Active Acid Research Articles

Glycerol 3-Phosphate Alters Trypanosoma brucei Hexokinase Activity in Response to Environmental Change

The African trypanosome, Trypanosoma brucei, compartmentalizes some metabolic enzymes within peroxisome-like organelles called glycosomes. The amounts, activities, and types of glycosomal enzymes are modulated coincident with developmental and environmental changes. Pexophagy (fusion of glycosomes with acidic lysosomes) has been proposed to facilitate this glycosome remodeling. Here, we report that, although glycosome-resident enzyme T. brucei hexokinase 1 (TbHK1) protein levels are maintained during pexophagy, acidification inactivates the activity. Glycerol 3-phosphate, which is produced in vivo by a glycosome-resident glycerol kinase, mitigated acid inactivation of lysate-derived TbHK activity. Using recombinant TbHK1, we found that glycerol 3-P influenced enzyme activity at pH 6.5 by preventing substrate and product inhibition by ATP and ADP, respectively. Additionally, TbHK1 inhibition by the flavonol quercetin (QCN) was partially reversed by glycerol 3-P at pH 7.4, whereas at pH 6.5, enzyme activity in the presence of QCN was completely maintained by glycerol 3-P. However, glycerol 3-P did not alter the interaction of QCN with TbHK1, as the lone Trp residue (Trp-177) was quenched under all conditions tested. These findings suggest potential novel mechanisms for the regulation of TbHK1, particularly given the acidification of glycosomes that can be induced under a variety of parasite growth conditions.

Role of some Factors Affecting Crown Gall Disease Development

The wounds, its depth and the time of inoculationof Agrobacterium tumefaciens on castor bean )Ricinus communis( plants affecting on the development of apricot crown gall disease whereas, the increase of wound depth and adding the pathogen after directly wounding plants increased the disease severity and gall formation. Where, the highest infection and gall formation resulted from inoculation of A. tumefaciens after directly wounding plants (zero time) at 3mm depth followed by 5mm depth, while the lowest gall formation and disease severity showed when adding A. tumefaciens suspension after 48 hrs. from wounding at one mm depth. On apricot plants (Prunus armeniaca), The plant hormones indole acetic acid (IAA) and cytokinin (CK) were decreased in the first stage of gall formation, while they were increased in the other stages of gall formation. Gibberellic acid (GA) was decreased in the first stage and then increased but it decreased again in the last stage. On the other hand, abscisic acid (ABA) inactivation plant hormone showed an opposite result whereas, ABA was increased in the first stage of gall formation and then it was decreased in the other stages of disease. Also, A. tumefaciens isolates obtained from different hosts having different gall index (different in virulence) produced different rates of plant hormones IAA, GA, ABA and CK. But the rate of CK was found to be correlated with virulence. Also, crown gall disease development dependent on the production amount of the three plant hormones (IAA, GA3 and CK) and the ratio between them. Fertilizers with (NPK) and compost resulted in decrease the severity of crown gall disease on apricot plants whereas; they reduced the number and weight of galls and root gall index in the treated plants compared with control. The best one treatment was (NPK) then (NP) and (NPK plus compost). Fertilizers with (NPK plus compost) due to increase the foliage of plants and in all fertilizer treatments increased the roots of plants compared with control except that treated with (NP). Also, no variation considered in the height of plants in all treatments, but the highest height recorded from (NPK plus compost) treatment. On the other hand, the uptake of (N) was increased in all fertilizers when used (N) alone or in combination with (P or K), also, (P) up take increased when used (P) alone or with (N or K). But (K) up take was increased only when used it with (N) or (P) and in combination with (NPK)

