Exercise-induced oxidative stress: the effects of ß-alanine supplementation in women.

The purpose of this study was to evaluate the effects of ß-alanine supplementation on markers of oxidative stress. Twenty-four women (age: 21.7±2.1 years; VO2max: 2.6±0.3 l min(-1)) were randomly assigned, in a double-blind fashion, to a ß-alanine (BA, 2×800 mg tablets, 3× daily; CarnoSyn®; n=13) or placebo (PL, 2×800 mg maltodextrin tablets, 3× daily; n=11) group. A graded oxygen consumption test (VO2max) was performed to evaluate VO2max, time to exhaustion, ventilatory threshold and establish peak velocity (PV). A 40-min treadmill run was used to induce oxidative stress. Total antioxidant capacity, superoxide dismutase, 8-isoprostane (8ISO) and reduced glutathione were measured. Heart rate and ratings of perceived exertion were recorded during the 40 min run. Separate three- [4×2×2; acute (base vs. IP vs. 2 vs. 4 h)×chronic (pre- vs. post-)×treatment (BA vs. PL)] and two- [2×2; time (pre-supplement vs. post-supplement)×treatment (BA vs. PL)] way ANOVAs were used for analyses. There was a significant increase in VO2max (p=0.009), independent of treatment, with no significant changes in TTE (p=0.074) or VT (p=0.344). Ratings of perceived exertion values were significantly improved from pre- to post-supplementation for the BA group only at 40 min (p=0.02). The ANOVA model demonstrated no significant treatment effects on oxidative stress. The chronic effects of BA supplementation demonstrated little antioxidant potential, in women, and little influence on aerobic performance assessments.

Ergolytic/ergogenic effects of creatine on aerobic power.

This study evaluated the effects of creatine (Cr) loading and sex differences on aerobic running performance. 27 men (mean±SD; age: 22.2±3.1 years, ht: 179.5±8.7 cm, wt: 78.0±9.8 kg) and 28 women (age: 21.2±2.1 years, ht: 166.0±5.8 cm, wt: 63.4±8.9 kg) were randomly assigned to either creatine (Cr, di-creatine citrate; n=27) or a placebo (PL; n=28) group, ingesting 1 packet 4 times daily (total of 20 g/day) for 5 days. Aerobic power (maximal oxygen consumption: VO2max) was assessed before and after supplementation using open circuit spirometry (Parvo-Medics) during graded exercise tests on a treadmill. 4 high-speed runs to exhaustion were conducted at 110, 105, 100, and 90% of peak velocity to determine critical velocity (CV). Distances achieved were plotted over times-to-exhaustion and linear regression was used to determine the slopes (critical velocity, CV) assessing aerobic performance. The results indicated that Cr loading did not positively or negatively influence VO2max, CV, time to exhaustion or body mass (p>0.05). These results suggest Cr supplementation may be used in aerobic running activities without detriments to performance.

Acute effects of a beverage containing bitter melon extract (CARELA) on postprandial glycemia among prediabetic adults.

