Intracellular compartmentation and control of alanine metabolism in rat liver parenchymal cells.
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Alanine↗
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Compartmentation of fatty acid oxidation in liver cells.
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Acetoacetates↗
Quantification of fatty acid activation of the uncoupling protein in brown adipocytes and mitochondria from the guinea-pig.
Brown adipocytes from cold-adapted guinea-pigs (C-cells) are more sensitive to uncoupling by exogenous palmitate than are cells from warm-adapted animals (W-cells) with much less uncoupling protein. Half-maximal respiratory stimulation of C-cells requires 80 nM free palmitate. Noradrenaline-stimulated lipolysis is not rate-limiting for the respiration of either C-cells or W-cells. Half-maximal stimulation of fatty acid oxidation by mitochondria from warm-adapted guinea-pigs (W-mitochondria) and cold-adapted guinea-pigs (C-mitochondria) both require 12 nM free palmitate. Palmitate uncouples C-mitochondria much more readily than M-mitochondria, paralleling its action on the adipocytes. The uncoupling is partially saturable, about 100 nM free palmitate being required for half-maximal response of C-mitochondria. W- and C-mitochondria show identical binding characteristics for palmitate. The respiratory increase of mitochondria is calculated as a function of bound palmitate. After correcting for the residual uncoupling protein present in W-mitochondria, palmitate is estimated to be almost ineffective as an uncoupler of brown fat mitochondria in the absence of the protein. It is concluded that fatty acids display characteristics required of a necessary and sufficient physiological activator of the uncoupling protein.
Adaptation, Physiological↗
Functional analysis of a GWAS pleiotropic hotspot suggests an auxin biosynthesis gene (AhPDS1), regulating pod development in peanut (Arachis hypogaea L.).
Peanut productivity and quality improvement rely on understanding the genetic factors influencing pod and seed size. This study aims to identify genetic factors and regulatory mechanisms influencing pod and seed size in peanuts. Herein, a genome-wide association study (GWAS) was conducted using 390 accessions from 15 peanut growing regions to analyze pod and seed traits across multiple planting seasons. A significant phenotypic variation was observed, with broad-sense heritability ranging from 53.6 to 85.4%. Strong correlations between pod and seed traits further suggest potential for co-selection in breeding efforts. A pleiotropic hotspot on chromosome B06 was strongly associated with six pod and seed traits. A peanut pod size regulator AhPDS1 (PODSIZE-1, Ahy_B06g085516) homolog of Arabidopsis thaliana YUCCA4 (AtYUC4, AT5G11320), involved in auxin biosynthesis, was selected as a candidate regulating pod and seed size. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) confirmed higher AhPDS1 expression in large pod as compared with the small pod genotypes. Subcellular localization showed AhPDS1 to be predominantly cytoplasmic, and GUS reporter assays indicated widespread expression in roots, stems, leaves, flowers, and pods, suggesting a broad functional role. Further overexpression of AhPDS1 in Arabidopsis and rice enhanced pod, seed, and grain sizes via the indole-3-pyruvic acid pathway in transgene lines. These findings highlight AhPDS1 as a potential target for peanut molecular breeding, offering opportunities to enhance pod size via auxin biosynthesis and support sustainable crop improvement.
Arachis↗
Specificity of the Westerfeld adaptation of the Voges-Proskauer test.
Aliphatic chain compounds at least four carbons long with vicinal carbonyl groups in the 2,3 positions were detected by the Westerfeld test. Acetoin, which has one carbonyl group and an adjacent hydroxyl group, gave positive results, but methyl action (3-hydroxy-3-methyl-2-butanone) was negative, and subsequent tests supported the conclusion that acetoin is oxidized to diacetyl by alpha-naphthol during the Westerfeld test in the absence or presence of air. 2,3-Pentanedione and 2,3-heptanedione gave positive results, but equimolar concentrations of these compounds gave maximal absorbancy readings that were only 35% (2,3-pentanedione) and 31% (2,3-heptanedione) of those obtained with diacetyl or acetoin. Negative results were obtained with pyruvic acid, 2,3-butylene glycol, and carbon ring compounds (1,2-cyclohexanedione, alloxan, and 3,4-dihydroxy-3-cyclobutene-1,2-dione). alpha-Naphtho could not be replaced in the test by beta-naphthol, 1,2,3,4,-tetrahydroxy-1-naphthol, or 5,6,7,8-tetrahydroxy-1-naphthol. Creatine could not be replaced by arginine, guanidine . HCl, or guanidinoacetic acid.