Effects of electrolytes on virus inactivation by acidic solutions

Acidic pH is frequently used to inactivate viruses. We have previously shown that arginine synergizes with low pH in enhancing virus inactivation. Considering a potential application of the acid inactivation of viruses for the prevention and treatment of superficial virus infection at body surfaces and fixtures, herein we have examined the effects of various electrolytes on the acid-induced inactivation of the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), the influenza A virus (IAV) and the poliovirus upon their incubation at 30˚C for 5 min. Eight electrolytes, i.e., phosphate, NaCl, glutamate, aspartate, pyrrolidone carboxylate, citrate, malate and acetate were tested. No detectable inactivation of the poliovirus was observed under the conditions examined, reflecting its acid-resistance. HSV-1 and HSV-2 responded similarly to the acid-treatment and electrolytes. Some electrolytes showed a stronger virus inactivation than others at a given pH and concentration. The effects of the electrolytes were virus-dependent, as IAV responded differently from HSV-1 and HSV-2 to these electrolytes, indicating that certain combinations of the electrolytes and a low pH can exert a more effective virus inactivation than other combinations and that their effects are virus-specific. These results should be useful in designing acidic solvents for the inactivation of viruses at various surfaces.

Thermal inactivation of acid, cold, heat, starvation, and desiccation stress-adapted Escherichia coli O157:H7 in moisture-enhanced nonintact beef.

This study was conducted to compare thermal inactivation of stress-adapted and nonadapted Escherichia coli O157:H7 in nonintact beef moisture enhanced with different brine formulations and cooked to 65°C. Coarsely ground beef was mixed with acid, cold, heat, starvation, or desiccation stress-adapted or nonadapted rifampin-resistant E. coli O157:H7 (eight-strain mixture, 5 to 6 log CFU/g) and a brine solution for a total moisture enhancement level of 10%. The brine treatments included distilled water (control), sodium chloride (0.5% NaCl) plus sodium tripolyphosphate (0.25% STP), or NaCl + STP combined with cetylpyridinium chloride (0.2% CPC), lactic acid (0.3% LA), or sodium metasilicate (0.2% SM). The treated meat was extruded into bags (15 cm diameter), semifrozen (-20°C for 4.5 h), and cut into 2.54-cm (1-in.)-thick portions. Samples were individually vacuum packaged, frozen (-20°C for 42 h), and tempered at 4°C for 2.5 h before cooking. Partially thawed (-1.8 ± 0.4°C) samples were pan broiled to an internal temperature of 65°C. Pathogen counts of partially thawed (before cooking) samples moisture enhanced with brines containing CPC, LA, or SM were 0.7 to 1.1, 0.0 to 0.4, and 0.2 to 0.4 log CFU/g, respectively, lower than those of the control. Compared with microbial count reductions obtained after pan broiling of beef inoculated with nonadapted E. coli O157:H7 cells, count reductions during cooking of meat inoculated with cold and desiccation stress-adapted, acid stress-adapted, and heat and starvation stress-adapted cells indicated sensitization, cross protection, and no effect, respectively, of these stresses on the pathogen during subsequent exposure to heat. Among all stressed cultures, CPC-treated samples (0.8 to 3.6 log CFU/g) and LA-treated samples (0.8 to 3.5 log CFU/g) had the lowest numbers of E. coli O157:H7 survivors after cooking.

A Single Amino Acid Substitution in the Capsid of Foot-and-Mouth Disease Virus Can Increase Acid Resistance

Foot-and-mouth disease virus (FMDV) particles lose infectivity due to their disassembly at pH values slightly below neutrality. This acid-dependent disassembly process is required for viral RNA release inside endosomes. To study the molecular determinants of viral resistance to acid-induced disassembly, six FMDV variants with increased resistance to acid inactivation were isolated. Infection by these mutants was more sensitive to drugs that raise the endosomal pH (NH(4)Cl and concanamycin A) than was infection by the parental C-S8c1 virus, confirming that the increase in acid resistance is related to a lower pH requirement for productive uncoating. Amino acid replacement N17D at the N terminus of VP1 capsid protein was found in all six mutants. This single substitution was shown to be responsible for increased acid resistance when introduced into an infectious FMDV clone. The increased resistance of this mutant against acid-induced inactivation was shown to be due to its increased resistance against capsid dissociation into pentameric subunits. Interestingly, the N17D mutation was located close to but not at the interpentamer interfaces. The mutants described here extend the panel of FMDV variants exhibiting different pH sensitivities and illustrate the adaptive flexibility of viral quasispecies to pH variations.

