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Synthesis of sparsomycin analogs as potential antitumor agents.

No information is available on the structural requirements for the antitumor activity of sparsomycin, an antibiotic obtained from the fermentation broth of Streptomyces sparsogenes. Its high in vivo and in vitro activity, novel structure, and uncommon mode of action have, therefore, suggested the synthesis of analogs. This report describes the preparation and screening of a series of N-substituted 3-aryl acrylamides which are closely related to sparsomycin. Three compounds exhibited some tumor inhibition but insufficient to warrant further testing.

Animals↗

Pancreatic lipase activity as influenced by unconjugated bile acids and pH, measured in vitro and in vivo.

The relation between pancreatic lipase activity, unconjugated bile acids and pH was studied in vitro and in vivo. Lipase activity was assayed in vitro using automatic titration, where the fatty acids liberated from the hydrolysis of glycerol tributyrate (GTB) were measured. The lipase activity was determined at different ratios of conjugated to unconjugated bile acids (100:0, 75:25, 50:50, 25:75, 0:100) in response to pH 6.6, 6.8, 7.0 and 7.5. The in vivo study involved 96 one-day-old male broiler chickens. The chickens were assigned randomly, in pens of six animals, into two dietary treatments (8 replicate blocks), composing a non-supplemented diet (A(-)) and a diet supplemented (A(+)) with avilamycin (10 mg/kg feed) and salinomycin (40 mg/kg feed). After 35 days, the chickens were killed and content of the proximal part of the small intestine was collected and analyzed for bacterial counts, pH, bile acid concentration, and lipase activity. Evidence for a significant pH-dependent inhibition of lipase activity by unconjugated bile acids was provided in vitro and confirmed in vivo. Due to a reduction in nutrient fermentation, the pH in the small intestine of antibiotic-fed chickens was significantly higher than in chickens fed the non-supplemented diet. The high pH in the small intestine of chickens fed the A(+)diet was accompanied by a significant increase in lipase activity, and coincided with a significantly lower concentration of unconjugated bile acids and a higher ratio of conjugated to unconjugated bile acids. This study emphasizes the important influence of unconjugated bile acids on lipase activity at physiological pH-values.

Animals↗

Mechanisms and experimental and epidemiological evidence relating dietary fibre (non-starch polysaccharides) and starch to protection against large bowel cancer.

The cause of human colo-rectal cancer is unknown, although international and racial comparisons suggest that diet may be important. Within populations, risk of cancer is also affected by genetic factors which remain to be elucidated. Dietary fibre and NSP consumption is not always high in populations at low risk of colo-rectal cancer, but rates are fast increasing with westernization (and meat and fat consumption) in Japan. The suggestion that dietary fibre is protective in colo-rectal cancer is based on the fact that cereal fibre from bran increases faecal weight, dilutes large intestinal contents, and speeds up transit time. In animal models, bran reduces the number of tumours induced by chemical carcinogens, and cellulose may have a similar effect. The faeces of some individuals contain mutagens, some of which have been identified as fecapentaenes and heterocyclic amines. Bran reduces faecal mutagenicity, although the mutagen concerned is unknown. Most dietary fibre is fermented in the large gut by anaerobic bacteria and little remains in faecal matter. Recent observations have shown that substantial amounts of starch survive digestion in the small bowel and are available also for fermentation in the large gut. The metabolic consequences of fermentation may be important in carcinogenesis via altered N metabolism, SCFA production, and pH reduction. Methane is also produced in some individuals, but, contrary to previous findings, is not a risk factor for large bowel cancer. Starch appears to be beneficial as a substrate for fermentation because yields of the SCFA butyrate are increased both in vitro and in vivo. Butyrate is an energy substrate for the colonic mucosa and an anti-proliferative and differentiating agent in cell culture lines. Possible mechanisms whereby starch and NSP may protect against colo-rectal cancer, therefore, exist. The majority of individual case-control epidemiological studies suggest that fibre-containing foods are protective in colo-rectal cancer, although this effect is largely due to vegetable, rather than cereal, consumption. Case-control studies of diet and large bowel cancer may, however, reflect the effect rather than the cause of the disease, so that confirmation of the possible protective effects of starch and NSP is needed from accurate prospective studies both of diet and associated risk factors.

