The enzymology of sulfur metabolism in phototrophic bacteria — A review

Summary The two functions of sulfur metabolism in phototrophic bacteria are to supply electrons for photosynthesis and to supply sulfur for biosynthetic purposes. The pathways for both functions may be partly identical. For the electron-supplying pathway the following enzymes are needed: a sulfide-oxidizing enzyme, a sulfur-oxidizing enzyme system, APS reductase, ADP sulfurylase and — in the case of thiosulfate utilization — thiosulfate reductase. Assimilatory reactions are catalyzed by ATP sulfurylase, APS kinase, sulfotransferases, PAPS reductase, sulfite reductase and o-acetylserine sulfhydrylase. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974.     

Subcellular distribution of enzymes involved in α-glucan utilization in Klebsiella pneumoniae

The double-isotopic labelling technique was used to identify comprehensively proteins involved in α-glucan catabolism in Klebsiella pneumoniae NCTC 9633. Cells were grown with either glycerol in the presence of ³H-leucine or with glycerol plus maltose in the presence of ¹⁴C-leucine. Each labelled culture was then fractionated into the main subcellular components, i.e. the cytoplasm, periplasm, cytoplasmic and outer membrane. Corresponding fractions derived from ³H-labelled and ¹⁴C-labelled cells were combined, and the proteins were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Gel slices were then counted for ³H- and ¹⁴C-radioactivity, a positive deviation from the standard ¹⁴C/³H ratio being evidence for the presence of a protein specifically induced by maltose in the culture medium. The protein pattern thus obtained was compared with the properties of proteins comprising a similar pathway for maltodextrin utilization in Escherichia coli K-12. Ample information which has been obtained mainly by genetic analysis is available about maltodextrin-utilizing enzymes in E. coli K-12.

1. Cytoplasm. Neither amylomaltase nor maltodextrin phosphorylase, well-known soluble enzymes, were identifiable by the double-labelling technique, presumably because these enzymes constitute only a very minor portion of all soluble proteins in the cytoplasm.
2. Periplasm. A prominent protein with a mass of 43000 daltons (43 kD) was found similar to the maltose-binding protein of E. coli K-12 (44 kD).
3. Cytoplasmic membrane. At least 2 proteins with a mass between 40 and 50 kD were detected, minor proteins were seen at ≈ 15 and ≈ 20 kD. One or 2 of the proteins may function as a permease catalyzing the active transport of maltodextrins.
4. Outer membrane. The major protein had a mass of 55 kD, other proteins were found with ≈ 18, ≈48, and ≈140 kD. The major protein may have the same function as the maltodextrin pore protein in E. coli K-12 (55 kD), because K. pneumoniae could grow on 10 μM maltose at practically the same rate as on 10 mM maltose. The 140 kD protein is pullulanase.

     

Recent advances in the development and applications of luminescent bacteria–based biosensors

Luminescent bacteria–based biosensors are widely used for fast and sensitive monitoring of food safety, water quality, and other environmental pollutions. Recent advancements in biomedical engineering technology have led to improved portability, integration, and intelligence of these biotoxicity assays. Moreover, genetic engineering has played a significant role in the development of recombinant luminescent bacterial biosensors, enhancing both detection accuracy and sensitivity. This review provides an overview of recent advances in the development and applications of novel luminescent bacteria–based biosensors, and future perspectives and challenges in the cutting-edge research, market translation, and practical applications of luminescent bacterial biosensing are discussed.     

Glycogen metabolism loss: a common marker of parasitic behaviour in bacteria?

We searched 55 completely sequenced bacterial genomes for glycogen synthesis and degradation enzymes. A significant proportion of these bacteria appears to lack glycogen metabolism capability. Interestingly, these bacteria are parasitic, symbiotic or fastidious (i.e. difficult to culture outside their normal environment). It is suggested that the lack of bacterial glycogen metabolism is a trait associated with parasitic behaviour in bacteria.     

Multiple forms of α-glucan phosphorylase in banana fruits: Properties and kinetics

Multiple forms of α-glucan phosphorylase isolated from mature banana fruit pulp differ from each other in several respects—pH optimun, temperature optimum, energy of activation, primer specificity, kinetics, and sensitivity towards metal ions, nucleotides, sugar mucleotides, amino acids, glycolytic intermediates, sulfhydryl binding agents, sulfhydryl reagents and phenolics.     

Recent advances in peptidomimetics antagonists targeting estrogen receptor α-coactivator interaction in cancer therapy

Estrogen receptor α (ERα) is a crucial target for ERα positive breast cancer treatment. Previous drug discovery efforts were focused on developing inhibitors that targeted the canonical ligand binding pockets of the ligand binding domain (LBD) of ERα. However, significant percentage of patients developed cancer relapse with drug-resistance. ERα peptidomimetic modulators have been considered as promising treatments for drug resistant breast cancers as they are targeting ERα-coactivator interacting interface instead of the ligand binding pocket of ERα. Herein, we reviewed the recent development of ERα peptidomimetics antagonists.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

The metabolism of α-tocopherol by plants

An enzyme system which metabolizes α-tocopherol has been identified in homogenates of etiolated pea shoots. Enzyme activity is considerably increased by the presence of 20% ethanol in the incubation mixture. The enzyme has an absolute requirement for phospholipid. The reaction utilizes molecular oxygen and it is proposed that the enzyme be called α-tocopherol oxidase.     