Efficacy and Safety of Hydroxychloroquine Sulphate In Chronic Lymphocytic Leukemia: Clinical Trial Experience In Untreated Patients

AbstractAbstract 1392 Background:Hydroxychloroquine (HCQ) has been used safely for many years for treatment of malaria, systemic lupus erythematosus and rheumatoid arthritis. It is known to have exerted immunomodulatory actions by affecting antigen presentation by macrophages and dendritic cells, DNA/RNA synthesis, and apoptosis in lymphocytes and endothelial cells. All of these actions can be attributed at least partially to the inactivation of endosomal acid proteases.CLL is the abnormal proliferation of a B lymphocyte clone. Based on the unique structural characteristics of the B-cell antigen receptor (BCR), at least some CLL cases probably results from ongoing antigen selection and stimulation. RNA and/or DNA from infection or cell apoptosis are candidate stimulants of this process. Binding of RNA and DNA to toll-like receptors (TLR 7 and 9) in endosomes is dependent on an acidic pH and can be prevented by HCQ due to its de-acidifiying effect. HCQ can also directly induce apoptosis in CLL cells through caspase 3 activation in vitro. We therefore hypothesized that administration of HCQ early in the disease would protect CLL patients from disease progression by eliminating these stimulatory TLR 7 and 9 signals. Methods:22 CLL patients with no urgent need for treatment were enrolled in the clinical trial. Patients took HCQ 400 mg/day for 52 weeks. Clinical response was measured every 4 weeks by history, physical exam, and absolute lymphocyte counts. Additional blood samples were taken every 8 weeks for companion laboratory studies. Statistical analyses used include Fisher's Exact Test, Slopes analysis and Repeated measures analysis of variance on the log-transformed absolute lymphocyte counts (ALCs). Results:In total, 8 IGHV unmutated (U-CLL) and 14 mutated (M-CLL) patients were enrolled; 3 U-CLL and 10 M-CLL completed the full year of HCQ treatment. Five U-CLL patients were dropped from the study due to disease progression requiring CLL therapy. One M-CLL patient left for personal reasons. Three M-CLL patients are still continuing on HCQ therapy.Patient outcomes can be separated into three patterns based on ALC changes: 7 patients (6 M-CLL and 1 U-CLL) had stable counts throughout the trial; 12 patients (8 M-CLL and 4 U-CLL) had a fall in ALCs after treatment; and 3 patients (all U-CLL) developed increased ALCs. The response to HCQ in CLL is associated with Ig V mutation status of patients (P<.03). With some U-CLL patients having a worsening of disease; overall U-CLL patients have worse outcome than M-CLL (P<.045). Nevertheless in most instances, U-CLL patients did respond to HCQ with stabilization or fall in ALC initially, although this was followed at an average of 18–22 weeks with an increase in ALCs and in some instances a need for therapy.Maximum HCQ blood concentrations in healthy individuals receiving 400 mg/day HCQ orally are reported to range from 67–221 ng/ml. In vitro assay using samples collected at trial initiation indicated that direct CLL cytotoxicity occurred at levels ≥10 μg/ml; this value was similar for U- and M-CLL clones. Using a non-cytotoxic concentration of 1 μg/ml of HCQ, in vitro TLR9 ligand ODN2006 enhanced expression of TLR9 was diminished. The proliferation and activation of both U- and M-CLL was blocked completely, although concentrations of 10–100 ng/ml had a superior blocking effect on M-CLL than U-CLL clones.In vivo administration of HCQ blocked CLL cell proliferation initiated by ODN2006. In comparison with samples taken before HCQ administration, patient samples obtained after trial initiation responded to ODN2006 poorly. Interestingly, the type of CLL cell response to ODN2006 closely correlated with patient's ALC change. The least blocking of HCQ on cell proliferation was again observed in U-CLL patients, especially with the samples taken from the timepoint at which elevated ALCs were observed. Conclusion:In this clinical trial, oral administration of HCQ (400 mg/day) stabilized or decreased ALCs in the initial 18–22 weeks (of a 46–52 week regimen) in 91% of patients (14 M-CLL and 6-U-CLL). Subsequently, rising ALCs were observed in most of the U-CLL cases (n=4), and ALCs in M-CLL patients remain stabilized. Our in vitro evaluation suggests that an increased dose of HCQ might target TLR9 signaling better and thus improve outcome especially in U-CLL cases. Another possibility that warrants further study is that HCQ may have several actions on CLL cells leading to worse outcome of the disease. Disclosures:No relevant conflicts of interest to declare.