Animals↗

Rapid microdilution-colorimetric assay for yeast susceptibility to fluorocytosine.

Acid production by certain yeast species through the fermentation of glucose was used as the basis of an in vitro test for measuring susceptibility of these organisms to 5-fluorocytosine. Serial dilutions of 5-fluorocytosine in yeast nitrogen base broth, with bromothymol blue indicator dye, were made on microtiter plates. A fixed-concentration suspension of yeast cells was added to successive wells of the plates, and the color change from blue to yellow, indicating generation of acid, was noted. Eighteen hours after inoculation the lowest concentration of 5-fluorocytosine that completely inhibited the production of acid was recorded as the minimum inhibitory concentration. The results were reproducible in multiple trials with organisms of the genera Candida, Torulopsis, and Saccharomyces. This test is a rapid, inexpensive alternative to current 48- to 72-h methods in which broth turbidity is used as the end point.

Candida↗

Pathway of glucose catabolism by strain VeGlc2, an anaerobe belonging to the verrucomicrobiales lineage of bacterial descent

Strain VeGlc2, an anaerobic ultramicrobacterium belonging to the Verrucomicrobiales lineage of bacterial descent, fermented glucose to acetate, propionate, succinate, and CO2. The distribution of radiolabel in the fermentation end products produced from position-labelled glucose and in vitro measurements of enzyme activities in crude cell extracts prepared from glucose-grown cells showed that glucose was metabolized via the Embden-Meyerhof-Parnas pathway. The 6-phosphofructokinase (EC 2.7.1.90) activity required pyrophosphate as the phosphoryl donor, and ATP could not replace pyrophosphate. The other enzyme activities were those of a classical Embden-Meyerhof-Parnas pathway. 14CO2 was incorporated into propionate and succinate, suggesting that a carboxylation reaction rather than a transcarboxylation reaction was involved in the reductive pathway leading to succinate and propionate. Difference spectra showed that a type b cytochrome was present, which could be involved in electron transport in the reductive pathway.

Journal Article↗

Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine.

Seven strains of Roseburia sp., Faecalibacterium prausnitzii, and Coprococcus sp. from the human gut that produce high levels of butyric acid in vitro were studied with respect to key butyrate pathway enzymes and fermentation patterns. Strains of Roseburia sp. and F. prausnitzii possessed butyryl coenzyme A (CoA):acetate-CoA transferase and acetate kinase activities, but butyrate kinase activity was not detectable either in growing or in stationary-phase cultures. Although unable to use acetate as a sole source of energy, these strains showed net utilization of acetate during growth on glucose. In contrast, Coprococcus sp. strain L2-50 is a net producer of acetate and possessed detectable butyrate kinase, acetate kinase, and butyryl-CoA:acetate-CoA transferase activities. These results demonstrate that different functionally distinct groups of butyrate-producing bacteria are present in the human large intestine.

Acetates↗

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.

Biological Transport↗

The art of fusion: from penams and cephems to penems.

This synopsis of published literature summarises the key chemical and bacteriological characteristics of penicillins, i.e. penams, cephalosporins, i.e. cephems, and their hybrid structure, i.e. the penems. Consequently, the antibacterial spectrum of a typical penem, e.g. faropenem, encompasses gram-positive as well as gram-negative species. Dependent from the substituents at position 1 of the five-membered saturated ring fused to the beta-lactam ring oxa-, carba-, or thiopenems can be differentiated. A major determinant of their antibacterial activity and CNS-excitatory potential, however, is the C-2 side chain. The excitatory potential correlates with the basicity of the C-2 side chain as does their antibacterial activity against gram-negative species and non-fermenters like P. aeruginosa. Lipophilicity is a determinant for good in vitro activity against gram-positive bacteria. Several investigational penems exhibit interesting antibacterial spectra, encompassing methicillin resistant staphylococci, enterococci and even P. aeruginosa due to their improved binding affinity to both wild-type and modified low-affinity penicillin binding proteins. The development of these agents may offer therapeutic alternatives for the management of infections.

