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PubMed1990

A novel enzymatic decarboxylation of oxalic acid by the lignin peroxidase system of white-rot fungus Phanerochaete chrysosporium.

Oxidation of veratryl alcohol by lignin peroxidase (LiP) was potently inhibited by oxalic acid. The inhibition analysis with Lineweaver-Burk plots clearly showed that the type of inhibition is non-competitive. The enzymatic oxidation of veratryl alcohol in the presence of 14C-oxalic acid yielded radioactive carbon dioxide. The results indicate that the apparent inhibition of LiP is caused by reduction of the veratryl alcohol cation radical intermediate back to the substrate level by oxalate, which is concomitantly oxidized to carbon dioxide.

Benzyl Alcohols↗

PubMed1983

Different biosynthetic pathways of the pyrimidine moiety of thiamin in procaryotes and eucaryotes.

[14C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-14C]glycine and [2-14C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [14C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.

Aspergillus nidulans↗

PubMed2006

Use of microdosing to predict pharmacokinetics at the therapeutic dose: experience with 5 drugs.

OBJECTIVES: A volunteer trial was performed to compare the pharmacokinetics of 5 drugs--warfarin, ZK253 (Schering), diazepam, midazolam, and erythromycin--when administered at a microdose or pharmacologic dose. Each compound was chosen to represent a situation in which prediction of pharmacokinetics from either animal or in vitro studies (or both) was or is likely to be problematic. METHODS: In a crossover design volunteers received (1) 1 of the 5 compounds as a microdose labeled with radioactive carbon (carbon 14) (100 microg), (2) the corresponding (14)C-labeled therapeutic dose on a separate occasion, and (3) simultaneous administration of an intravenous (14)C-labeled microdose and an oral therapeutic dose for ZK253, midazolam, and erythromycin. Analysis of (14)C-labeled drugs in plasma was done by use of HPLC followed by accelerator mass spectrometry. Liquid chromatography-tandem mass spectrometry was used to measure plasma concentrations of ZK253, midazolam, and erythromycin at therapeutic concentrations, whereas HPLC-accelerator mass spectrometry was used to measure warfarin and diazepam concentrations. RESULTS: Good concordance between microdose and therapeutic dose pharmacokinetics was observed for diazepam (half-life [t((1/2))] of 45.1 hours, clearance [CL] of 1.38 L/h, and volume of distribution [V] of 90.1 L for 100 microg and t((1/2)) of 35.7 hours, CL of 1.3 L/h, and V of 123 L for 10 mg), midazolam (t((1/2)) of 4.87 hours, CL of 21.2 L/h, V of 145 L, and oral bioavailability [F] of 0.23 for 100 microg and t((1/2)) of 3.31 hours, CL of 20.4 L/h, V of 75 L, and F of 0.22 for 7.5 mg), and development compound ZK253 (F = <1% for both 100 microg and 50 mg). For warfarin, clearance was reasonably well predicted (0.17 L/h for 100 microg and 0.26 L/h for 5 mg), but the discrepancy observed in distribution (67 L for 100 microg and 17.9 L for 5 mg) was probably a result of high-affinity, low-capacity tissue binding. The oral microdose of erythromycin failed to provide detectable plasma levels as a result of possible acid lability in the stomach. Absolute bioavailability for the 3 compounds examined yielded excellent concordance with data from the literature or data generated in house. CONCLUSION: Overall, when used appropriately, microdosing offers the potential to aid in early drug candidate selection.

Administration, Oral↗

PubMed1978

Intraocular penetration of gentamicin after subconjunctibal and retrobulbar injection .

We compared the ocular penetration of labeled with radioactive carbon gentamicin in squirrel monkeys after subconjunctival and retrobulbar administration. In both normal and infected (Staphylococcus aureus endophthalmitis) eyes, high concentrations of drug were achieved in the sclera and choroid-retina by both routes, while corneal levels were markedly higher after subconjunctival injection than after retrobulbar injection. Regional variations in concentration were evident in these tissues; the highest levels were clustered about the site of injection. Aqueous humor concentrations were lowest in the group with normal eyes treated by the retrobulbar route; vitreous humor levels were extremely low in normal eyes injected subconjunctivally. These data differ from those in rabbits, especially with regard to penetration of the vitreous humor of normal eyes. Interspecies differences were less marked in inflamed eyes. The two species were similar in demonstrating maximum access to the cornea and aqueous humor with subconjunctival injection, and equivalence of the two routes in penetrating the vitreous humor of the inflamed eyes.