Pancreatic enzyme replacement therapy in chronic pancreatitis

Exocrine pancreatic insufficiency (EPI) is a serious condition which occurs in several diseases including chronic pancreatitis (CP), cystic fibrosis, pancreatic cancer, and as a result of pancreatic surgery. The lack or absence of pancreatic enzymes leads to an inadequate absorption of fat, proteins, and carbohydrates, causing steatorrhoea and creathorrhea which results in abdominal discomfort, weight loss, and nutritional deficiencies. To avoid malnutrition related morbidity and mortality, it is pivotal to commence pancreatic enzyme replacement therapy (PERT) as soon as EPI is diagnosed. Factors as early acidic inactivation of ingested enzymes, under dosage, and patient incompliance may prevent normalisation of nutrient absorption, in particular of fat digestion. This review focuses on the current status of how to diagnose and treat EPI.

Lipid droplet analysis in caveolin-deficient adipocytes: alterations in surface phospholipid composition and maturation defects

Caveolins form plasmalemnal invaginated caveolae. They also locate around intracellular lipid droplets but their role in this location remains unclear. By studying primary adipocytes that highly express caveolin-1, we characterized the impact of caveolin-1 deficiency on lipid droplet proteome and lipidome. We identified several missing proteins on the lipid droplet surface of caveolin-deficient adipocytes and showed that the caveolin-1 lipid droplet pool is organized as multi-protein complexes containing cavin-1, with similar dynamics as those found in caveolae. On the lipid side, caveolin deficiency did not qualitatively alter neutral lipids in lipid droplet, but significantly reduced the relative abundance of surface phospholipid species: phosphatidylserine and lysophospholipids. Caveolin-deficient adipocytes can form only small lipid droplets, suggesting that the caveolin-lipid droplet pool might be involved in lipid droplet size regulation. Accordingly, we show that caveolin-1 concentration on adipocyte lipid droplets positively correlated with lipid droplet size in obese rodent models and human adipocytes. Moreover, rescue experiments by caveolin- green fluorescent protein in caveolin-deficient cells exposed to fatty acid overload demonstrated that caveolin-coated lipid droplets were able to grow larger than caveolin-devoid lipid droplets. Altogether, these data demonstrate that the lipid droplet-caveolin pool impacts on phospholipid and protein surface composition of lipid droplets and suggest a functional role on lipid droplet expandability.

Anorectal and urinary anomalies and aberrant retinoic acid metabolism in cytochrome P450 oxidoreductase deficiency