Anti-Bacterial Agents↗

In vitro assessment of the plaque-removing ability of hydrodynamic shear forces produced beyond the bristles by 2 electric toothbrushes.

BACKGROUND: The aim of this study was to compare the efficacy of interproximal plaque removal by the hydrodynamic shear forces produced by 2 electric toothbrushes (toothbrush A, Sonicare Plus and toothbrush B, Braun Oral B) using laboratory-grown plaque biofilms in an anatomically correct in vitro brushing model. METHODS: In vitro oral biofilms were grown in a constant-depth film fermenter on hydroxyapatite (HA) discs. Brushing experiments were conducted in a specially constructed machine designed to simulate the brushing of interproximal plaque between mandibular molar teeth; the bristles of the toothbrush did not make contact with the biofilm at any time. The load force between the brushes and teeth were in accordance with typical use and an exposure time of 5 seconds was used throughout. The efficacy of brushing was assessed by enumeration of the percentage of viable bacteria removed from the HA discs. These experiments were conducted with the electronic effect of the toothbrushes either activated or inactivated. RESULTS: Activation of toothbrush A significantly (P = 0.004) increased the median percentage of bacteria removed from 0.47% to 48.45%. Likewise, activation of toothbrush B significantly (P = 0.015) increased the median percentage of bacteria removed from 2.85% to 15.86%. The median percentage of plaque bacteria removed by the active toothbrush A was significantly (P = 0.009) greater than that removed by toothbrush B in this model system. CONCLUSION: These data imply that toothbrush A would be more effective than toothbrush B at removing interproximal dental plaque in vivo.

Adult↗

Inhibition of pinocytosis by hygrolidin family antibiotics: possible correlation with their selective effects on oncogene-expressed cells.

A fermentation broth of Streptomyces sp. SIPI-A4-0044 inhibited in vitro growth of src or ras oncogene-expressed (onc+) cells more strongly than that of oncogene-unexpressed (onc-) counterparts. The active components were isolated and identified as hygrolidin family antibiotics (HGL). In mixed cultures consisting of onc+ and onc- cells, at an appropriate ratio, HGL showed selective toxicity to focus like structures of onc+ cells, leaving monolayer areas of onc- cells little damaged. HGL rapidly inhibited pinocytosis, or the influx of neutral red into the cells, at concentrations partially inhibitory to the cell growth. In contrast, HGL only slightly inhibited the influx of 2-deoxyglucose, nucleosides and leucine and the syntheses of DNA, RNA and protein even at high concentrations. Upon prolonged exposure to sublethal concentrations of HGL, onc- cells but not onc+ cells recovered pinocytotic activity and resumed growth.

Animals↗

Pyrroindomycins, novel antibiotics produced by Streptomyces rugosporus LL-42D005. II. Biological activities.

The pyrroindomycins, a complex of novel antibiotics identified in fermentation broths of "Streptomyces rugosporus" LL-42D005, demonstrated excellent in vitro activity against Gram-positive bacteria. The semisynthetic diacetyl derivative of pyrroindomycin B (pyrroindomycin B-Ac2) was bactericidal for exponential-phase cells, but not for stationary-phase cells. This compound also exhibited marginal protection against a lethal Staphylococcus aureus challenge in mice. The poor in vivo activity of this antibiotic complex may be related to binding to blood components, as suggested by elevated MICs observed in blood-containing media. Incorporation of radiolabeled precursors into DNA, RNA, and protein was inhibited in an exponential-phase culture of Bacillus subtilis within ten minutes of exposure to pyrroindomycin B-Ac2. Microscopic examinations of drug-treated cells revealed lysis within the same ten minute period. These data are consistent with an effect of pyrroindomycin B-Ac2 on the integrity of the bacterial membrane.

Animals↗

[Fibrinolytic properties and thrombolytic effect of a novel fibrinolytic enzyme from Streptomyces sp. Y405].