Animals↗

PubMed1993

Site specificity and meal stimulation of the intestinal absorption of water, electrolytes, and bile acids.

Ileal water and electrolyte absorption exceed jejunal absorption in both the basal and meal-stimulated states. The purposes of these experiments were to determine: (1) if luminal bile acids alter basal or meal-stimulated intestinal absorption, and (2) if there is site specificity or meal stimulation of intestinal bile acid absorption. Twenty-five centimeters of canine proximal jejunal and distal ileal Thiry-Vella fistulas were constructed. Simultaneous jejunal and ileal absorption studies (n = 88) were performed with a luminal perfusate containing polyethylene glycol labeled with radioactive carbon-14 to calculate the absorption of water, electrolytes, and the bile acid taurocholate (TC). In group 1, there was no TC in the luminal perfusate, whereas in group 2, 10 mM of TC was present in the luminal perfusate. Half of the observations were performed after a meal stimulus, which consisted of an orally ingested, 480-kcal mixed nutrient meal. Intraluminal TC did not affect basal or meal-stimulated water or electrolyte absorption. In both the basal and meal-stimulated states, ileal absorption of water, electrolytes, and TC significantly exceeded jejunal absorption (p < 0.05). A meal significantly stimulated water and electrolyte absorption in both the jejunum and ileum, but it stimulated absorption of TC in the ileum only (p < 0.05). Intraluminal TC does not alter basal or meal-stimulated intestinal water and electrolyte absorption. A meal stimulates increased water and electrolyte absorption in both the jejunum and the ileum, but it stimulates bile acid absorption in the ileum only. Bile acid absorption is site specific and responsive to a meal stimulus.

Animals↗

PubMed2002

Composition of particulate and dissolved organic matter in a disturbed watershed of southeast Brazil (Piracicaba River basin).

The elemental and isotopic composition of particulate and dissolved organic matter was investigated in the Piracicaba River basin, São Paulo State, Brazil. Comparison of riverine organic matter from the Piracicaba River basin, a region where rivers and streams receive urban sewage and industrial effluents, with data reported for the pristine Amazon system revealed significant differences associated with anthropogenic impacts. One important difference was N enrichment in the particulate organic material of the Piracicaba basin rivers, due to (a) urban and industrial effluents, and (b) enhanced phytoplankton growth, which results from the combination of nutrient enrichment and damming of sections of the rivers. Radiocarbon concentrations were overall more depleted (older 14C age) in the Piracicaba basin rivers than in the Amazon, which may reflect the importance of soil erosion in the former. Analyses of stable and radioactive carbon isotopes and lignin-derived compounds indicated that coarse particulate organic material is composed of a mixture of soil particles and degraded organic matter from C3 and C4 vascular plants. Fine particulate organic material was composed mainly of soil particles and phytoplankton cell remains, the latter especially during low water. Ultrafiltered dissolved organic matter was the most degraded fraction according to its lignin oxidation products, and showed the greatest influence of C4 plant sources.

Brazil↗

PubMed1976

The absolute configuration of the amino acids in delta-(alpha-aminoadipyl)cysteinylvaline from Penicillium chrysogenum.

Radioactive carbon-14 L-alpha-aminoadipic acid, L-cysteine, or L-valine were readily incorporated into the intracellular tripeptide, delta-(alpha-aminoadipyl)cysteinylvaline (ACV), by washed starved cells of Penicillium chrysogenum. The labeled ACV in each case was oxidized with performic acid and isolated as its corresponding sulfonic acid derivative. After acid hydrolysis, the configuration of the component acids was determined by L- and D-amino acid oxidases, which showed the tripeptide (ACV) from P. chrysogenum to be delta-(L-aminoadipyl)-L-cysteinyl-D-valine.

2-Aminoadipic Acid↗

PubMed1975

Oral inhalation system for exposing rabbits to cigarette smoke.

An oral inhalation system for exposing rabbits to cigarette smoke has been developed. The essential components of the system comprise a smoke delivery device that adapts to the oral cavity, a molded face mask, and a sealable body enclosure. The functional capability of the system has been validated by detecting the presence of nicotine in plasma and by measuring the deposition of dotricontane labeled with radioactive carbon (14C) in lungs after exposure to cigarette smoke.

Animals↗

PubMed1977

Metabolism of pentachlorophenol by a soil microbe.