Context: Cytochrome P450 oxidoreductase (POR) is an electron donor for all microsomal P450 enzymes including CYP26 involved in inactivation of all-trans retinoic acid (atRA). Although previous studies in Por knockout mice suggest that atRA accumulation is relevant to various posterior organ abnormalities, a systematic analysis has not been performed for anorectal and urinary anomalies in patients with POR deficiency (PORD). Objective: To report the frequencies of anorectal and urinary anomalies and plasma atRA values in PORD patients. Patients: We studied 37 Japanese patients with PORD, consisting of 15 homozygotes for R457H (group A), 15 compound heterozygotes for R457H and one apparently null mutation (group B), and seven patients with other combinations of mutations (group C). Since R457H is a severe hypomorphic mutation, the residual POR function is predicted to be higher in group A than in group B. Results: Imperforate anus was observed in four patients (10.8%) and vesicoureteral reflux was found in three patients (8.1%), with no significant difference in the frequencies of such anomalies between groups A and B. In addition, a complex urogenital malformation including penile agenesis was identified in one patient. Plasma atRA values were above the reference range in nine of 12 patients examined, and were similar between groups A and B and between patients with and without anomalies. Conclusions: The results imply that aberrant atRA metabolism due to CYP26 deficiency underlies various anorectal and urinary anomalies in patients with PORD. Clinical phenotypes may be primarily determined by maternal oral retinol intake during pregnancy, and plasma atRA values may be largely influenced by the amount of postnatal oral retinol intake in such patients.

Influences of simultaneous physicochemical stress exposures on injury and subsequent responses of E. coli O157:H7 to resuscitative and inactivative challenges

E. coli O157:H7 responses namely, sublethal injury (% injury), subsequent resuscitation (lag) and growth parameters (k(G) and max population), and inactivation rates (D(25) and D(55)) were investigated following exposures to simultaneous pH (3.0-8.0), a(w) (0.93-0.99) and temperature stresses (3.0-62 degrees C). Multiple regression analyses showed that % injury was significantly influenced by a(w), a(w)(2), and temperature while the lag times were influenced by pH, pH-temperature interaction, and a(w)-temperature interaction. The measured growth parameters were however, not significantly influenced by any of the stressors. Acid inactivation rates (D(25)) in apple juice were influenced by a(w), pH-temperature interaction, and a(w)-temperature interaction; while the thermal inactivation rates (D(55)) in apple juice were influenced by pH and a(w) stresses. Results also showed that % injury alone may not correctly predict the subsequent resuscitation rates of the test organism at favorable growth conditions. The nature of the injury contributed by a specific stressor also influenced resuscitation. The occurrence of injury and loss of cell viability did not induce acid stress adaptation to the organism. However, exposures to very low- and low pH conditions that resulted in injury and loss in cell viability induced thermal cross protection in E. coli O157:H7. The results obtained in the study contribute in further understanding the behavior of the test organism after exposure to combinations of stresses commonly encountered in food and food processing ecologies.

Effect of sonication on colour, ascorbic acid and yeast inactivation in tomato juice

Power ultrasound is recognised as a potential non-thermal technique to inactivate micro-organisms pertinent to fruit juices. In this study tomato juice was sonicated at different amplitude levels (24.4–61.0 μm) at a constant frequency of 20 kHz for treatment times (2–10 min) and pulse durations of 5 s on and 5 s off. Hunter colour values ( L∗, a∗ and b∗), pH, °Brix, titratable acidity, ascorbic acid and yeast inactivation were measured. No significant differences ( p < 0.05) in pH, °Brix or titratable acidity were observed. Regression modelling was used to investigate the main effects of amplitude level and treatment time. Prediction models were found to be significant ( p < 0.05) with low standard errors and high coefficients of determination ( R 2). Model predictions for critical quality parameters of Hunter colour values ( L∗, a∗ and b∗), ascorbic acid and yeast inactivation were closely correlated with the experimental results obtained.

Oncogenic and cell survival properties of the retinoic acid metabolizing enzyme, CYP26A1

Vitamin A deficiency (VAD) is associated with increased susceptibility to carcinogenesis in animal models and elevated risk for a number of human cancers. Here, we found that CYP26A1, the gene encoding a cytochrome P450 enzyme specifically involved in metabolic inactivation of retinoic acid (RA), the most active vitamin A derivative, is highly expressed in 42% (27/65) of primary breast cancers. We also showed that enhanced expression of CYP26A1 suppresses cellular responses to anoikis and consequently promotes anchorage-independent growth. This transformed phenotype was sufficient to markedly increase tumorigenic and metastatic potential. Suppression of CYP26A1 significantly reversed the CYP26A1-mediated oncogenic characteristics, suggesting a direct link between intracellular RA status and tumorigenicity. Our observations provide strong evidence for oncogenic and cell survival properties of CYP26A1 in carcinogenesis, and suggest mechanisms whereby VAD might promote cancer development.