Acetoin↗
Chemical Composition of Two Exopolysaccharides from Bacillus thermoantarcticus.
The thermophilic bacterium Bacillus thermoantarcticus produces two exocellular polysaccharides (EPS 1 and EPS 2), which can be obtained from the supernatant of liquid cultures by cold-ethanol precipitation, in yields as high as 400 mg liter(sup-1). The EPS fraction was produced with all substrates tested, although a higher yield was obtained with mannose as the carbon and energy source. The EPS content was proportional to the total biomass. On a weight basis, EPS 1 and EPS 2 represented about 27 and 71%, respectively, of the total carbohydrate fraction. EPS 1 is a sulfate heteropolysaccharide containing mannose and glucose in a relative molar proportion of 1.0 and 0.7, respectively. EPS 2 is a sulfate homopolysaccharide containing mannose as the major component. The absolute configurations of hexoses were shown to be d for both EPSs. Nuclear magnetic resonance spectra confirmed the presence of (alpha)-d-mannose and (beta)-d-glucose in EPS 1 and only (alpha)-d-mannose in EPS 2. In addition, (sup1)H nuclear magnetic resonance analysis and chemical analysis indicated the presence of pyruvic acid in EPS 2.
Journal Article↗
Variable expressions of Staphylococcus aureus bicomponent leucotoxins semiquantified by competitive reverse transcription-PCR.
A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/10(4) CFU to 10(2) mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system in S. aureus, except that expression of hlgA was not affected in the agr mutant.
Bacterial Proteins↗
Inhibition of growth by erythritol catabolism in Brucella abortus.
The growth of Brucella abortus (US-19) in a complex tryptose-yeast extract medium containing D-glucose is inhibited by 10 mM erythritol. The enzymes of the erythritol pathway, except for D-erythrulose 1-phosphate dehydrogenase (D-glycero-2-tetrulose 1-phosphate:nicotinamide adenine dinucleotide (NAD+) 4-oxidoreductase) were detected in the soluble and membrane fractions of cell extracts. Glucose catabolism by cell extracts was inhibited by erythritol, whereas, phosphorylated intermediates of the hexose monophosphate pathway were converted to pyruvic acid with oxygen consumption. Erythritol kinase (EC 2.7.1.27; adenosine 5'-triphosphate (ATP): erythritol 1-phosphotransferase) was found to be eightfold higher in activity than the hexokinase in cell extracts. In vivo, ATP is apparently consumed with the accumulation of D-erythrulose 1-phosphate (D-glycero-2-tetrulose 1-phosphate) and no substrate level phosphorylation. ATP levels dropped 10-fold in 30 min after addition of erythritol to log phase cells in tryptose-yeast extract medium with D-glucose as the carbon source. These data suggest bacteriostasis in the presence of erythritol results from the ATP drain caused by erythritol kinase.
Adenosine Triphosphate↗
Bacitracin and protease production in relation to sporulation during exponential growth of Bacillus licheniformis on poorly utilized carbon and nitrogen sources.
Growth of Bacillus licheniformis in a chemically defined medium containing glucose and ammonium chloride yielded a doubling time of 1.00 h. Examination of the culture during exponential growth revealed a lack of heat-resistant spores together with a complete absence of detectable concentrations of bacitracin or extracellular serine protease. Replacement of glucose as the sole carbon source by glycerol, pyruvic acid, citric acid, or lactic acid resulted in doubling times of 1.13, 2.00, 3.16, and 3.95 h, respectively. Bacitracin, protease, and heat-resistant spores were produced during exponential growth in amounts related to these doubling times. A qualitatively similar pattern was observed when ammonium chloride was replaced by sodium nitrate, alanine, or glutamic acid which gave doubling times of 1.65, 1.77, and 1.90 h, respectively. Protease, but not bacitracin, concentrations were substantially higher when the growth rate was restricted by use of poor nitrogen rather than poor carbon sources. The relationships between bacitracin production, protease production, and the sporulation process are discussed.
Amino Acids↗
Development and trifoliin A-binding ability of the capsule of Rhizobium trifolii.