A novel protease (designated as SW-1) purified from the fermentation broth of Streptomyces sp. strain Y405 showed fibrinolytic activity. In vitro, on plasminogen-free fibrin plate which had been heated at 85 degrees C for 30 minutes to eliminate plasminogen, SW-1 showed the same fibrinolytic activity as the mixture of SW-1 and plasminogen. Without adding plasminogen into the fibrin clot tube SW-1 showed the same effect as with plasminogen (P > 0.05). The results might indicate that SW-1 is a fibrinolytic enzyme which degrades fibrin directly, but not a plasminogen activator which degrades fibrin by activating plasminogen. SW-1 was found also to degrade fibrinogen. In vivo, the thrombolytic effect of SW-1 (4000 u.kg-1) on venous thrombus in rat was most remarkable versus that of control(P < 0.001), but was not divergent versus that of urokinase at the some dose(P > 0.05). SW-1 was shown to enhance plasminogen and plasmin activity but did not show significant effect on endogenous t-PA or alpha 2-PI. SW-1 also caused decrease of fibrinogen level in plasma, indicating that it degrades the fibrin in thrombus and at the same time degrades the circulating fibrinogen to certain extent. The changes of these biochemical data demonstrated the thrombolytic properties of SW-1 in vivo.

Animals↗

Differential multisite phosphorylation of the trehalose-6-phosphate synthase gene family in Arabidopsis thaliana: a mass spectrometry-based process for multiparallel peptide library phosphorylation analysis.

Multisite protein phosphorylation plays a fundamental role in metabolic regulation. To detect and quantify in vitro kinase phosphorylation activities, we developed a highly selective LC-MS/MS-based method using high resolution multiple reaction monitoring on a triple quadrupole mass spectrometer. This method eliminates the need for stable isotope labeling and enables multiparallel kinase target assays. Using these assays, we made the first observation of in vitro phosphorylation of different trehalose-6-phosphate synthase (TPS) isozymes. TPSs possess putative Ca2+-independent, sucrose non-fermenting 1-related protein kinase 1 (SnRK1) phosphorylation sites. Sixteen synthetic peptides from six different Arabidopsis thaliana TPS isozymes containing the SnRK1 consensus recognition motif were phosphorylated simultaneously in vitro, and their phosphorylation dynamics were determined. We achieved absolute quantification of TPS peptide phosphorylation by tuning the mass spectrometer to the corresponding synthetic standard phosphopeptides. The selectivity of the mass spectrometer in the multiple reaction monitoring mode compensates for the low ionization efficiency of phosphopeptides in the presence of a complex matrix. Results are in close agreement with recent in vivo studies of TPS phosphorylation and regulation and reveal significant differences in the phosphorylation levels of different TPS members within the TPS gene family ranging over 3 orders of magnitude. Substituting EGTA for CaCl2 in the reaction mixture reduced the formation of some of the phospho-TPS peptides drastically, indicating that Ca2+-dependent kinases are active in the presence of Ca2+-independent SnRKs. This agrees with the proposed overlap of the consensus motifs of these kinases and enables delineation between Ca2+-independent and Ca2+-dependent phosphorylation. Results demonstrate that multiparallel kinase target assays are sensitive enough to provide evidence for differential multisite phosphorylation of homologous TPS proteins and their highly conserved putative phosphorylation sites.

Amino Acid Sequence↗

An in vitro method for making repeated pH measurements on human dental plaque.

A combination pH micro-electrode inserted into human plaque centrifuged to the base of a closely-fitting fine-mesh wire "basket" has been used to make continuous recordings of the pH responses produced by exposure to various carbohydrate substrates. The pH depressions given by continued immersion in sugar and starch solutions resemble, both in depth and duration, those obtained by telemetric pH measurements of approximal plaque samples. In contrast, brief exposure to sugar solutions will produce a Stephan type of pH curve. Measurements of pH can be made using a succession of different substrates if, after each substrate is tested, the electrode-plaque assembly is washed with water and its acidity neutralized in circulating saliva. It is suggested that this in vitro pH method could be used instead of the telemetric measurements of approximal plaque for comparing acid production from foods or for other purposes.