Metabolism of pentachlorophenol (PCP)-ring-14C(U) by a microorganism (Pseudomonas sp.) isolated from soil was examined. The microorganism degraded PCP-14C rapidly, and released 14CO2 equivalent to approximately 50% of the PCP-14c added to the bacterial cell suspension in 1 hour of incubation. The results of amino acid analysis of the bacterial cells incubated with PCP-14C showed that radioactive carbon derived from PCP-14C was incorporated rapidly into the cell constituents, and that the pattern of 14C-amino acids in the cell constituents was not much different between the 15 minute and 24 hour incubation periods. Intermediate metabolities of PCP isolated from the incubation medium were identified as tetrachlorocatechol and tetrachlorohydroquinone by spectral analyses.

Biodegradation, Environmental↗

PubMed1989

[Nitrogen metabolism in the large intestine of ruminants. 6. Metabolism of intracecally administered 14C and 15N urea in sheep with simultaneous intracecal doses of heat-damaged hay].

Two wethers (28 kg and 33 kg) were supplied with ileocaecal re-entrance cannulae and received a straw pellet ration rich in crude fibre (70.5% straw, 12% chopped sugar beet, 10% cereals, 2% urea, 3% NH4HCO3 and 2.5% of a mineral mixture). In a preliminary period 50% of the digesta flow was collected on 6 successive days for 18 h each. An amount of digesta sufficient for 24 h was apportioned for hourly application and stored at a temperature of -20 degrees C for the main trial. In the main trial the two animals received intracaecally the collected digesta with a supplement of ca. 6 g hay damaged by heat/kg LW(0.75) in hourly portions over 24 h (hay made up ca. 15 and 20% resp. of the DM amount). In addition, each digesta sample was supplemented with 14C and 15N labelled urea (19.7.10(6) Bq 14C urea and 364 mg 15N excess from 15N urea). About 9% of the applied 15N amount was microbially utilized; the utilization quota was thus lower than after the application of partly hydrolyzed straw meal (16% in a previous trial). The 14C activity from 14C urea was quickly eliminated in the form of CO2 in the respiratory gases (at the 18th hour after the end of the infusion 70% excreted as CO2). The half-lives for the urea resulting from the semi-logarithmic decrease of the atom-% 15N excess in the blood plasma were 7.9 and 7.7 resp. 23% and 34% resp. of the applied 15N excess were excreted in urine. The excretion of radioactive carbon in urine, however, was at 2.8% and 4.3% resp. of the applied amount very low 120 h after the beginning of the trial (96 h after the end of the infusion). On the whole one can conclude from this trial that hay damaged by heat has only a low stimulating effect on microbial activity in the large intestine.

Animal Feed↗

PubMed2002

Subarachnoid hemorrhage induces dynamic changes in regional cerebral metabolism in rats.

Following a subarachnoid hemorrhage (SAH), adult rats exhibit dynamic regional changes in cerebral glucose metabolism characterized by an increase in metabolic rates and a subsequent upregulation of cytochrome oxidase (CO). We evaluated both local cerebral metabolic rates for glucose (ICMRglc: (mol/100 g/min) and CO in 23 brain regions of interest (ROI). Sham animals underwent anesthesia and superficial surgery; saline-controls received an injection of 0.9% saline into the cisterna magna; and SAH rats received an injection of autologous blood into the cisterna magna. This blood, measured by albumin labeled with radioactive carbon 14, distributed throughout the brain but predominated ventrally. After experimental animals were sacrificed at day 0 (3 h), 1, 3, and 7 days postinjection, ROI were analyzed using [14C]2-deoxy-D-glucose autoradiography and CO histochemistry. ICMRglc in SAH rats increased in many regions (ranging from 0.7% to 32.2% above sham levels). Cytochrome oxidase also increased from 1% to 9% above sham levels, peaking on day 3. Conversely, saline-controls exhibited prolonged depression of ICMRglc (ranging from 11% to 35% below sham levels) and CO (ranging from 4% to 11% below sham levels) from day 0 through day 7. All saline-control ROI for all time points showed this metabolic depression, and between 91% and 95% of saline-control ROI presented lower CO levels as compared to sham. Overall, ICMRglc and CO levels were greater in SAH than in saline-control ROI. However, when considering the influence of subarachnoid blood on metabolic changes in SAH animals, both CO and 2DG levels did not correlate well with the amount of 14C-albumin binding. While previous studies have measured both metabolic rates of glucose and CO soon after SAH, this is the first to simultaneously conduct these measurements in the same SAH rat model.