A Comparative Study of Thermal and Acid Inactivation Kinetics in Fruit Juices ofSalmonella entericaSerovar Typhimurium andSalmonella entericaSerovar Senftenberg Grown at Acidic Conditions

Acid and heat inactivation in orange and apple juices of Salmonella enterica serovar Typhimurium Colección Española de Cultivos Tipo (i.e., Spanish Type Culture Collection) 443 (CECT 443) (Salmonella Typhimurium) and S. enterica serovar Senftenberg CECT 4384 (Salmonella Senftenberg) grown in buffered brain heart infusion (pH 7.0) and acidified brain heart infusion up to pH 4.5 with acetic, citric, lactic, and hydrochloric acids was evaluated. Acid adaptation induced an adaptive response that increased the subsequent resistance to extreme pH conditions (pH 2.5) and to heat, although the magnitude of these responses differed between the two isolates and fruit juices. The acid resistance in orange juice for acid-adapted cells (D-values of 28.3-34.5 min for Salmonella Senftenberg and 30.0-39.2 min for Salmonella Typhimurium) resulted to be about two to three times higher than that corresponding to non-acid-adapted cells. In apple juice, acid-adapted Salmonella Senftenberg cells survived better than those of Salmonella Typhimurium, obtaining mean D-values of 114.8 +/- 12.3 and 41.9 +/- 2.5 min, respectively. The thermotolerance of non-acid-adapted Salmonella Typhimurium in orange (D(58)-value: 0.028 min) and apple juices (D(58)-value: 0.10 min) was approximately double for acid-adapted cells. This cross-protection to heat was more strongly expressed in Salmonella Senftenberg. D(58)-values obtained for non-acid-adapted cells in orange (0.11 min) and apple juices (0.19 min) increased approximately 10 and 5 times, respectively, after their growth in acidified media. The conditions prevailing during bacterial growth and heat treatment did not significantly influence the z-values observed (6.0 +/- 0.3 degrees C for Salmonella Typhimurium and 7.0 +/- 0.3 degrees C for Salmonella Senftenberg). The enhanced acid resistance found for both isolates could enable them to survive for prolonged time periods in the gastrointestinal tract, increasing the risk of illness. Further, it should be taken into account that microbial growth in acidified media also induces a cross-protection response against heat that should also be considered for the design of pasteurization processes for acid foods.

Arginine and lysine decarboxylases and the Acid Tolerance Response of Salmonella Typhimurium

Salmonella Typhimurium CECT 443 inactivation at pH 2.5 in Mineral Medium (MM) and MM supplemented with 0.01% (w/v) arginine, lysine or glutamic acid was studied using stationary-phase cells grown in buffered BHI pH 7.0 (non-acid adapted cells) and acidifed BHI up to pH 4.5 with acetic, citric, lactic and hydrochloric acids (acid adapted cells). In all cases, acid adapted cells, with D-values ranging from 23.34 to 86.90 min, showed a significantly higher acid resistance than non-acid adapted cells, with D-values between 8.90 and 10.29 min. Whereas the conditions used for acid adaptation did not exert a significant effect on the acid resistance of the S. Typhimurium CECT 443 resulting cells, the inclusion of lysine and arginine in the challenge medium protected them against acid inactivation, reaching D-values of about 2 and 3 times higher, respectively, than those found in MM or MM supplemented with glutamic acid. None of these three amino acids significantly modified the acid resistance of non-acid adapted cells. The relative expression level of adiA (encoding the arginine decarboxylase), adiY (encoding the transcriptional activator of adiA), cadA (encoding the lysine decarboxylase) and cadB (encoding the lysine/cadaverine transport protein) was examined by quantitative PCR. Acid adapted cells showed higher relative expression levels for both systems, arginine decarboxylase and lysine decarboxylase, which demonstrates that the induction of specialized pH-homeostatic systems plays an important role in S. Typhimurium CECT 443 protection against acid stress. However, the increased acid resistance showed by acid adapted cells challenged in MM arginine or lysine free suggests the existence of other microbial survival strategies.