BACKGROUND: Acute ingestion of bitter melon (BM) has been shown to suppress the postprandial glycemic response in diabetics, but its impact on glucose regulation among individuals with impaired glucose tolerance is unclear. Moreover, one's glucose tolerance level may influence the effectiveness of BM. This study aimed to examine the acute effects of a beverage containing BM extract on blood glucose regulation during an oral glucose tolerance test (OGTT) among prediabetics. METHODS: Ten prediabetic adults completed two OGTTs-glucose only (D2) and glucose+BM (D3). Responders were identified as subjects whose area under the glucose curve (AUCglu) during D3 was lower than D2. To compare the acute effects of the beverage among individuals with varying glucose tolerance levels, subjects were grouped by their glucose response pattern-Fastpeak (peak glucose (Glupeak) at 30 min postglucose (30P)) and Slowpeak (Glupeak after 30P). RESULTS: During D3, responders (n=5) experienced a 13.2% reduction in AUCglu (95% confidence interval (CI): -18.1% to -8.3%), 12.2% reduction in mean glucose (95% CI: -17.3% to -7.0%) and 10.6% reduction in Glupeak (95% CI: -17.5% to -3.7%); plasma glucose was reduced by 9.1% at 30P (95% CI: -15.6% to -2.6%), -24.0% at 60P (95% CI: -36.8% to -11.2%) and -20.0% at 90P (95% CI: -35.8% to -4.2%) during D3. No between-trial differences were noted for Fastpeak or Slowpeak. CONCLUSIONS: Acute ingestion of BM prior to the second OGTT (D3) led to a reduced postprandial glucose response in 50% of the subjects but did not affect the insulin response. Furthermore, the effectiveness of the beverage was seemingly uninfluenced by the subjects' glucose tolerance level. Although BM has shown to aid blood glucose management in diabetics, it remains uncertain why only a portion of subjects responded positively to the BM extract in the current study.

Studies on the mechanism of action of A80915A, a semi-naphthoquinone natural product, as an inhibitor of gastric (H(+)-K+)-ATPase.

A semi-naphthoquinone natural product, A80915A, produced by Streptomyces aculeolatus was found to be a potent inhibitor of gastric (H(+)-K+)-ATPase, the enzyme responsible for acid secretion in the stomach. Enzyme activity was measured by potassium-stimulated hydrolysis of ATP or p-nitrophenolphosphate with enzyme prepared from the stomach fundic mucosa of pigs. Concentration-dependent inhibition was observed with an IC50 of about 2-3 microM for both ATPase and p-nitrophenylphosphatase. A Hill plot indicated that the enzyme has two binding sites for A80915A. Inhibition was not affected by the presence of the reducing agent dithiothreitol, indicating a lack of involvement of enzyme sulfhydryl groups. A 30-min incubation of enzyme with increasing drug concentrations followed by a 10-fold dilution did not alter the IC50, indicating that A80915A does not covalently modify the enzyme. Coincubation of enzyme with 3.8 microM A80915A resulted in time-dependent inhibition. The rate of inhibition was slowed significantly by the presence of 20 mM potassium, rubidium and ammonium but not by 20 mM sodium, lithium and choline, or by 40 mM sucrose. The level of inhibition was influenced by the order of addition of potassium and drug to the enzyme. Taken together, these studies indicate that inhibition by A80915A is dependent on the conformation of gastric (H(+)-K+)-ATPase and that potassium slows the rate of inhibition by converting the enzyme to a conformation where the drug binding site is not as accessible. The mode of action of A80915A is distinct from that of two well characterized proton pump inhibitors, omeprazole and SCH 28080.

Deacylation of A21978C, an acidic lipopeptide antibiotic complex, by Actinoplanes utahensis.

A21978C, produced by Streptomyces roseosporus NRRL 11379, is an acidic lipopeptide antibiotic complex that inhibits Gram-positive bacteria. Individual factors of the complex possess an identical peptide core or "nucleus", and are differentiated by the distinctive fatty acid acyl group attached to the N-terminus of the nucleus. Certain members of the family Actinoplanaceae deacylated A21978C to yield the unaltered nucleus, which was then reacylated to form new analogs. Actinoplanes utahensis NRRL 12052 was the most efficient of these cultures, producing up to 500 micrograms of nucleus per ml of culture broth per hour. Eacylation was also accomplished with semi-pure and tert-butoxycarbonyl (tert-BOC)-A21978C. In the latter, the ornithine amino group was blocked to prevent formation of diacyl analogs during reacylation. The acylase was an endoenzyme present in submerged cultures of A. utahensis from less than 18 to greater than 168 hours of incubation. Whole cells suspended in phosphate buffer or entrapped in polyacrylamide gel also deacylated A21978C efficiently.

