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PubMed1985

Comparison of four hippurate hydrolysis methods for identification of thermophilic Campylobacter spp.

The test for hippurate hydrolysis is critical for separation of Campylobacter jejuni and C. coli strains. Glycine and benzoic acid are formed when hippurate is hydrolyzed by C. jejuni. The test used in most laboratories is one of several variations of the ninhydrin tube test described by Hwang and Ederer (M. Hwang and G. M. Ederer, J. Clin. Microbiol. 1:114-115, 1975) for detection of glycine. We evaluated three modifications of the Hwang and Ederer method and the gas-liquid chromatographic (GLC) method described by Kodaka et al. (H. Kodaka, G. L. Lombard, and V. R. Dowell, Jr., J. Clin. Microbiol. 16:962-964, 1982) for detecting benzoic acid. Campylobacter strains comprised 22 C. jejuni, 11 C. coli, and 8 C. laridis strains. The species identification of each strain was confirmed by DNA relatedness. All strains of C. jejuni were positive and all strains of C. coli and C. laridis were negative by the GLC method for detecting hippurate hydrolysis, whereas three strains of C. jejuni gave negative or variable results in the tube tests. The GLC method is more sensitive than the tube methods for detecting hippurate hydrolysis and should be used on cultures yielding variable or questionable test results.

Bacteriological Techniques↗
PubMed1986

Two-dimensional thin-layer chromatography for the specific detection of hippurate hydrolysis by microorganisms.

Glycine, one of the end products of hippurate hydrolysis by microorganisms, was detected by a rapid, specific technique utilizing two-dimensional thin-layer chromatography. A loopful of growth of each organism from its suitable agar medium was washed, suspended, and incubated with 0.1% sodium hippurate for 30 min at 37 degrees C. The supernatant of the incubated suspension from each organism was then dansylated, and the dansyl derivatives were separated by two-dimensional thin-layer chromatography on polyamide sheets. Glycine, a product of hippurate hydrolysis, was detected under UV light. This technique does not require prolonged incubation and was found to be more specific and reliable than the standard ninhydrin reaction. In addition, it is inexpensive and can be easily conducted in a clinical microbiological reference laboratory. By this method, 100% (22/22) of Campylobacter jejuni and 0% (0/9) of Campylobacter coli reference strains were positive. In addition, 100% (13/13) of group B streptococci, 100% (24/24) of group D streptococci, and 90% (18/20) of Gardenerella vaginalis clinical isolates were positive for hippurate hydrolysis. This method is useful for the identification to the species level of Campylobacter organisms and the biotyping of Gardnerella organisms and for the detection of hippurate hydrolysis by unknown microorganisms.

Bacteria↗
PubMed1998

Development of the phenylketonuria screening programme in Estonia.

OBJECTIVE: To develop the phenylketonuria (PKU) screening programme in Estonia. METHOD: All data about patients with PKU, born during 1980-92, were documented to establish its prevalence at birth in Estonia. Newborn screening for the diagnosis and treatment of PKU was started in Estonia in 1993 and the prevalence at birth established by screening. Phenylalanine was determined from filter paper blood by a modified fluorometric method based on enhancement of the fluorescence of a phenylalanine-ninhydrin reaction product by L-leucyl-L-alanine. RESULTS: During three years (1993-5) 36,074 newborns (85% of the total) were screened for PKU. PKU was diagnosed in six cases during the first four to six weeks of life. All investigated cases could be classified as classical PKU. No cases of mild forms of hyperphenylalaninaemia were diagnosed. The retrospective study showed an average incidence of PKU of 1 in 8090, the prospective study identified a comparable incidence of 1 in 6010 live births. CONCLUSION: The prevalence at birth of classic PKU in Estonia is higher than the average in Europe and similar to that of some eastern and middle European countries.

Estonia↗
PubMed1977

Lipids of antibiotic-resistant and -susceptible members of the Enterobacteriaceae.

Lipids of antibiotic-resistant and related -susceptible strains of the Enterobacteriaceae were extracted with chloroform-methanol and characterized by thin-layer chromatography, densitometry, and fatty acid analysis using gas chromatography. Quantitative differences which correlated with antibiotic resistance existed among the phospholipids and fatty acids. A relatively higher concentration of a ninhydrin-positive phospholipid concentration with a lower amount of phosphatidylethanolamine was observed in antibiotic-resistant strains of serratia marcescens. Bacterial strains which harbored R-factor 222 had a higher ratio of phosphatidylglycerol to diphosphatidylglycerol than their respective parent strains while those strains which were resistant to the polymyxins had a lower ratio of these phospholipids. Differences in the relative amounts of certain unsaturated and cyclopropane fatty acids were observed between susceptible and resistant strains. Such differences, however, were dependent upon a particular genus and species.