Effects of organic acids on thermal inactivation of acid and cold stressed Enterococcus faecium

In this study the adaptative response to heat (70 °C) of Enterococcus faecium using fresh and refrigerated (at 4 °C for up to 1 month) stationary phase cells grown in Brain Heart Infusion (BHI) buffered at pH 7.4 (non-acid-adapted cells) and acidified BHI at pH values of 6.4 and 5.4 with acetic, ascorbic, citric, lactic, malic and hydrochloric acids (acid-adapted cells) was evaluated. In all cases, the survival curves obtained were concave upward. A mathematical model based on the Weibull distribution accurately described the inactivation kinetic. The results indicate that previous adaptation to a low pH increased the bacterial heat resistance, whereas the subsequent cold storage of cells reduced E. faecium thermal tolerance. Fresh acid-adapted cells showed t 2.5-values (time needed to obtain an inactivation level of 2.5 log 10 cycles) ranging from 2.57 to 9.51 min, while non-acid-adapted cells showed t 2.5-values of 1.92 min. The extent of increased heat tolerance varied with the acid examined, resulting in the following order: citric ≥ acetic > malic ≥ lactic > hydrochloric ≥ ascorbic. In contrast, cold storage progressively decreased E. faecium thermal resistance. The t 2.5 values found at the end of the period studied were about 2–3-fold lower than those corresponding to non-refrigerated cells, although this decrease was more marked (about 5-fold) when cells were grown in buffered BHI and BHI acidified at pH 5.4 with hydrochloric acid. These findings highlight the need for a better understanding of microbial response to various preservation stresses in order to increase the efficiency of thermal processes and to indicate the convenience of counterbalancing the benefits of the hurdle concept.

The Desensitization Gating of the MthK K+ Channel Is Governed by Its Cytoplasmic Amino Terminus

The RCK-containing MthK channel undergoes two inactivation processes: activation-coupled desensitization and acid-induced inactivation. The acid inactivation is mediated by the C-terminal RCK domain assembly. Here, we report that the desensitization gating is governed by a desensitization domain (DD) of the cytoplasmic N-terminal 17 residues. Deletion of DD completely removes the desensitization, and the process can be fully restored by a synthetic DD peptide added in trans. Mutagenesis analyses reveal a sequence-specific determinant for desensitization within the initial hydrophobic segment of DD. Proton nuclear magnetic resonance (1H NMR) spectroscopy analyses with synthetic peptides and isolated RCK show interactions between the two terminal domains. Additionally, we show that deletion of DD does not affect the acid-induced inactivation, indicating that the two inactivation processes are mutually independent. Our results demonstrate that the short N-terminal DD of MthK functions as a complete moveable module responsible for the desensitization. Its interaction with the C-terminal RCK domain may play a role in the gating process.

Enzymatic esterification of lactic acid, utilizing the basicity of particular polar organic solvents to suppress the acidity of lactic acid