A54145, a new lipopeptide antibiotic complex: microbial and chemical modification.

A54145 is a complex of acidic lipopeptide antibiotics produced by Streptomyces fradiae NRRL 18158, NRRL 18159, and NRRL 18160. Each antibiotic factor consists of a peptide core bearing an N-terminal acyl substituent. N-Lys-tert-BOC-protected A54145 complex was deacylated by Actinoplanes utahensis; three protected core peptides were isolated. A54145 antibiotic analogs were synthesized by acylation of the tryptophan N-terminus with 2,4,5-trichlorophenyl active esters, followed by deblocking with trifluoroacetic acid.

Deacylation of echinocandin B by Actinoplanes utahensis.

Echinocandin B (ECB) is a lipopeptide antifungal agent produced by several species of Aspergillus. The lipid side chain of cyclic lipopeptides is known to be an important determinant of their antibiotic activity and toxicity. Deacylation of another lipopeptide antibiotic, A21978C, had formerly been accomplished with Actinoplanes utahensis. In spite of the structural dissimilarities between the peptide cores and acyl side chains of A21978C and ECB, A. utahensis also removed the linoleoyl acyl unit from the amino terminus of ECB to yield the bioinactive cyclic peptide core, or "nucleus". The ECB nucleus, which contained a new titratable group at the N-terminus, was subsequently employed for chemical reacylation with other side chains to yield a variety of novel ECB analogs. One of these, cilofungin (LY121019), containing an N-(4-n-octyloxybenzoyl)acyl unit, is currently undergoing clinical evaluation.

De-esterification of cephalosporin para-nitrobenzyl esters by microbial enzymes.

Bacteria and actinomycetes were screened for esterase enzymes capable of removing the para-nitrobenzyl ester from cephalosporins. An esterase preparation from Bacillus subtilis was used to prepare cephalexin and 7-ADCA from the corresponding para-nitrobenzyl esters.

A54145, a new lipopeptide antibiotic complex: isolation and characterization.

A54145 is a complex of acidic lipopeptide antibiotics which are produced by Streptomyces fradiae and are active against Gram-positive bacteria. The A54145 complex was isolated by adsorption on Diaion HP-20 nonfunctionalized macroreticular resin and/or ion exchange on Amberlite IRA-68 anion exchange resin. Antibacterial factors A, A1, B, B1, C, D, E, and F were obtained in purified form by repeated preparative reverse phase HPLC on C8 and/or C18 bonded-phase supports. The molecular formulae of the factors are C72H109N17O27 (factors A and A1), C73H111N17O27 (factors B, B1, C, and D), C74H113N17O27 (factor E), and C71H107N17O27 (factor F). The identities of the acyl side chains were established as 8-methylnonanoyl (factors F, A, and B1), n-decanoyl (factors A1 and B), and 8-methyldecanoyl (factors C, D, and E).

Isolation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors, were isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098. A58365A and A58365B are homologous nitrogen-containing bicyclic structures of molecular formulae C12H13NO6 and C13H15NO6.

A54145, a new lipopeptide antibiotic complex: discovery, taxonomy, fermentation and HPLC.

A54145 is a complex of new lipopeptide antibiotics that inhibits Gram-positive bacteria and acts as a growth promotant for broiler chicks. Eight factors; A, B, C, D, E, F, A1 and B1; have been isolated and characterized. They contain four similar peptide nuclei, each of which is acylated with either an 2-decanoyl, n-decanoyl, or undecanoyl side chain. Taxonomic studies ascertained that the producing microorganism was a strain of Streptomyces fradiae. Fermentation studies determined that superior antibiotic yields were obtained in stirred bioreactors in a soybean flour-molasses medium employing a continuous glucose feed. These findings, interwoven with the selection of hyper-productive mutants, increased fermentation yields from less than 50 micrograms/ml to more than 1 mg/ml. An analytical HPLC system was developed for the identification and subsequent quantitation of each factor of the A54145 complex.