The age-dependent lectin-binding ability of Rhizobium trifolii 0403 capsular polysaccharide (CPS) was examined by following the development of the capsule and its ability to interact with the white clover lectin trifoliin A. Bacteria grown on agar plates for 3, 5, 7, 14, and 21 days were examined by electron microscopy and immunofluorescence microscopy with antibodies prepared against either R. trifolii 0403 CPS or trifoliin A after pretreatment with the lectin. The capsule began to develop at one pole by day 3 and completely surrounded the cells in cultures incubated for 5 days or longer. The capsular polysaccharide on cells cultured for 3 and 5 days was completely reactive with trifoliin A, became noticeably less reactive by day 7, and was only reactive with the lectin at one pole of a few cells after that time. The quantity and location of lectin receptors on bacteria of different ages directly correlated with their attachment in short-term clover root hair-binding studies. Cells from 3- or 21-day-old cultures attached almost exclusively in a polar fashion, whereas cells grown for 5 days attached to root hairs randomly and in the highest numbers. CPS isolated from a 5-day-old culture had higher lectin-binding ability than CPS from 3- and 7-day-old cultures, whereas the CPS from a 14-day-old culture had the lowest. Chemical analyses of the isolated CPS showed changes in the levels of uronic acids (as glucuronic acid), pyruvate, and O-acetyl substitutions with culture age, but the neutral sugar composition remained relatively constant. These results provide evidence that the age-dependent distribution of lectin receptors dictates the level and orientation of attachments of R. trifolii 0403 to clover root hairs.
Agglutination↗
Genetic analysis of carbohydrate transport-deficient mutants of Salmonella typhimurium.
Mutants (car) isolated from Salmonella typhimurium were unable to utilize or ferment the following carbohydrates (all d-configuration): glucose, fructose, mannose, N-acetylglucosamine, sorbitol, mannitol, maltose, melibiose, and glycerol. The mutants did utilize galactose, glucose 6-phosphate, gluconic acid, glucuronic acid, pyruvate, and l-lactate. Biochemical analysis showed that there were two classes of mutants, each lacking one component of a phosphotransferase system. CarA mutants were deficient in enzyme I; carB lacked the phosphate carrier protein, HPr. Mapping experiments showed that the carA gene was located near pro; the carB gene mapped near purC.
Biological Transport↗
Antimicrobial susceptibility testing of Bilophila wadsworthia isolates submitted for routine laboratory examination.
MICs of antibiotics against Bilophila wadsworthia isolates were measured by agar and broth microdilution with pyruvic acid and by Etest. The inoculum size influenced greatly agar dilution. Despite discrepancies in MICs depending on the measurement method used, clindamycin consistently showed potent activity. Broth microdilution and Etest appear to be candidates for laboratory susceptibility testing.
Aged↗
Blood pyruvate concentration measured by a specific method in control subjects.
A specific method of estimating pyruvate, depending upon the oxidation of reduced nicotinamide-adenine dinucleotide in the presence of lactic dehydrogenase, has been modified to improve its accuracy. The precision of the modification, measured by duplicate analysis, was represented by a standard deviation of 0.03 mg./100 ml. The mean recovery of added pyruvate was 99%. The mean blood pyruvate concentration in 30 fasted subjects, who had no illness known to affect pyruvate metabolism, was 0.44 mg./100 ml. (S.D. 0.05). Blood pyruvate levels were measured at 30-minute intervals for two hours after oral or intravenous administration of glucose. The highest levels obtained in each case were about 0.4 mg./100 ml. higher than the initial level, and were found 60 to 90 minutes after oral administration and 30 to 60 minutes after intravenous administration.
Fasting↗
Insulin secretion by isolated perfused rat and mouse pancreas.
A method for isolation and perfusion of a pancreas preparation consisting of pancreas, stomach, proximal duodenum, and spleen is described. Basic characteristics of regulation of insulin secretion from the perfused pancreas isolated from rats, albino mice, obese mice (ob/ob), and black mice were identical. Viability and stability of the pancreas preparation during perfusion were maintained as documented by measurements of oxygen consumption of the pancreas preparation, perfusion pressure, and pH of the perfusion medium. The insulin-secretory capacity of the pancreas of different animal species was compared. Insulin secretion by the perfused rat and obese (ob/ob) mouse pancreas was much more potent than that by the pancreas of lean albino and lean black mice. D-Glucose-induced insulin secretion from the pancreas was decreased after fasting of the animals and was dependent on glucose concentration and presence of calcium in the perfusion medium. D-Glyceraldehyde, tolbutamide, D-mannose, dihydroxyacetone, L-leucine, and L-arginine also induced insulin secretion from the pancreas. D-Fructose, D-galactose, L-glucose, 3-O-methyl-D-glucose, N-acetylglucosamine, D-xylose, D,L-glyceric acid, pyruvate, L-lactate, and theophylline did not provoke insulin secretion.
