Metabolic syndrome is associated to high-sensitivity cardiac troponin T elevation

Introduction: Metabolic syndrome (MetS) and high-sensitivity cardiac troponin T (hs-TnT) are associated with higher risk for cardiovascular diseases (CVD). Our aim was to assess the relation between hs-TnT elevation and MetS in a general population sample.

Materials and methods: Individuals participating in an annual health survey program between 2010 and 2016 were included in the study. Blood samples including hs-TnT levels were collected. The study population was divided into three groups based on hs-TnT levels – undetectable (<5?ng/L), intermediate (5–14?ng/L) and elevated (>14?ng/L).

Results: A total of 5994 subjects were included in the study, the mean age was 48.5 and 4336 (72%) were males. Compared with subjects with undetectable hs-TnT the prevalence of MetS was higher in those with detectable and elevated levels – 392 (10%) vs. 270 (15%) and 51 (33%), respectively (p?<?0.001). In a multivariate model adjusted for age, gender and multiple co-morbidities, the number of MetS components and presence of MetS were significantly associated with an increased risk for detectable hs-TnT levels (OR?=?1.02 {for each component}; 95% CI [1.00–1.05], p?=?0.04) and (OR?=?1.13; 95% CI [1.07–1.2], p?<?0.001) respectively. Only the waist, glucose and hypertension components of the MetS were significantly associated with elevated troponin.

Conclusions: The MetS and its distinct components have a cumulative impact on hs-TnT levels in apparently healthy subjects.     

Comet assay for assessment of DNA damage in greenhouse workers exposed to pesticides

Purpose: The main goal of the present study was to determine DNA damage in pesticide-exposed greenhouse workers and pesticides non-exposed controls.

Materials and methods: The DNA damage was measured by alkaline comet assay method (pH?>?13) in 41 greenhouse workers and 45 non-exposed individuals as the control. Pesticide exposure was assessed by duration of working in the greenhouse and pesticide application in the greenhouse time. DNA damage was estimated by arbitrary unit and damage frequency.

Results: Arbitrary unit and damage frequency were consistently significantly higher in greenhouse workers than those of the controls (p?=?0.001). In terms of gender in greenhouse, DNA damage of female workers was significantly higher than those in male workers (p?<?0.05). We found significant correlation between DNA damage and working hours spent. Multiple linear regression analysis showed that working hours in the greenhouse as an indication of pesticide exposure were significantly associated with the DNA damage, which can be attributed to the genotoxic potential of the pesticide mixture.

Conclusions: The comet assay is sensitive to detect the damage exposed to chronic effect of pesticides in greenhouse workers. Significant DNA damage was obtained for the exposed group, which was associated with the pesticide exposure.     

N-Acylhomoserine lactone-mediated quorum sensing regulates biofilm structure in Methylobacterium populi P-1M,an isolate from a pink-pigmented household biofilm

Numerous gram-negative bacteria have quorum-sensing systems and produce AHL as a quorum-sensing signal molecule. In this study, we demonstrated that Methylobacterium populi P-1M, an isolate from a pink-pigmented household biofilm, produced two AHLs, C14:1-HSL as a predominant product and 3OHC14-HSL as a minor product. The complete genome sequence of M. populi P-1M revealed the presence of genes that are predicted to encode an AHL synthase (mpoI) and AHL receptor (mpoR). M. populi P-1M formed a pellicle-like biofilm, which had a flat surface and was easily removable. In contrast, biofilms formed by mpoI and/or mpoR deletion mutants had a wavy surface structure and strongly adhered to the glass tube. When C14:1-HSL was added to the mpoI mutant culture, the biofilm structure resembled that of the wild-type strain. These results demonstrated that the structure and adhesion strength of M. populi P-1M biofilms are determined in part by AHL-mediated quorum sensing.

Abbreviations: AHL: N-acyl-l-homoserine lactone; C14:1-HSL: N-tetradecenoyl-l-homoserine lactone; 3OHC14-HSL: N-(3-hydroxytetradecanoyl)-l-homoserine lactone; SAM: S-adenosyl-l-methionine; ACP: acyl-acyl carrier protein; EPS: extracellular polysaccharide; DMSO: dimethyl sulfoxide     

Phytochemicals,antioxidant activity and hepatoprotective effect of ginger (Zingiber officinale) on diethylnitrosamine toxicity in rats

Context: Chronic liver damage has serious medical consequences.