Method for the detection and quantitation of chitinase inhibitors in fermentation broths; isolation and insect life cycle effect of A82516.

A new method of screening for chitinase inhibitors in crude fermentation broths as a means of discovering new insecticidal leads has been developed. In this procedure soluble Remazol brilliant violet 5R dye-coupled chitin degradation products released from insoluble chitin azure substrate by hydrolysis with Streptomyces griseus chitinase are filtered in 0.45 micron Millititer HA 96 well filtration plates and collected in 96 well microtiter plates. Inhibitors of this reaction are detected by a decrease in absorbance (570 nm) of the filtrate. A chitinase inhibitor, designated A82516, produced by culture A82516 was discovered using this screen. Purified A82516 was found to have an IC50 of 3.7 X 10(-6) M for S. griseus chitinase. At a test concentration of 0.27 mg/ml, A82516 was 100% effective in preventing development of house fly larvae to pupae. Allosamidin, a recently reported chitinase inhibitor in vitro, has spectral properties identical to A82516.

Structure elucidation of A58365A and A58365B, angiotensin converting enzyme inhibitors produced by Streptomyces chromofuscus.

A58365A and A58365B, angiotensin converting enzyme inhibitors isolated from the culture filtrate of Streptomyces chromofuscus NRRL 15098, are homologous compounds of molecular formulas C12H13NO6 and C13H15NO6. The molecular similarities of the two inhibitors were established by comparison of their 1H NMR, 13C NMR, and UV spectra. Catalytic hydrogenation of A58365A led to a tetrahydro-deoxy derivative, C12H17NO5; extensive 1H NMR decoupling studies at 360 MHz allowed all the non-exchangeable protons of the derivative to be connected in a continuous substructure. This fragment was combined with information from other spectroscopic methods to suggest the structures for A58365A (1) and A58365B (2); the conclusions were confirmed by an X-ray crystallographic analysis of A58365A-dimethyl ester.

A54145 a new lipopeptide antibiotic complex: microbiological evaluation.

A54145 complex is made up of eight factors; A, A1, B, B1, C, D, E, and F which were active in vitro (MIC 0.25 approximately greater than 32 micrograms/ml) against Gram-positive aerobic organisms. The complex, factor B and B1 were found to be active against two strains of Clostridium perfringens. A calcium dependence study on some of the factors showed that their in vitro antibacterial activity was greatly enhanced by the presence of calcium (50 mg/liter) in the media. Resistance build-up was seen when Staphylococcus sp. and Streptococcus sp. were passed seven times in the presence of sublethal concentrations of A54145 antibiotics. This resistance disappeared immediately when the resistant organisms were passed in the absence of the antibiotics. Factor A was very effective against Staphylococcus aureus and Streptococcus pyogenes infections in mice (sc ED50s of 3.3 approximately 2.4 mg/kg x 2, respectively). Factor B was more active against S. pyogenes in vivo (sc ED50, 0.9 mg/kg x 2). Acute mouse toxicities were determined with these antibiotics. Semisynthetic derivatives were evaluated.

Synthesis of new analogs of echinocandin B by enzymatic deacylation and chemical reacylation of the echinocandin B peptide: synthesis of the antifungal agent cilofungin (LY121019).

The antifungal antibiotic, echinocandin B (ECB), was modified by a sequential procedure in which the initial step involved enzymatic removal of the native N-linoleoyl group from the N-terminus using an Actinoplanes utahensis culture. The resulting product, ECB nucleus, was reacylated using active esters or acid halides of various substituted acids to give a series of ECB analogs. These analogs possessed anti-Candida activity both in vitro and in vivo (mice). Other studies have shown that one of these, cilofungin, the 4-n-octyloxybenzoyl-ECB analog (LY121019), has excellent anti-Candida activity, low toxicity and is superior to other available antifungal antibiotics.