Anti-Bacterial Agents↗
PubMed1984

Preliminary characterization of inhibitors of Neisseria gonorrhoeae produced in vitro by Eubacterium limosum.

Among anaerobic bacteria normally found in the urogenital flora, Eubacterium limosum was found to inhibit the in vitro growth of Neisseria gonorrhoeae. The antigonococcal activity produced by E. limosum was soluble in methanol and in a chloroform--methanol mixture (30:70). The fraction soluble in chloroform--methanol (30:70) yielded eight absorbance peaks when chromatographed on Bio-Gel P-2 and the inhibitory activity was found in the first two peaks. This activity was not absorbed on DEAE Sephacel and was eluted with distilled water in a peak considered as peak 1, on which preliminary characterization was done. The inhibitory activity of peak 1 was found to be heat and pH resistant and not susceptible to proteases, lipase, or amylases. When peak 1 was chromatographed on cellulose paper using a butanol--acetic acid (4:1) solvent system, eight different spots were detected upon spraying the paper with ninhydrin. No spot was detected with anthrone, bromothymol, nor Sudan black reagents used for the detection of carbohydrates and lipids. Based on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and gel chromatography on Sephadex G-25, peak 1 appeared either as a diffuse band and as a single peak, respectively. The molecular weight of the inhibitory complex was estimated to be 2400. All these results suggest that the antigonococcal activity produced by E. limosum is composed of more than one low molecular weight amino compound.

Bacteriocins↗
PubMed1978

Recharacterization of fungal dinucleoside polyphosphate (HS3).

Three polyphosphorylated dinucleosides given the pseudonyms of HS3, HS2, and HS1 that were erroneously described as diguanosine polyphosphates (LéJohn, H. B., Cameron, L. E., McNaughton, D. R. & Klassen, G. R. (1975) Biochem, Biophys, Res, Commun. 66, 460-467) have been repurified and partially recharacterized. They have proved to be extremely complex molecules; chemical (HCl and KOH hydrolysis), physical (ultraviolet-light spectral analysis and ion-exchange chromatography), and enzymic (nucleotide pyrophosphatase and bacterial alkaline phosphatase hydrolysis) studies showed that (i) all three HS compounds are uracil rich and (ii) only HS3 contains a purine nucleoside and glutamate. The partial structure of HS3 was deciphered as a moiety of ADP--sugar X--glutamate (the mode of attachment of glutamate is obscure) that is covalently linked to another moiety composed of UDP, mannitol, and four phosphates. Sugar X had chromatographic characteristics of ribitol, but the chromatographic isolate also contained a ninhydrin-sensitive entity presumed to be an amino group. Sugar X, THEREFore, may be an amino sugar polyol. Only the general chemical compositions of HS2 and HS1 were determined. Each contained two uridines and HS2 had 10 phosphates whereas HS1 had 12.

Adenosine Diphosphate↗
PubMed1978

A rapid chromatographic method for the determination of omega-N-methylarginines in protein and muscle tissues.

A rapid and sensitive chromatographic method is described for the complete separation and determination of NG-methylarginine, NG,NG-dimethlarginine, NG,N'G-dimethylarginine, and related compounds in protein and muscle tissue hydrolysates. This method is designed to be used both with conventional amino acid analyzers using a single column (10 X 0.9 cm) of Durrum type DC-6A resin at 51 degrees C, one buffer system (0.21 M sodium citrate pH 5.40), and one buffer flow rate (70 ml/h) and with the accelerated and fully automatic Beckman Spinco model 121M amino acid analyzer using a 10 X 0.28 cm microcolumn packed with the Beckman type AA-10 resin and operated with the same buffer at 7.9 ml/h. This procedure was successfully applied to the determination of omega-N-methylarginines in various animal tissues, and the presence of four as yet unidentified ninhydrin-positive compounds is reported.

Amino Acids↗
PubMed1987

An endogenous peptide that stimulates lanthanum-resistant calcium uptake in vascular tissue.