Objective: To investigate the hepatoprotective effect of dry Zingiber officinale (ginger) and its essential (volatile) oil against diethylnitrosamine (DEN) toxicity in rats.

Materials and methods: Phenols and flavonoids components were characterized in dry ginger using HPLC-UV instrument while ginger essential oil (E.O.) was investigated via GC-MS technique. Antioxidant activity was determined in vitro. In rat model, ginger was administrated for 2 months. Lipid profile, antioxidant biomarkers, liver functions and histopathology were assessed.

Results: Chlorogenic acid (63.85?ppm) and hesperidin (156.91?ppm) are among the major phenolic and flavonoid constituents in dry ginger. Curcumene (15.21%) and linalool (13.47%) represent the main E.O. constituents. In rats treated with ginger E.O., a significant elevation in serum HDL (31.14%) was accompanied by a decrease in LDL (55.14%). A significant decrease in serum ALT and ALP was reported (56.85% and 53.84%, respectively). Serum GSH-Px activity has significantly increased 75.06%. Meanwhile, E.O. showed anticancer potential against HepG2 cell line (IC₅₀?=?40?µg/mL). Liver histopathological examinations confirmed the protective effect against abnormalities.

Conclusion: Ginger was able to reduce the severity of DEN-cytotoxicity in rats, which suggests a novel antioxidant role originating from this medicinal plant.     

Identifying membrane-bound quinoprotein glucose dehydrogenase from acetic acid bacteria that produce lactobionic and cellobionic acids

Acetic acid bacteria are used in the commercial production of lactobionic acid (LacA). However, the lactose-oxidizing enzyme of these bacteria remains unidentified. Lactose-oxidizing activity has been detected in bacterial membrane fractions and is strongly inhibited by d-glucose, suggesting that the enzyme was a membrane-bound quinoprotein glucose dehydrogenase, but these dehydrogenases have been reported to be incapable of oxidizing lactose. Thus, we generated m-GDH-overexpressing and -deficient strains of Komagataeibacter medellinensis NBRC3288 and investigated their lactose-oxidizing activities. Whereas the overexpressing variants produced ~2–5-fold higher amounts of LacA than the wild-type strains, the deficient variant produced no LacA or d-gluconic acid. Our results indicate that the lactose-oxidizing enzyme from acetic acid bacteria is membrane-bound quinoprotein glucose dehydrogenase.

Abbreviations: LacA: lactobionic acid; AAB: acetic acid bacterium; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection     

Elimination,Replacement and Isomerization Reactions by Intact Cells Containing Tyrosine Phenol Lyase

Tyrosine phenol lyase catalyzes a series of α,β-elimination, β-replacement and racemization reactions. These reactions were studied with intact cells of Erwinia herbicola ATCC 21434 containing tyrosine phenol lyase.

Various aromatic amino acids were synthesized from l-serine and phenol, pyrocatechol, resorcinol or pyrogallol by the replacement reaction using the intact cells. l(d)-Tyrosine, 3,4-dihydroxyphenyl-l(d)-alanine (l(d)-dopa), l(d)-serine, l-cysteine, l-cystine and S-methyl-l-cysteine were degraded to pyruvate and ammonia by the elimination reaction. These amino acids could be used as substrate, together with phenol or pyrocatechol, to synthesize l-tyrosine or l-dopa via the replacement reaction by intact cells. l-Serine and d-serine were the best amino acid substrates for the synthesis of l-tyrosine or l-dopa. l-Tyrosine and l-dopa synthesized from d-serine and phenol or pyrocatechol were confirmed to be entirely l-form after isolation and identification of these products. The isomerization of d-tyrosine to l-tyrosine was also catalyzed by intact cells.

Thus, l-tyrosine or l-dopa could be synthesized from dl-serine and phenol or pyrocatechol by intact cells of Erwinia herbicola containing tyrosine phenol lyase.     

Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine

Pyrrolothiazolate formed by the Maillard reaction between l-cysteine and d-glucose has a pyrrolothiazole skeleton as a chromophore. We searched for a Maillard pigment having a pyrrolooxazole skeleton formed from l-threonine or l-serine instead of l-cysteine in the presence of d-glucose. As a result, two novel yellow pigments, named pyrrolooxazolates A and B, were isolated from model solutions of the Maillard reaction containing l-threonine and d-glucose, and l-serine and d-glucose, respectively, and identified as (2R,3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-2,5,7a-trimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid and (3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-5,7a-dimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid by instrumental analyses. These compounds were pyrrolooxazole derivatives carrying a carboxy group, and showed the absorption maxima at 300–360 nm under acidic and neutral conditions and at 320–390 nm under alkaline conditions.     

Tumor delivery of liposomal doxorubicin prepared with poly-L-glutamic acid as a drug-trapping agent

Context: Poly-l-glutamic acid (PGA) is an anionic polymer with a large number of carboxyl groups that can interact electrostatically with cationic drugs such as doxorubicin (DOX).

Objective: For stable encapsulation of DOX into liposomes, we prepared triethylamine (TEA)-PGA-liposomes using PGA as an internal trapping agent.

Methods: We prepared TEA-PGA-liposomes by remote loading of DOX with a TEA gradient into preformed liposomes prepared with 1, 2, or 4?mg/mL PGA (molecular weights 4800, 9800, and 20 500), and evaluated their biodistribution and antitumor effects on Lewis lung carcinoma (LLC) tumor-bearing mice.

Results: TEA-PGA-liposomes using the higher the molecular weight or concentration of PGA showed a slower release of DOX from the liposomes. TEA-PGA-liposomes prepared with a high concentration of PGA could enhance DOX accumulation in tumors and prolonged DOX circulation in the serum, indicating that DOX may be retained stably in the liposomal interior by interaction with PGA. Furthermore, injection of TEA-PGA-liposomes prepared with 4?mg/mL of PGA₉₈₀₀ or 2?mg/mL PGA₂₀₅₀₀ strongly inhibited tumor growth in LLC tumor-bearing mice.

Conclusions: PGA may be a potential trapping agent for liposomal DOX for tumor drug delivery.     

Synthesis of Certain Disaccharides Containing a α-or β- Rhamnopyranosidic Group and the Substrate Specificity of α-l-Rhamnosidase from Aspergillus niger

To investigate the substrate specificity of α-l-rhamnosidase from Aspergillus niger, the following seven substrates were synthesized: methyl 3-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (1), methyl 3-O-α-l-rhamnopyranosyl-α-l-xylopyranoside (2), methyl 3-0-α-l-rhamnopyranosyl-α-l-rhamnopyranoside (3), methyl 4-0-α-l-rhamnopyranosyl-α-d-galactopyranoside (4), methyl 4-O-α-l-rhamnopyranosyl-α-d-mannopyranoside (5), methyl 4-0-α-l-rhamnopyra-nosyl-α-d-xylopyranoside (6), and 6-0-β-l-rhamnopyranosyl-d-mannopyranose (7). Compounds 1~6 were well-hydrolyzed by the crude enzyme, but 7 was unaffected.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

Regulatory Properties of Prephenate Dehydrogenase and Prephenate Dehydratase from Corynebacterium glutamicum

Regulatory properties of the enzymes in l-tyrosine and l-phenyalanine terminal pathway in Corynebacterium glutamicum were investigated. Prephenate dehydrogenase was partially feedback inhibited by l-tyrosine. Prephenate dehydratase was strongly inhibited by l-phenylalanine and l-tryptophan and 100% inhibition was attained at the concentrations of 5 × 10^?2mm and 10^?1mm, respectively. l-Tyrosine stimulated prephenate dehydratase activity (6-fold stimulation at 1 mm) and restored the enzyme activity inhibited by l-phenylalanine or l-tryptophan. These regulations seem to give the balanced synthesis of l-tyrosine and l-phenyl-alanine. Prephenate dehydratase from C. glutamicum was stimulated by l-methionine and l-leucine similarly to the enzyme in Bacillus subtilis and moreover by l-isoleucine and l-histidine. C. glutamicum mutant No. 66, an l-phenylalanine producer resistant to p-fluorophenyl-alanine, had a prephenate dehydratase completely resistant to the inhibition by l-phenylalanine and l-tryptophan.     