Animals↗
Immunohistochemical analysis of morules in colonic neoplasms: morules are morphologically and qualitatively different from squamous metaplasia.
Morules develop in several neoplasms and have been considered as a type of squamous metaplasia despite the absence of keratinization and intercellular bridges. The objective of this study was to clarify the pathological significance of morules and to distinguish morules from squamous metaplasia in colonic neoplasms. Ten cases of morule-associated colonic neoplasms (4 adenocarcinomas, 1 adenoma with carcinoma in situ, and 5 adenomas), and 3 cases of squamous metaplasia in colonic adenocarcinoma were examined morphologically and immunohistochemically. Morules were well-defined structures composed of small, oval to short-spindled cells with bland nuclei, and frequently associated with intranuclear inclusions that were positive for biotin and biotin-binding enzymes (pyruvic acid carboxylase and propionyl CoA carboxylase). On immunohistochemical examination, morules characteristically showed nuclear overexpression of beta-catenin, cyclin D1 and p63, low Ki-67 labeling index (<1%), cytoplasmic overexpression of CD10, and no expression of cytokeratin 20. These molecules were useful for the differentiation of morules. Furthermore, p63 and 34betaE12 positivities in morules suggested that they have a basal/stem cell phenotype. Thus, morules were morphologically and qualitatively different from squamous metaplasia. We consider that morules in colonic neoplasms are cell clusters with a basal/stem cell phenotype, and have less proliferative and less invasive potential than other cancer cells.
Adenocarcinoma↗
Infantile mitochondria encephalomyopathies: report on 4 cases.
Four muscle biopsies from the quadriceps femoris muscle of children with symptoms suggesting mitochondrial encephalomyopathy were examined for morphological and biochemical differences. From a biochemical point of view, our patients showed a derangement of mitochondrial metabolism and in particular a marked deficit in the activity of cytochrome c oxidase. In all patients there was an abnormally high rise of lactate and pyruvic acid levels in blood before and after glucose loading. Results of morphological and enzyme histochemical studies showed smaller muscle fibre diameters than in normal children. Typical red ragged fibres and ultrastructural abnormalities in muscle mitochondria were present in 1 case only. Three patients showed focal changes in the distribution of some sarcoplasmic oxidative enzyme activities. The present findings suggest that the main metabolic disorder, pointed out by biochemical studies, is scarcely reflected by morphological and ultrastructural studies in the early stages of mitochondriopathies.
Biopsy↗
A case of Kearns-Shy syndrome with later onset.
We report a case of Kearns-Shy syndrome in a 44-year-old woman. She complained of bilateral ptosis, exotropia and gait disturbance. Diffuse chorioretinal degeneration and numerous punctate whitish spots were observed in both fundi. Eye movements were severely disturbed. An electroretinogram was almost nonrecordable, while visually evoked cortical potentials to pattern stimulation were normal. The dark adaptation curve showed an elevation of rod threshold. Besides such ophthalmological findings, muscle weakness, extinguished tendon reflex and healing difficulty were observed. Blood lactate, pyruvic acid and serum creatinine kinase were at high levels. A muscle biopsy showed ragged-red fibers and partial deficiency of cytochrome c oxidase. The patient is being treated with coenzyme Q, and we are now following up the therapeutic effects of this treatment.
Adult↗
Desulfuration of cysteine and methionine by Fusobacterium nucleatum.
Fusobacterium nucleatum is a Gram-negative anaerobic rod-shaped bacterium frequently isolated from human dental plaque. It is capable of the desulfuration of cysteine and methionine, resulting in the formation of sulfide and thiol volatiles, respectively. Intact cells, as well as cell-free extracts produced by French pressure cell lysis of F. nucleatum, hydrolyzed radiolabeled cysteine to produce sulfide, pyruvic acid, and ammonia. The hydrolysis products of radiolabeled methionine were a volatile thiol, ketobutyrate, and ammonia. Both activities were associated with the cytoplasmic component, not the membrane. The desulfuration mechanisms are heat-labile, inhibited by the presence of excess substrate, and rates are dependent upon substrate concentration. These dissimilar pathways by F. nucleatum can account in part for the presence of sulfur-containing volatile products that occur in the mouth.
Cysteine↗