A recent report has described the preparation of an extract from hemolyzed erythrocytes that has a stimulatory effect on lanthanum-resistant calcium uptake by vascular tissue in vitro and a hypertensive effect when injected into normotensive rats. The compound having a stimulatory effect on calcium uptake was further fractionated by molecular sieve and ion exchange chromatography, precipitation with CaCl2, high voltage paper electrophoresis, and high performance liquid chromatography (HPLC). HPLC yielded only a single fraction containing biological activity. This fraction was ninhydrin positive and acid labile. The amino acid composition was as follows: Asp/Asn (1.41), Ser (1.02), Glu/Gly (1.00), and Gly (2.00). Based on the assumption that the compound contains a single glutamic acid or glutamine residue, concentration-response data indicated that only nanomolar amounts of material were necessary to achieve significant stimulation. There was a marked increase in stimulatory activity of the resolubilized compound following calcium precipitation. The compound became inactive or showed a reduction in activity after being applied to a cation exchange column to remove calcium. Subsequent reprecipitation with CaCl2 and resolubilization restored the lost activity. Thus, we conclude that the compound is a small, acidic, calcium-dependent peptide that is extremely potent in stimulating lanthanum-resistant calcium uptake in vascular tissue.

Amino Acids↗
PubMed1975

Free amino acids of sex organs of the mouse: regulation by androgen.

Male mice were castrated at 2 mo and a pellet of testosterone propionate was implanted subcutaneously 2 wk later. Mice were killed after 11 days and tissue extracts were analyzed. All the common 20 amino acids were present in widely varying concentrations. Castration uniformly increased the concentration of all free amino acids of the seminal vesicle, excepy proline, cystine/2, and tryptophan; androgen restored values to normal. These changes were not entirely due to changes in quantity of seminal vesicle fluid. Concentrations of amino acids of the prostate were not significantly changed by castration or testosteronepropionate. Fifteen other ninhydrin-postive compounds were detected. Hypotaruine, taurine alpha-aminobutyric acid, and cysteic acid in seminal vesicles were greatly decreased and several other compounds were slightly decreased by castration and restored tp normal by testosterone propionate. In the prostate, hypotaurine, alpha-aminobutyric acid, cysteic/cysteinessulfinic acids, glycerophosphoethaniomine, cystathionine, and phosphoethanolamine were decreased and alpha-amino-n-butyric acid was increased by castration and restored to normal by testosterone propionate. Concentrations of taurine and 5 other compounds were not affected. Epidiymis and testis also contained appreciable pools of amino acids and the other compounds. Amino acids concentrations were lower in cauda than in caput epididymis; in the testis values were intermidate, with much higher concentrations of glutamic and aspartic acids. Taurine concentrations were twice as great in cauda as in caput epidiymis; concentrations of hypotaurine and the other sulfur-containing compounds were similar in both parts of the epidiymis. The most striking result in the testis was the high concentration of reduced glutathione.

Amino Acids↗
PubMed1975

Monosodium glutamate metabolism in the neonatal monkey.

Monosodium glutamate (MSG) administered by gastric tube to 10 infant monkeys at doses of 1-4 g/kg produced rapid increases in plasma glutamate (17- to 33-fold) and aspartate (50- to 90-fold) levels. The degree of elevation was proportional to the dose administered. Levels of other amino acids were unaffected. Two of the monkeys exhibited high fasting glutamate levels and abnormal glutamate tolerance curves. Despite this apparent decreased ability to metabolize glutamate, neither these animals nor any of the others for whom morphologic studies have been previously reported demonstrated neurotoxicity. Studies using 14-C-labeled glutamated indicated conversion of administered glutamate to two ninhydrin-negative compounds identified as glucose and lactate, as well as to asparate.

Amino Acids↗
PubMed1976

Intracellular osmoregulatory role of amino acids and urea in marine elasmobranchs.

Little skates, Raja erinacea, and stingrays, Dasyatis americana, were gradually transferred over a period of 4-5 days from full strength to approximatley 50% seawater. Plasma and muscle osmolarity fell. Hematocrits were essentially unchanged. Extracellular fluid volume (ECF) of muscle, estimated as the chloride space, increased 70% during this period. Regulation of muscle cell volume was associated with sharp declines in cellular concentrations of total amino acids (ninhydrin-positive materials) and urea. The osmoregulatory importance of the free amino acid pool in erythrocytes and muscle was a particularly prominent feature in both species. Intracellular amino acid concentration in R. erinacea muscle fell from 214 to 144 mmol/liter during transfer to 50% seawater, urea from 398 to 264, and trimethylamine oxide (TMAO) dropped from 63.9 to 35.8 mmol/liter. TMAO plasma levels were similar in stingray and skate, but muscle TMAO concentrations were much higher in the former. Urea content in stingray plasma greatly exceeded that in R. erinacea-630 and 574 mmol/liter in two specimens-perhaps the highest recorded.