Effect of α-p-chlorophenoxyisobutyrate on the metabolism of isoprenoid compounds in the rat

1. Feeding of alpha-p-chlorophenoxyisobutyrate (CPIB) to rats increased ubiquinone concentration in the liver but not in other tissues. The increase was progressive with the time of feeding and related to the concentration of CPIB in the diet. 2. Incorporation of [1-(14)C]acetate, but not of [2-(14)C]mevalonate, into sterols in the liver in vivo or by liver slices in vitro was decreased on feeding the rats with CPIB. However, incorporation of mevalonate into ubiquinone increased. 3. CPIB, when added in low concentrations to liver slices, had no effect on isoprene synthesis from acetate; higher concentrations, however, were inhibitory. 4. No activation of ubiquinone synthesis from mevalonate was observed when CPIB was added to the liver slices synthesizing ubiquinone. 5. The increase in ubiquinone in CPIB-fed animals appears to be due to increased synthesis in the initial stages and to decreased catabolism in the later stages. 6. An inverse relationship was found between the concentration of ubiquinone in the liver and the serum sterol concentration in CPIB-fed rats.     

β-glucan degradation in malting sorghum

In all of four malting sorghum varieties, -glucan contents decreased by more than 50% 2 days after germination, due to enzymic digestion. The variety that had the least -glucan content in the malt gave the highest filterable volume of sweet wort while the variety with the highest -glucan content had the lowest volume of filterable wort.A.C. Ogbonna is with the Department of Brewing Science and Technology, University of Uyo, P.M.B. 1017, Uyo, Nigeria, A.L. Egunwu was with the University of Nigeria, Nsukka, Nigeria, and is now with Nigerian Breweries PLC, Aba, Abia State, Nigeria.     

Effect of α-ketobutyrate on palmitic acid and pyruvate metabolism in isolated rat hepatocytes

α-Ketobutyrate, an intermediate in the catabolism of threonine and methionine, is metabolized to CO₂ and propionyl-CoA. Recent studies have suggested that propionyl-CoA may interfere with normal hepatic oxidative metabolism. Based on these observations, the present study examined the effect of α-ketobutyrate on palmitic acid and pyruvate metabolism in hepatocytes isolated from fed rats. α-Ketobutyrate (10 mM) inhibited the oxidation of palmitic acid by 34%. In the presence of 10 mM carnitine, the inhibition of palmitic acid oxidation by α-ketobutyrate was reduced to 21%. These observations are similar to those previously reported using propionate as an inhibitor of fatty acid oxidation, suggesting that propionyl-CoA may be responsible for the inhibition. α-Ketobutyrate (10 mM) inhibited ¹⁴CO₂ generation from [¹⁴C]pyruvate by more than 75%. This inhibition was quantitatively larger than seen with equal concentrations of propionate. Carnitine (10 mM) had no effect on the inhibition of pyruvate oxidation by α-ketobutyrate despite the generation of large amounts of propionylcarnitine during the incubation. α-Ketobutyate inhibited [¹⁴C]glucose formation from [¹⁴C]pyruvate by more than 60%. This contrasted to a 30% inhibition caused by propionate. These results suggest that α-ketobutyrate inhibits hepatic pyruvate metabolism by a mechanism independent of propionyl-CoA formation. The present study demonstrates that tissue accumulation of α-ketobutyrate may lead to disruption of normal cellular metabolism. Additionally, the production of propionyl-CoA from α-ketobutyrate is associated with increased generation of propionylcarnitine. These observations provide further evidence that organic acid accumulation associated with a number of disease states may result in interference with normal hepatic metabolism and increased carnitine requirements.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Isolation of α-glucan and lipopolysaccharide fractions from Acetobacter xylinum

A cellular phenol-water extract of Acetobacter xylinum NRC 17007 was fractionated on Sepharose 4 B. The fraction eluting with the void volume consisted to about 95% of glycogen-like material. The lipopolysaccharide fraction was of lower molecular weight and had the following composition (%, w/w): Mannose, 42; glucose, 7; galactose, 3.8; heptose, 2; 2-keto-3-deoxy-octonate, 1.2; glucosamine, 3.3; phosphate, 4.5; total fatty acids, 3.9. Among the fatty acids, 3-hydroxy-tetradecanoic acid was present, and 2-hydroxy-hexadecanoic acid predominated.     

Intestinal microflora and homeostasis of the mucosal immune response: implications for probiotic bacteria?