Animals↗

PubMed1977

Xylitol infusion and oxalate formation in rabbits.

Oxalate levels in the plasma and urine fractions of fasted normal, oxythiamin treated (20 mg/kg) and 4-deoxypyridoxine treated (300 mg/kg) rabbits were determined following infusion with either xylitol or glucose at a dose of 2 g/kg body weight. Biochemical determinations showed that transient thiamin or pyridoxine deficient states had been induced in the antivitamin treated rabbits. In the first 24 hour following infusion with either carbohydrate, urinary oxalate levels remained within the normal range for all groups. Oxythiamin hastened the appearance of the transient, elevation in plasma oxalate concentrations seen in rabbits after infusion with glucose. After xylitol infusion, the elevation of plasma oxalate was not significnatly above normal. 4-Deoxypyridoxine enhanced peak plasma oxalate levels above those of controls for both sugars. Glucose, at an equivalent dose to xylitol, resulted in higher plasma oxalate levels than xylitol for all groups. Infusions of [U-14C]xylitol and [U-14C]glucose solutions into 4-deoxypyridoxine treated rabbits demonstrated a conversion of the administered radioactive carbon into 14C oxalate of 0.01% with a high dilution of the specific activity. The results suggest that oxalate production from xylitol is negligible; any toxicity related to xylitol administration is not a consequence of oxalate production.

Animals↗

PubMed1990

Secretion of Ricinus communis glyceraldehyde-3-phosphate dehydrogenase by Escherichia coli.

A plasmid, pWEH1, was constructed containing a fusion of the DNA encoding the signal sequence of the Escherichia coli outer-membrane protein A to the 5'-end of a glyceraldehyde-3-phosphate dehydrogenase cDNA from Ricinus communis. When expressed in E. coli, the fusion protein was secreted by the normal membrane-potential-dependent pathway. Processing by signal peptidase was inhibited by low concentrations of phenethyl alcohol. Quantitative cell fractionation was used to show that the mature plant protein was associated with the bacterial outer membrane. The protein could not be released from the membrane by washing with alkaline sodium carbonate. Radioactivity from [U-14C]-palmitate was incorporated into the heterologous protein. These results suggest that the sequence of this normally cytoplasmic enzyme contains a cryptic lipid-modification site, and the combination of a signal sequence plus a lipid-modification sequence results in specific targeting to the bacterial outer membrane.

Blotting, Western↗

PubMed1983

Regional pulmonary clearance of inhaled C15O and C15O2 in man at rest and during exercise.

The regional clearance of radioactive carbon monoxide (C15O) and carbon dioxide (C15O2), after a single maximal inspiration, was investigated in six normal subjects seated on a bicycle, at rest and during exercise at 50 W. The clearances were measured sequentially during breath-holding by four pairs of scintillation counters vertically aligned over the right lung. The clearance rate (k) for C15O and C15O2 increased from apex to base and from rest to exercise. On exercise, the apex base gradient (calculated over 11 cm vertical distance) for C15O decreased by 52% because of a larger increase in the upper zones but the C15O2 gradient did not change. Thus the increase in blood flow (approximately equal to kC15O2) on exercise was accompanied by recruitment and/or distension of pulmonary capillary blood volume (approximately equal to kC15O2), most marked in the upper regions. The ratio of the clearance (C15O/C15O2) decreased in the lower zones on exercise by 25% without significant change in the upper zone. This ratio reflects the product of capillary mean transit times and peripheral vascular and extravascular volumes. Since the latter increase on exercise, capillary transit times must have shortened considerably at a moderate level of exercise at all levels in the lung.

Adult↗

PubMed1963

Gamma irradiation of polypeptides: transformation of amino acids.

The formation of aspartic acid from glutamic acid, of aspartic and glutamic acids from proline, of alpha-amino-n-butyric acid from methionine, of aspartic acid from histidine, of dihydroxyphenylalanine from tyrosine, of tyrosine and dihydroxyphenylalanine from phenylalanine, of alanine from cysteine, and of glycine from alanine was observed when aqueous solutions of these amino acids in the form of peptides or polyamino acids were irradiated. When poly-L-glutamic acid or poly-L-proline was irradiated in the presence of C(14)-labeled NaHCO(3), the radioactive carbon was fixed by the aspartic and the glutamic acid.

