermentation, pH 7 and salt concentration 2% to S aureus and pH 6 salt concentration 1% to E coli. Cell free extract had no potency as antifungi to Candida albicans except CFE yielded by thermophylic bacteria fermented in medium with pH 7 and salt concentration 1% in 24 hr with inhibition zone index 1,17. Keywords: cell free extract, antimicrobial, pH, salt concentration, fermentation period

Assessment of hydrogen metabolism in commercial anaerobic digesters.

Degradation of biomass in the absence of exogenous electron acceptors via anaerobic digestion involves a syntrophic association of a plethora of anaerobic microorganisms. The commercial application of this process is the large-scale production of biogas from renewable feedstock as an alternative to fossil fuels. After hydrolysis of polymers, monomers are fermented to short-chain fatty acids and alcohols, which are further oxidized to acetate. Carbon dioxide, molecular hydrogen (H2), and acetate generated during the process are converted to methane by methanogenic archaea. Since many of the metabolic pathways as well as the syntrophic interactions and dependencies during anaerobic digestion involve formation, utilization, or transfer of H2, its metabolism and the methanogenic population were assessed in various samples from three commercial biogas plants. Addition of H2 significantly increased the rate of methane formation, which suggested that hydrogenotrophic methanogenesis is not a rate-limiting step during biogas formation. Methanoculleus and Methanosarcina appeared to numerically dominate the archaeal population of the three digesters, but their proportion and the Bacteria-to-Archaea ratio did not correlate with the methane productivity. Instead, hydrogenase activity in cell-free extracts from digester sludge correlated with methane productivity in a positive fashion. Since most microorganisms involved in biogas formation contain this activity, it approximates the overall anaerobic metabolic activity and may, thus, be suitable for monitoring biogas reactor performance.

Characterization of a trehalose-degrading enzyme from the hyperthermophilic archaeon Sulfolobus acidocaldarius

We purified a cytosolic trehalase (TreH) from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Enzyme activity in cell-free extracts indicated that trehalose degradation in the cell occurred via the hydrolytic activity of TreH, and not via TreP (phosphorolytic activity) or TreT (transfer activity). TreH was purified to near-homogeneity by DEAE anion-exchange chromatography, followed by size exclusion and HiTrap Q anion-exchange chromatography, and its molecular mass was estimated as 40kDa. Maximum activity was observed at 85°C and pH 4.5. The half-life of TreH was 53 and 41minat 90°C and 95°C, respectively. TreH was highly specific for trehalose and was inhibited by glucose with a Ki of 0.05mM. Compared with TreH from other trehalases, TreH from S.acidocaldarius is the most thermostable trehalase reported so far. Furthermore, this is the first trehalase characterized in the Archaea domain.

In vivo and in vitro inhibition of cholinesterase activity in Colossoma macropomum (tambaqui) fingerlings by the herbicide trifluralin

The Amazonian fish Colossoma macropomum (tambaqui) is farmed intensively in rice paddies around the São Francisco River delta in northeast Brazil, where the herbicide trifluralin is regularly used. The aims of this study were to evaluate the inhibitory effects of trifluralin on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of brain and muscle from C. macropomum in order to assess the value of this species as a sentinel for herbicide contamination. Trifluralin was highly toxic to fingerlings in vivo (96 h-LC50 = 0.42 mg L-1). Cholinesterase activity in cell-free extracts of brain was associated with two isoforms, AChE and BChE, although the former predominated. The specific activity of brain AChE was reduced significantly (p &lt; 0.05) following 96.h exposure of fingerlings to trifluralin at 0.5 and 0.75 mg L-1, but increased by 30% after exposure to 1.0 mg L-1 of herbicide. Muscle AChE was not affected by exposure to trifluralin. Km and Vmax values of brain AChE were 0.043 ±.0.015 mmol L-1 and 0.301.±.0.014 mmol min-1 mg-1protein, respectively. Brain AChE was moderately sensitive to trifluralin (IC50 = 0.78 mg L-1), but was very sensitive to the anticholinesterase agent eserine (IC50 = 0.043 mg L-1). AChE inhibition in C. macropomum may be employed as a biomarker for biomonitoring trifluralin contamination in water bodies.

Deciphering the Regulatory Circuitry That Controls Reversible Lysine Acetylation in Salmonella enterica.