The intestinal microflora can be considered a postnatally acquired organ that is composed of a large diversity of bacteria that perform important functions for the host and can be modulated by environmental factors, such as nutrition. Specific components of the intestinal microflora, including lactobacilli and bifidobacteria, have been associated with beneficial effects on the host, such as promotion of gut maturation and integrity, antagonisms against pathogens and immune modulation. Beyond this, the microflora seems to play a significant role in the maintenance of intestinal immune homeostasis and prevention of inflammation. The contribution of the intestinal epithelial cell in the first line of defense against pathogenic bacteria and microbial antigens has been recognized. However, the interactions of intestinal epithelial cells with indigenous bacteria are less well understood. This review will summarize the increasing scientific attention to mechanisms of the innate immune response of the host towards different components of the microflora, and suggest a potential role for selected probiotic bacteria in the regulation of intestinal inflammation.     

Sex difference in cellular calcium metabolism of rat hepatocytes and in α-adrenergic activation of glycogen phosphorylase

Three topics were the subject of these investigations: (i) the difference between males and females in the basal calcium metabolism of hepatocytes; (ii) the source of the calcium which triggers the phosphorylase a stimulation induced by epinephrine through alpha-adrenergic receptors; (iii) the time relation between the rise in phosphorylase activity and the increase in calcium efflux. We found that there was no difference between males and females in total or exchangeable cell calcium. However, there were significant differences in the mitochondrial calcium pool and fluxes measured by steady-state kinetic analyses: they were smaller and the rate constants of mitochondrial calcium influx and efflux were lower in males than in females. The ⁴⁵Ca content of isolated mitochondria and microsomes was also significantly lower in males than in females. In both males and females, epinephrine stimulated phosphorylase activity and calcium efflux even in the absence of extracellular calcium, indicating that the principal source of calcium which triggers the enzyme stimulation is intracellular. During the first 10 min following stimulation by 10^?6 M epinephrine, the total cell calcium, ⁴⁵Ca and the mitochondrial calcium were significantly depressed in male hepatocytes. After 10 min, these changes were reversed and the cell or mitochondrial calcium content was greater than in controls. In females, on the other hand, changes could only be detected if the cells were transferred to calcium-free media before the stimulation. In both males and females, there was a good temporal relationship between the stimulation of calcium efflux and the rise in phosphorylase a activity when hepatocytes were exposed to increasing concentrations of epinephrine: both rose at least 75% in less than 15 s. We conclude that there are important differences in cellular calcium metabolism between males and females. The rise in cytosolic calcium induced by alpha-adrenergic activation is principally due to a mobilization of calcium from an intracellular pool, probably the mitochondria.     

Photodynamic inactivation of bacteria on immobilized α-terthienyl film

Summary The photodynamic inactivation by near UV and -terthienyl (T) of bacteria immobilized in poly(methyl methacrylate) (PMMA) was observed. Killing curves for Escherichia coli strains exhibited multi-hit kinetics, whereas curves for Pseudomonas aeruginosa and Staphylococcus aureus exhibited single-hit kinetics. Cell inactivation by irradiated T film at an immobilized concentration of 20 g/cm² was substantially caused by generated singlet oxygen (¹O₂), but not by that from free T molecules released from the film after irradiation with near UV. The inactivation rate was found to depend on the surface density of T in the film. A linear relationship between the concentration of ¹O₂ generated on the film surface and the apparent rate of cell death was obtained. Offprint requests to: M. Takano     

Dietary calcium phosphate content and oat β-glucan influence gastrointestinal microbiota, butyrate-producing bacteria and butyrate fermentation in weaned pigs

This study aimed to evaluate the effects of oat β-glucan in combination with low- and high-dietary calcium phosphate (CaP) content on gastrointestinal bacterial microbiota, prevalence of butyrate-production pathway genes and fermentation end-products in 32 weaned pigs allocated to four diets: a cornstarch-casein-based diet with low [65% of the calcium (Ca) and phosphorous (P) requirement] and high CaP content (125% and 115% of the Ca and P requirement, respectively); and low and high CaP diets supplemented with 8.95% of oat β-glucan concentrate. Pigs were slaughtered after 14 days, and digesta were collected for quantitative PCR analysis, and quantification of short-chain fatty acids and lactate. The high CaP content reduced gastric lactate and streptococci and propionate in the large intestine. Oat β-glucan distinctly raised gastric bacterial numbers, and colonic lactobacilli and bifidobacteria. Although not reflected by gene copies of butyrate-production pathway genes, oat β-glucan also increased gastric, caecal and colonic butyrate concentrations, which may be favourable for intestinal development in weaned pigs. Thus, a high CaP content negatively affected the intestinal abundance of certain fermentation end-products, whereas oat β-glucan generally enhanced bacterial numbers and activity. The results emphasize the importance of the stomach for bacterial metabolism of oat β-glucan in weaned pigs.