Mechanism of l-Threonine and l-Lysine Production by Analog-resistant Mutants of Corynebactemum glutamicum

Homoserine dehydrogenases and aspartokinases in l-threonine- or l-threonine and l-lysine-producing mutants derived from Corynebacterium glutamicum KY 9159 (Met^?) were studied with respect to the sensitivity to the inhibition by end products, l-threonine and l-lysine. The activities of homoserine dehydrogenases in the mutants which produced l-threonine or l-threonine and l-lysine were slightly less susceptible to the inhibition by l-threonine than the activity in the parent strain, KY 9159. The aspartokinases in the threonine-producing mutants, KY 10484 and KY 10230, which were resistant to α-amino-β-hydroxylvaleric acid (AHV, a threonine analog) and more sensitive to thialysine (a lysine analog) than the parent, were sensitive to the concerted feedback inhibition by l-lysine and l-threonine by about the same degree as KY 9159. The aspartokinase in an AHV- and thialysine-resistant mutant, KY 10440, which was derived from KY 10484 and produced about 14 mg/ml of l-threonine in a medium containing 10% glucose was less susceptible to the concerted feedback inhibition than KY 10484 or KY 9159, although the activity was still under the feedback control. In the parent strain, l-threonine activated aspartokinase activity in the absence of ammonium sulfate, an activator of the enzyme, but partially inhibited the activity in the presence of the salt. On the other hand, the enzyme of KY 10440 was activated by l-threonine either in the presence or in the absence of the salt. In another AHV- and thialysine-resistant mutant, KY 10251, which was derived from KY 10230 and produced both 9 mg/ml of l-threonine and 5/5 mg/ml of l-lysine, l-threonine and l-lysine simultaneously added hardly inhibited the activity of aspartokinase.

Implications of these results are discussed in relation to l-threonine or l-lysine production, AHV or thialysine resistance and regulation of l-threonine biosynthesis in these mutants.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Production of l-allose and d-talose from l-psicose and d-tagatose by l-ribose isomerase

l-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert l-psicose and d-tagatose to l-allose and d-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce l-allose and d-talose. Conversion reaction was performed with the reaction mixture containing 10% l-psicose or d-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of l-allose and d-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert l-psicose to l-allose without remarkable decrease in the enzyme activity over 7 times use and d-tagatose to d-talose over 37 times use. After separation and concentration, the mixture solution of l-allose and d-talose was concentrated up to 70% and crystallized by keeping at 4 °C. l-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% l-allose and 7.30% d-talose that were obtained from l-psicose and d-tagatose, respectively.     

On the Mechanism of l-Prolyl Diketopiperazine Formation by Streptomyces

Since l-prolyl diketopiperazines, l-prolyl-l-valine anhydride and l-leucyl-l-proline anhydride, had been isolated from the culture filtrate of Streptomyces sp. S-580, the mechanism of l-prolyl diketopiperazine formation by Streptomyces has been studied. These two l-prolyl diketopiperazines were not formed from their constituent amino acids incubated with intact cell or cell free homogenate of this strain in buffered salt solution containing energy source. However, from milk casein, poly peptone or gelatin, the former two were components of the culture medium of this strain, hydrolyzed with the pure streptomyces-protease, these l-prolyl diketopiperazines were obtained (only from gelatin, glycyl-l-proline anhydride were obtained in addition to these two). Furthermore, in hydrolysis of some synthetic l-prolyl peptides with this enzyme, l-prolyl diketopiperazine formation were also studied, and as the result, glycyl-l-proline anhydride was obtained from glycyl-l-prolyl-l-leucine but no l-prolyl diketopiperazine was formed from l-prolyl-l-leucyl-glycine. From these evidences, the possible route of l-prolyl diketopiperazine formation by Streptomyces has been discussed.     

Solubilities Studies of Basic Amino Acids

The solbilities of l-basic amino acids in the type of free, monohydro-chloride and dihydrochloride in water were determined, and the results were formulated as follows.

l-Arginine: log S = 0.9770+0.01345t (t is from 0°~70°C)

l-Histidine: log S = 0.3627+0.00905t (t is from 0°~70°C)

l-Arginine monohydrochloride: log S = 1.6532+0.01301t (t is from 0°~70°C)

l-Lysine monohydrochloride dihydrate: log S = 1.6990+0.01294t (t is from 0°~55°C)

l-Lysine monohydrochloride monohydrate: log S = 1.7404+0.01256t (t is from 55°~70°C)

l-Lysine dihydrochloride: log S = 2.2138+0.00409t (t is from 0°~70°C)

l-Histidine dihydrochloride: log S = 1.9085+0.00265t (t is from 0°~70°C)

Three component systems (basic amino acid, hydrochloric acid, water) were studied and the solubilities in mixed solution system of alcohol-water were also investigated.     