A21978C, a complex of new acidic peptide antibiotics: isolation, chemistry, and mass spectral structure elucidation.

A21978C, produced by Streptomyces roseosporus, NRRL 11379, is a complex of new acidic lipopeptolide antibiotics which inhibits Gram-positive bacteria. HPLC separation of the various components from the purified complex resulted in the isolation of A21978C1, -C2 and -C3 (major components) and -C4, -C5, and -C0 (minor components). Each of these components was fermented with cultures of Actinoplanes utahensis (NRRL 12052) to give the identical inactive peptide ("A21978C nucleus") by removal of the fatty acid acyl groups from the N-terminus. This peptide was composed of 13 amino acids: L-kynurenine, L-threo-3-methylglutamic acid, L-asparagine, L-aspartic acid (3 residues), glycine (2 residues), L-tryptophan, L-ornithine, D-alanine, D-serine and L-threonine. The amino acid sequence was determined using a combination of the Edman degradation and gas chromatography mass spectrum (GC-MS) analysis of appropriately derivatized peptides obtained from partial hydrolysis. Each major component was shown to be acylated with a branched chain fatty acid at the N-terminus and the structure of this fatty acid was determined by 1H NMR and mass spectral methods. A structure for A21978C was assigned on the basis of this degradative and physico-chemical information.

Preparation and evaluation of 3,4''-ester derivatives of 16-membered macrolide antibiotics related to tylosin.

A large group of ester derivatives of tylosin-related macrolides was prepared in which the hydroxyl groups at C-3 and C-4'' were acylated by either chemical or biochemical methods. Most of the derivatives exhibited excellent in vitro antimicrobial activity. However, only the 3,4''-diacyl derivatives of tylosin and macrocin showed any significant improvements of in vivo efficacy against experimental infections in rodents.

Enzymatic and chemical modifications of lipopeptide antibiotic A21978C: the synthesis and evaluation of daptomycin (LY146032).

The novel lipopeptide antibiotic A21978C complex is active against Gram-positive organisms. This complex consists of a common peptide nucleus with various lipid acyl groups at the N-terminus characteristic of each individual factor. The fatty acid acyl group is removed by incubation of the A21978C complex with Actinoplanes utahensis to give the peptide nucleus. This peptide nucleus has the same amino acid sequence as A21978C. New analogs of A21978C were synthesized by acylation of the N-terminus of a tert-butoxycarbonyl (tert-BOC)-protected nucleus and subsequent deprotection. 1H NMR showed that the newly introduced acyl group was at the desired N-terminus. Three major groups of analogs were synthesized bearing fatty acid acyl, amino-aroyl and extended peptide side chains. Each analog was evaluated for antimicrobial activity and acute toxicity. Of these analogs, the n-decanoyl analog of A21978C (LY146032) gave the best survival in the mouse acute toxicity test at a high dose of 1,000 mg/kg, iv and was chosen for further study. This analog has been named daptomycin.

A80915, a new antibiotic complex produced by Streptomyces aculeolatus. Discovery, taxonomy, fermentation, isolation, characterization, and antibacterial evaluation.

New semi-naphthaquinone antibiotics A80915A, B, C, and D were isolated from the fermented broth of Streptomyces aculeolatus A80915 (NRRL 18422). Factors A and C, present in both the broth filtrate and mycelial methanol extract, and factors B and D, found predominantly in the broth filtrate, were recovered by extraction with ethyl acetate. Purification of the individual factors was accomplished by preparative reverse phase high performance liquid chromatograph on C18 bonded silica supports. Factors A through D show antimicrobial activity against Gram-positive aerobic and anaerobic organisms in vitro. Mechanism of action studies demonstrated nearly complete inhibition of macromolecular biosynthesis (protein, RNA, DNA, and cell wall) by A80915 factors A through D. A less highly cyclized semi-naphthaquinone, A80915 factor G, was isolated from the broth of the strain fermented in an alternate medium.