AbstractBACKGROUND: Lipase‐catalyzed esterification of lactic acid with ethanol was investigated; however, some difficulties exist with such a system. Because of its high polarity, lactic acid is immiscible with non‐polar organic solvents, which have generally been used for non‐aqueous enzyme reactions. In addition, the strong acidity of lactic acid causes acid inactivation of enzymes and acid‐catalyzed, non‐enzymatic esterification.RESULT: In the present study, particular polar organic solvents, such as 1,4‐dioxane, were found to suppress the enzyme inactivation and non‐enzymatic esterification caused by the acidity. The magnitude of this effect varied with solvents and strongly correlated with the Kamlet‐Taft parameter β, which indicates the basicity of the solvents in non‐aqueous systems. An immobilized lipase from Candida antarctica was found to be the most active and stable enzyme for the reaction. Based on the findings, lipase‐catalyzed esterification of lactic acid (1.0 mol L−1) could be continued for up to 4 weeks without any loss of enzyme activity.CONCLUSION: In addition to miscibility with lactic acid, the effect of these polar solvents, which is to suppress the acidity of lactic acid and is presumably due to the basicity, appears to play a very important role in the efficient enzymatic reaction of lactic acid. Copyright © 2008 Society of Chemical Industry

Stabilization of enzyme activity during the esterification of lactic acid in hydrophobic ethers and ketones as reaction media that are miscible with lactic acid despite their high hydrophobicity

Lipase-catalyzed esterification of lactic acid has thus far achieved only limited success due to high acidity and polarity of lactic acid: acid inactivation of enzymes, and immiscibility of lactic acid with hydrophobic organic solvents that are commonly used for non-aqueous enzyme reactions. Herein, we investigated enzymatic esterification of lactic acid with ethanol by using hydrophobic ethers and ketones as reaction media. Both of the solvents are miscible with lactic acid, and have the basicity to suppress the acid inactivation of enzymes, as well as polar solvents. Nevertheless, these solvents would be less harmful to enzymes rather than polar solvents because of their high hydrophobicity. Using the ethers and ketones, ethyl lactate was successfully synthesized enzymatically even at higher lactic acid concentrations, and the enzyme activity was effectively stabilized even in repeated batch-wise reactions (2.0 M lactic acid, 4 weeks). This effect of some ethers and ketone to stabilize the enzyme was significantly higher than that of polar solvents in the presence of polar alcohols, the other substrate. In addition, more various kinds of lipase could esterify 1.0 M lactic acid in the hydrophobic ether and ketone rather than those in hydrophobic solvents, polar solvents, and a solvent-free system.

Inactivation of Calcium-Dependent Lactic Acid Bacteria Phages by Phosphates

The capacity of three phosphates to interrupt the lytic cycle of four specific autochthonal bacteriophages of lactic acid bacteria used as starters was assayed. The phosphates used (polyphosphates A and B and sodium tripolyphosphate–high solubility [TAS]) were selected on the basis of their capacity to sequester divalent cations, which are involved in the lytic cycle of certain bacteriophages. The assays were performed in culture media (deMan Rogosa Sharpe and Elliker broths) and reconstituted (10%, wt/vol) commercial skim milk to which phosphates had been added at concentrations of 0.1, 0.3, and 0.5% (wt/vol). Phosphate TAS was the most inhibitory one, since it was able to inhibit the lytic cycle of all bacteriophages studied, in both broths and milk. In broth, polyphosphates A and B inhibited the lytic cycle of only two bacteriophages at the maximal concentration used (0.5%), whereas in milk, they were not capable of maintaining the same inhibitory effect.

A single M protein mutation affects the acid inactivation threshold and growth kinetics of a chimeric flavivirus

Numerous viruses of the Flaviviridae family, including dengue, yellow fever, Japanese encephalitis, and West Nile, cause significant disease in humans and animals. The structure and function of the molecular components of the flavivirus envelope are therefore of significant interest. To our knowledge, a membrane (M) protein mutation which affects the pH at which flavivirus particles are inactivated in vitro has never been reported. Here we show that substitution of proline for glutamine at residue M5 (M Q5P) of a Japanese encephalitis–yellow fever chimera (ChimeriVax-JE) increases its acid sensitivity in vitro by 0.3 pH units (i.e., increases the pH at which virus titer is reduced by 50% from 6.08 to 6.38). In addition, growth kinetics of this mutant virus are accelerated in Vero cells, while neurovirulence and neuroinvasiveness measured in a mouse model are unaffected. A possible interpretation of these observations is that M can modulate the envelope (E) protein function during cell infection.