Dental Plaque↗

FK409, a novel vasodilator isolated from the acid-treated fermentation broth of Streptomyces griseosporeus. I. Taxonomy, fermentation, isolation, and physico-chemical and biological characteristics.

FK409, a novel vasodilator with anti-platelet aggregation activity, has been isolated from the acid-treated fermentation broth of Streptomyces griseosporeus No. 16917, which was cultured on a medium containing NaNO3 for 4 days. FK409 was purified from the culture-filtrate by extraction with ethyl acetate after adjusting the pH to 3.0 with HC1, followed by silica gel chromatography. The molecular formula of this compound was determined to be C8H13N3O4. In vitro, FK409 showed a potent relaxation activity on noradrenaline induced contraction of rat aorta. In addition to the vasodilating activity, this compound also showed potent anti-aggregation activities towards rabbit platelets. In vivo, intravenously administered FK409 resulted in marked blood pressure lowering in rats.

Animals↗

Evaluation of Mannich bases and related compounds as inhibitors of mitochondrial function in yeast and inhibition of blood platelet aggregation, blood clotting, and in vitro metabolism of 5-dimethylamino-1-phenyl-1-penten-3-one hydrochloride.

Dimethylamino-1-phenyl-1-penten-3-one hydrochloride (Ia) and 32 analogs were tested for inhibition of respiratory-dependent growth in Saccharomyces cerevisiae. Thirteen of the 33 compounds tested appeared to affect mitochondrial function, since the inhibition of respiratory-dependent growth was statistically greater than the inhibition of growth on fermentable energy sources. Inhibition of mitochondrial function in yeast and growth inhibition of an in vitro culture of human epidermoid carcinoma (KB) were positively correlated since 83% of the compounds tested either had mitochondrial-inhibiting properties and significant activity in the KB test or were inactive in both tests. Similarly, 78% of compounds tested showed murine toxicity and mitochondrial inhibition or had no effect on murine toxicity and yeast mitochondrial function. Injection of Ia into rats resulted in the appearance of blood in the urine and feces. Compound Ia inhibited adenosine diphosphate and collagen-induced aggregation of rat platelets but had no effect on blood clotting. TLC, following incubation of Ia with a rat liver extract, showed that the structure of Ia was not enzymatically modified and indicated activity per se on platelet aggregation and mitochondrial function.

Adenosine Diphosphate↗

Stimulatory effect of menaquinone-7 (vitamin K2) on osteoblastic bone formation in vitro.

Menaquinone-7, which is vitamin K2 (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10(-7)-10(-5) M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10(-7)-10(-5) M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.

Alkaline Phosphatase↗

Production of African breadfruit (Treculia africana) and soybean (Glycine max) seed based food formulations, 1: Effects of germination and fermentation on nutritional and organoleptic quality.

Germination and fermentation were investigated as methods of improving the nutritional and organoleptic properties of soybean and African breadfruit seed based food formulations. Four products consisting of germinated-fermented soy-breadfruit seeds (GFSB), nongerminated-fermented soy-breadfruit seeds (NGFSB), germinated-nonfermented soy-breadfruit seeds (GNFSB) and nongerminated-nonfermented soy-breadfruit seeds (NGNFSB) were prepared. Phytic acid contents, in vitro protein digestibilities, protein efficiency ratios (PER), net protein ratios (NPR), flavor, appearance and overall acceptability were evaluated. Germination followed by natural lactic fermentation significantly (p < 0.05) reduced the phytic acid by a factor of 11.6 in NGNFSB compared to reduction factors of 2.1 and 1.5 in GNFSB and NGFSB, respectively. The in vitro protein digestibility (%), PER and NPR values of 73.4, 2.46 and 3.62 for GFSB; 71.1, 2.35 and 3.46 for NGFSB; 68.7, 2.16 and 3.41 for GNFSB were significantly (p < 0.05) higher than the 64.7, 1.82 and 2.11 for NGNFSB. The mean sensory scores were 5.26-5.67 for GNFSB, 4.66-4.94 for NGNFSB, 4.33-4.80 for GFSB and 4.27-4.34 for NGFSB on a 7-point rating scale.

Africa↗