Amino Acids↗
PubMed1982

Free amino acids in the blood of fetal and maternal Weddell seals.

The content of free amino acids in whole blood was measured in near-term gravid female Weddell seals (Leptonychotes weddelli) and compared with fetal amino acid profiles during rest and during experimental diving. With the exception of taurine and glutathione, Ninhydrin-reactive components of acid extracts of blood occurred in higher concentrations on the fetal side of the placenta than on the maternal side. Compared with humans the Weddell seal displayed higher ratios of fetal arterial to maternal arterial levels for aspartate, glycine, alanine, valine, tyrosine, phenylalanine, and total branched-chain amino acids. In the resting state the total free amino acid concentration in maternal blood was only about 70% as large as the total amino acid concentration in fetal blood, compared with a value of over 80% for humans. Only modest changes in the concentrations of specific amino acids occurred during simulated awake diving, but the overall maternal pools of glycine, glutamate, and glutamine were augmented, creating favorable conditions for uptake by the fetus.

Amino Acids↗
PubMed1993

Response of tissues of the rat to anisosmolality in vivo.

Rats were exposed to osmotic stress either acutely, over periods of 1 or 4 h, or chronically, over several days. In acute experiments, hyposmolality was induced by intraperitoneal infusion of dilute glucose or mannitol solutions, whereas hyperosmolality was induced by use of sodium chloride, concentrated glucose or mannitol solutions, or urea. Chronic hypernatremia was induced by daily administration of sodium chloride to water-deprived animals; chronic hyponatremia was induced by daily injection of antidiuretic hormone supplemented with glucose. Animals were made hyperglycemic using streptozotocin or uremic by ureteral ligation. Where appropriate, animals were anesthetized with thiobutabarbital (Inaktin) or ether. In acute experiments, analysis of the composition of the cardiac ventricle, diaphragm, liver, and renal cortex showed no evidence of cell volume regulatory processes involving transmembrane movement of potassium ions. There was a small but significant increase in free amino acids [measured as ninhydrin-positive substance (NPS)] in cardiac muscle exposed to hypertonic solutions of sodium chloride and glucose but not when plasma osmolality was raised using mannitol. In cerebral cortical tissue, after 4 h of exposure to acute hypertonicity by infusion of sodium chloride or glucose, there was a significant increase in tissue potassium content and a slight increase in NPS content. In chronic experiments, tissue analysis revealed good evidence for cellular volume readjustment only in cerebral cortex and heart. In the cortex, levels of free amino acids, principally taurine and glutamate (plus glutamine), showed large increases during hypernatremia and hyperglycemia and corresponding decreases during hyposmolality. In heart the principal amino acid present was taurine, and it, together with aspartate and glutamate (plus glutamine), showed large changes under osmotic stress. Other tissues analyzed showed only small changes in composition.

Animals↗
PubMed1988

Accumulation of major organic osmolytes in rat renal inner medulla in dehydration.

Osmotically active organic solutes, osmolytes, exist at high concentrations in the renal inner medulla; however, their modulation during antidiuresis remains largely undefined. Renal osmolyte levels were measured by nuclear magnetic resonance spectroscopy and biochemical assays in perchloric acid extracts from normal and dehydrated (3 days) rats. Dehydration increased urine osmolality from 1,503 to 3,748 mosmol/kg and inner medullary urea content from 2,036 to 4,405 nmol/mg protein. In addition, inner medullary trimethylamines [glycerophosphorylcholine (GPC) and betaine] and polyhydric alcohols (inositol and sorbitol) significantly increased by 95 and 78%, respectively. Ninhydrin-positive substances (amino acids), although abundant, were unchanged. Renal cortex also contained GPC, betaine, and inositol but only inositol increased with dehydration. Analysis of correlations among inner medullary osmolytes showed that only GPC was consistently elevated by dehydration and was not directly correlated with the other osmolytes. In contrast, betaine and inositol contents were linearly related to each other and both tended to rise only when sorbitol content was unchanged. In conclusion, the major osmolytes in the rat renal inner medulla can increase during antidiuresis but they are regulated in a complex manner.

Animals↗
PubMed1981

Biogenic monoamines in early embryos of sea urchins.