Alanine↗

PubMed1986

Biodegradation of chlorinated ethenes by a methane-utilizing mixed culture.

Chlorinated ethenes are toxic substances which are widely distributed groundwater contaminants and are persistent in the subsurface environment. Reports on the biodegradation of these compounds under anaerobic conditions which might occur naturally in groundwater show that these substances degrade very slowly, if at all. Previous attempts to degrade chlorinated ethenes aerobically have produced conflicting results. A mixed culture containing methane-utilizing bacteria was obtained by methane enrichment of a sediment sample. Biodegradation experiments carried out in sealed culture bottles with radioactively labeled trichloroethylene (TCE) showed that approximately half of the radioactive carbon had been converted to 14CO2 and bacterial biomass. In addition to TCE, vinyl chloride and vinylidene chloride could be degraded to products which are not volatile chlorinated substances and are therefore likely to be further degraded to CO2. Two other chlorinated ethenes, cis and trans-1,2-dichloroethylene, were shown to degrade to chlorinated products, which appeared to degrade further. A sixth chlorinated ethene, tetrachloroethylene, was not degraded by the methane-utilizing culture under these conditions. The biodegradation of TCE was inhibited by acetylene, a specific inhibitor of methane oxidation by methanotrophs. This observation supported the hypothesis that a methanotroph is responsible for the observed biodegradations.

1-Propanol↗

PubMed1992

Role of heterotrophic bacteria in complete mineralization of trichloroethylene by Methylocystis sp. strain M.

Biodegradation experiments with radioactively labeled trichloroethylene showed that 32% of the radioactive carbon was converted to glyoxylic acid, dichloroacetic acid and trichloroacetic acid and that the same percentage was converted to CO2 and CO after 140 h of incubation by a pure culture of a type II methane-utilizing bacterium, Methylocystis sp. strain M, isolated from a mixed culture, MU-81, in our laboratory. In contrast, these water-soluble (14C)trichloroethylene degradation products were completely or partially degraded further and converted to CO2 by the MU-81 mixed culture. This phenomenon was attributed to the presence of a heterotrophic bacterium (strain DA4), which was identified as Xanthobacter autotrophicus, in the MU-81 culture. The results indicate that the heterotrophic bacteria play an important role in complete trichloroethylene degradation by methanotrophs.

Biodegradation, Environmental↗

PubMed1974

Lipid peroxidation in the killing of phagocytized pneumococci.

To directly examine the role of hydrogen peroxide in the killing of bacteria after ingestion by granulocytes, we have studied some of the events of phagocytosis of a mutant strain of pneumococci which is relatively deficient in peroxide production. The hydrogen peroxide-deficient pneumococci and the otherwise identical wild type were grown with [(14)C]arachidonic and [(3)H]palmitic acid labels to label their lipids with unsaturated and saturated fatty acids, respectively. They were then incubated with both normal and chronic granulomatous disease granulocytes. The rates of ingestion and bacterial killing and the stability of fatty acids in the cell-bacteria complex were followed. Radioactive carbon dioxide released from glucose was also independently followed to measure glucose oxidation. Ingestion was similar for all cell-bacteria combinations. Chronic granulomatous disease cells killed the peroxide-positive wild pneumococci much more effectively (20-fold) than the peroxide-deficient mutant. Normal cells killed both peroxide-positive and -negative strains effectively. A considerable loss of [(14)C]arachidonic acid ( approximately 40%) consistent with lipid peroxidation of this unsaturated fatty acid was observed in all normal cells and in chronic granulomatous disease cells with peroxide-positive pneumococci. However, no loss of [(14)C]arachidonic acid occurred in chronic granulomatous disease cells with the peroxide-deficient pneumococci. No loss tritiated palmitic acid occurred in any cell-bacteria combination. Glucose oxidation was impaired in the chronic granulomatous disease cells in comparison to normal cells at rest and was especially impaired in chronic granulomatous disease cells ingesting the peroxide-deficient mutant pneumococci. This defect was partially corrected after phagocytosis of the peroxide-positive strain. These data directly support the hypothesis that bacterial killing is partially dependent upon an intact peroxide-generating system in the leukocyte-bacteria complex. Moreover, they indicate that bacterial lipid peroxidation is associated with the generation of peroxide during phagocytosis. Finally, they suggest that such peroxidation may contribute to effective phagocytic bacterial killing.

Animals↗