ABSTRACTIn Salmonella enterica, the reversible lysine acetylation (RLA) system is comprised of the protein acetyltransferase (Pat) and sirtuin deacetylase (CobB). RLA controls the activities of many proteins, including the acetyl coenzyme A (acetyl-CoA) synthetase (Acs), by modulating the degree of Acs acetylation. We report that IolR, a myo-inositol catabolism repressor, activates the expression of genes encoding components of the RLA system. In vitro evidence shows that the IolR protein directly regulates pat expression. An iolR mutant strain displayed a growth defect in minimal medium containing 10 mM acetate, a condition under which RLA function is critical to control Acs activity. Increased levels of Pat, CobB, or Acs activity reversed the growth defect, suggesting the Pat/CobB ratio in an iolR strain is altered and that such a change affects the level of acetylated, inactive Acs. Results of quantitative reverse transcription-PCR (qRT-PCR) analyses of pat, cobB, and acs expression indicated that expression of the genes alluded to in the IolR-deficient strain was reduced 5-, 3-, and 2.6-fold, respectively, relative to the levels present in the strain carrying the iolR+ allele. Acs activity in cell-free extracts from an iolR mutant strain was reduced ~25% relative to that of the iolR+ strain. Glucose differentially regulated expression of pat, cobB, and acs. The catabolite repressor protein (Crp) positively regulated expression of pat while having no effect on cobB.

Activity of Na+/K+-activated Mg2+-dependent ATP-hydrolase in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9

The aim of our work was to study Na+/K+-activated Mg2+-dependent ATPase activity in cell-free extracts of the sulfate-reducing bacteriaDesulfovibrio pigerVib-7 andDesulfomicrobiumsp. Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme reaction. The maximum ATPase activity for both bacterial strains at +35 ºC was determined. The highest activities of the studied enzyme in the cell-free extracts ofD. pigerVib-7 at pH 7.0 andDesulfomicrobiumsp. Rod-9 at pH 6.5 were measured. Based on experimental data, the analysis of kinetic properties of the ATP-hydrolase reaction by the studied bacteria was carried out. The enzyme activity, initial (instantaneous) reaction rate (V0) and maximum rate of the ATPase reaction (Vmax) was significantly higher inD. pigerVib-7 cells than inDesulfomicrobiumsp. Rod-9. Michaelis constants (Km) of the enzyme reaction for both bacterial strains were determined.

Effect of vanadium on nitrate reductase activity in tomato leaves

The activity of nitrate reductase in cell-free extracts from tomato leaves is completely inhibited by 100 μM NaVO&lt;sub&gt;3&lt;/sub&gt; or VOCl&lt;sub&gt;2&lt;/sub&gt;. In experiments in vivo vanadium ions inhibit the activity of the enzyme in 50 to 60 per cent. Addition of l mM vanadium to the medium on which tomato seedlings are grown causes after 24 h almost complete inhibition of nitrate reductase activity in cell-free extracts of the enzyme. Inhibition with vanadium may be abolished in experiments &lt;i&gt;in vitro&lt;/i&gt; if the extract is treated with a rather concentrated EDTA solution or Sephadex G-25. The data obtained in this study indicate that vanadium ions in relatively low concentrations inhibit the activity of nitrate reductase in tomato seedlings both &lt;i&gt;in vivo&lt;/i&gt; and &lt;i&gt;in nitro&lt;/i&gt;.

Antimicrobial Activity of a Novel Pichia membranifaciens Strain Isolated from Naturally Fermented Cashew Apple Juice

Yeasts form a natural and dominant surface flora on the cashew apple and fermented cashew apple juice due to its rich nutritional content. In the present study, yeast flora from naturally fermented cashew apple juice was studied. Based on different colony characters on potato dextrose agar, 11 different types of yeasts were isolated. The yeast strain ICARG 16 was further characterized and based on morphological, biochemical and 26S rDNA sequencing. The strain was identified as Pichia membranifaciens. The antimicrobial activity of cell free extract of the strain ICARG 16 was studied against Staphylococcus aureus MTCC 1144 and found to inhibit the growth of the pathogen. The isolate could tolerate 10 % of ethanol, showed growth at 27 °C, in 50 % glucose. The strain did not exhibit amylase, protease, pectinase and cellulase activity, however, it showed urease activity. Thus the naturally occurring yeast isolates P. membranifaciens ICARG16 may be of pharmaceutical interest.