Indolylalkylamine determination was performed on the eggs and embryos of six species of sea urchins, using several fluorometric techniques, including the fluorescence of a substance itself and the fluorescence of its condensation products after treatment with orthophthaldialdehyde and ninhydrin. The serotonin-like substance of sea urchin embryos as well as of adults was shown to consist of at least two components, of which the major one is tryptamine or its derivative with a substituting group at aminonitrogen. Further, serotonin was found to be present at all developmental stages investigated. although at much lower concentrations. The results of this study suggest a regulatory role of 'prenervous' tryptamine in the early embryogenesis of the sea urchin.

Animals↗
PubMed1978

Studies on the histomorphology and histochemistry of the saccus vasculosus of Cyprinion macrostomus.

The saccus vasculosus of Cyprinion macrostomus is made up of coronet and supporting cells. Neurones are not observed. The luminal surface of the coronet cells exhibits hair-like protrusions. The saccus vasculosus is mainly concerned with a secretory function. Plenty of secretory material is seen in the lumen. PAS and alcian blue tests are positive for the secretory material in the lumen and the apical protrusions of the coronet cells. Some cells also exhibit small PAS- and alcian blue-positive granules. The possibility of the elaboration of glycogen by the coronet cells is ruled out. The toluidine blue test is also positive for the secretory material in the lumen and the apical protrusions. Small metachromatic granules are also seen in some of the coronet cells. The acetone-Sudan black B and Sudan black B tests point to lipid moiety of the coronet cells, the apical protrusions and the secretory material in the lumen. The acrolein-Schiff and ninhydrin-Schiff tests point to a protein moiety of the coronet cells and the secretory material in the lumen. The performic acid-alcian blue test showed cystine to be present in small amount.

Animals↗
PubMed1985

Hemoglobin Hofu associated with beta 0-thalassemia.

A mild anemia (hemoglobin 9 g/dl) was found in a patient from Seville (Spain) with marked morphological abnormalities in the peripheral blood smear. The red cell osmotic fragility showed a mild resistance curve with a mean cell fragility (MCF) of 0.375% NaCl (normal = 0.450). Chemical Chemical and thermal instability test and search for inclusion bodies gave positive results. Hemoglobin electrophoresis at pH 8.9 revealed absence of Hb A, a major component of fast mobility (94%), and increased Hb F and Hb A2 levels (1.5% and 4.6%, respectively). The fast fraction, isolated and purified by means of cellulose acetate electrophoresis, precipitated in acid acetone and treated with urea 8 M and mercaptoethanol, revealed an anomalous beta chain. Trypsin-digested globin peptides were separated by high-voltage electrophoresis at pH 6.4 and ascendant chromatography. With differential staining, an extra peptide was detected in an unusual site, more anodic than alpha Tp4 but in lower position. Peptide map of the fast beta chain, stained with ninhydrin, and also for Tyr, confirmed the position of the new peptide and the absence of the usual beta Tp13. The new peptide, separated by high-voltage electrophoresis at pH 3.5, revealed absence of Val and the presence of an additional Glu residue, which should appear only in position beta 126. The diagnosis of Hb Hofu (alpha 2 beta 2 126 Val----Glu; H4) was reached, thus interpreting its increase and the absence of Hb A, as an association with beta o-thalassemia, producing a mild hemolytic anemia. Evidence was obtained that Hb Hofu is a mild unstable hemoglobin variant.

Adult↗
PubMed1978

Identification of beta-aspartylglycine in uremic serum and its toxicity.

An unidentified ninhydrin-positive substance of acidic nature was found in the serum of uremic patients. This substance was isolated from hemodialysate by the methods of ion-exchange chromatography, gel-filtration and paper electrophoresis, and identified as beta-aspartylglycine by amino acid analysis, N-terminal amino acid determination and comparison with authentic sample synthesized in this laboratory. The quantitative determination of beta-aspartylglycine in serum revealed that the serum concentrations of beta-aspartyl-glycine in uremic patients increased much higher than those in normal subjects. The toxicity of beta-aspartylglycine in mice with acute renal failure induced by uranyl acetate was investigated. The mice given more than 1,0 g/kg body weight of beta-aspartylglycine showed behavioral alterations: low response to the stimuli and low activity, and some mice died by the injection of 4.0 g/kg body weight of the peptide. These results suggested that beta-aspartyl-glycine might be a possible factor which influences the development of uremic toxaemia.

Acute Kidney Injury↗