Formations of Oligopeptides from Dipeptides by the Protease from Streptomyces cellulosae

The protease from Streptomyces cellulosae formed more turbidity in a 16% soybean protein hydrolysate in the initial stage of the reaction than α-chymotrypsin did, when the proteolytic activity of the protease was same as that of α-chymotrypsin. In highly concentrated solutions (2.5%) of various dipeptides, oligopeptides were produced by condensation by the protease. The oligopeptides formed were (l-Leu-Gly)₂ and (l-Leu-Gly)₃ from l-Leu-Gly, (l-Phe-l-Val)₂ from l-Phe-l-Val, (l-Val-l-Phe)₂ and (l-Val-l-Phe)₃ from l-Val-l-Phe, and (l-Leu-l-Met)₂ and (l-Leu-l-Met)₃ from l-Leu-l-Met.     

Effects of Methionine,Cystine and Taurine on Plasma Cholesterol Level in Rats Fed a High Cholesterol Diet

l-Methionine γ-lyase (EC 4.4.1.11) catalyzes α,β-elimination of l-2-amino-3-(N-methylamino)propionic acid and l-2-amino-3-(N-hydroxyethylamino)propionic acid to yield pyruvate, ammonia, and the corresponding amines. These amino acids also undergo the enzymatic β-replacement reaction with thiols to produce the corresponding S-substituted cysteines. Thus, l-methionine γ-lyase cleaves a C-N bond in addition to C-S, C-Se, and C-O bonds at the β position of amino acids by elimination and replacement reactions. A linear relationship between the reactivity, (log(V_max/Km) and the pKa value of the conjugated acid of the leaving group has been found for Se-methyl-l-selenocysteine, S-methyl-l-cysteine, and O-methyl-l-serine. However, l-2-amino-3-(N-methylamino)propionic acid has shown lower reactivity than that expected from the pKa value of methylammonium ions.     

Purification and Crystallization of d-Arabinose (l-Fucose) Isomerase from Aerobacter aerogenes by Polyethylene Glycol

d-Arabinose(l-fucose) isomerase (d-arabinose ketol-isomerase, EC 5.3.1.3) was purified from the extracts of d-arabinose-grown cells of Aerobacter aerogenes, strain M-7 by the procedure of repeated fractional precipitation with polyethylene glycol 6000 and isolating the crystalline state. The crystalline enzyme was homogeneous in ultracentrifugal analysis and polyacrylamide gel electrophoresis. Sedimentation constant obtained was 15.4s and the molecular weight was estimated as being approximately 2.5 × 10⁵ by gel filtration on Sephadex G-200.

Optimum pH for isomerization of d-arabinose and of l-fucose was identical at pH 9.3, and the Michaelis constants were 51 mm for l-fucose and 160 mm for d-arabinose. Both of these activities decreased at the same rate with thermal inactivation at 45 and 50°C. All four pentitols inhibited two pentose isomerase activities competitively with same Ki values: 1.3–1.5 mm for d-arabitol, 2.2–2.7 mm for ribitol, 2.9–3.2 mm for l-arabitol, and 10–10.5 mm for xylitol. It is confirmed that the single enzyme is responsible for the isomerization of d-arabinose and l-fucose.     

Production of l-Valine by 2-Thiazolealanine Resistant Mutants Derived from Glutamic Acid Producing Bacteria

Potent l-valine producers were screened among 2-thiazolealanine resistant mutants derived from three typical l-glutamic acid producing bacteria: Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Arthrobacter citreus. By strain No. 487, the best producer derived from Brevibacterium, 31 mg/ml of l-valine was produced after 72 hr when 10% glucose was supplied as a carbon source, thus giving the yield of 31% from glucose. Accumulation of the other amino acids was negligible. The addition of l-isoleucine and l-leucine in the culture medium did not reduce the l-valine production, indicating that the l-valine biosynthesis is insensitive to these end products in the l-valine producer.