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Multiple chromosomes in bacteria: structure and function of chromosome II of Rhodobacter sphaeroides 2.4.1T.

Although multiple chromosomes occur in bacteria, much remains to be learned about their structural and functional interrelationships. To study the structure-function relationships of chromosomes I and II of the facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1T, auxotrophic mutants were isolated. Five strains having transposon insertions in chromosome II showed requirements for p-aminobenzoic acid (pABA)-dihydroxybenzoic acid (dHBA), serine, thymine, uracil, or histidine. The His, Thy, and pABA-dHBA mutants reverted to prototrophy at low frequency and concordantly lost their transposon insertions from the genome. The Ser, Ura, and pABA-dHBA mutants were complemented by cosmids that carried the region of chromosome II where the transposon insertions were located. The cosmids used for complementation analysis were selected, on the basis of map position, from a set of overlapping clones that had been ordered by a combination of hybridization and restriction endonuclease mapping. These experiments provide the basis for detailed studies of the structure, function, and interaction between each chromosome, and they demonstrate at this early stage of investigation that no fundamental differences exist between each chromosome.

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Study on the effect of electromagnetic impulse on neurotransmitter metabolism in nerve cells by high-performance liquid chromatography-electrochemical detection coupled with microdialysis.

In this paper, a reverse-phase high-performance liquid chromatographic method using a chemically modified electrode coupled with microdialysis was developed to study the effect of electromagnetic impulse (EMI) on monoamine neurotransmitter metabolism in nerve cells. To detect the monoamines and their metabolites, a poly (para-aminobenzoic acid) (P-pABA)-modified electrode was prepared. The modified electrode exhibited efficiently electrocatalytic oxidation for monoamines and their metabolites with relatively high sensitivity, stability, and long life. Nerve cells were primarily cultured. EMI was radiated to three experimental model nerve cells: (i) on mature nerve cells, (ii) on the culture medium, and (iii) on juvenile nerve cells for various periods of time. Then the levels of monoamines in the culture medium were detected by high-performance liquid chromatography-electrochemical detection. The data indicated that electromagnetic fields could influence neurotransmitter metabolism by direct effect on nerve cells or effect on the nutrient medium and that the effect was not only relevant with the length of radiation time, but also with the growing state of the nerve cells.

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Metabolic activation, DNA adducts, and H-ras mutations in human neoplastic and non-neoplastic laryngeal tissue.

Metabolic activation, DNA adducts, and H-ras mutations were examined in human laryngeal tissue (n = 16) from both smoker and non/ex-smoker patients with laryngeal cancer. DNA adducts detected by 32P-postlabelling were evident only in smokers (n = 13); in fact, smoking cessation for as little as 10 months resulted in no DNA adducts detected (n = 3). Total DNA adduct levels in these samples were significantly correlated with levels of cytochromes P-4502C and 1A1 in laryngeal microsomes. Moreover, the P-4501A1 levels represent the highest yet found in human tissues. In contrast, laryngeal microsomes did not have detectable P-4501A2 activity, while laryngeal cytosols showed appreciable N-acetyltransferase activity for p-aminobenzoic acid (NAT1) but not sulfamethazine (NAT2). DNA was extracted from laryngeal specimens and amplified by PCR. Nylon filter dot or slot blots were hybridized with 32P-labelled probes for codons 12, 13, and 61 of the H-ras gene. Sixty percent of specimens demonstrated mutations in either codon 12, 13, or 61; a single common and specific mutation was a Gln-->Glu transversion in codon 61. This mutation appeared in 5 laryngeal specimens, all from smokers. These results implicate cigarette smoke components, bioactivated by CYP1A1 and/or CYP2C, in DNA adduct formation. These results also demonstrate a probable smoking-related H-ras Gln-->Glu transversion in codon 61.

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Sodium and blood pressure: positive associations in a north London population with consideration of the methodological problems of within-population surveys.

Blood pressure (BP) and 24-hr urinary sodium (Na) and potassium (K) excretion were measured in 58 men and women aged 40 and above who were selected randomly from the age-sex register of a general practice in North London. All 58 urine collections were reported as complete, but only 28/56 (50%) were classified as 'complete' by the excretion of a biological marker, p-aminobenzoic acid (PABA). Reliability of Na excretion estimated from repeated urine collections was 0.86, indicating that variability of Na excretion within individuals in this middle-aged and elderly population was low. Systolic blood pressure (SBP) was significantly related to 24-hr Na excretion and to 24-hr Na/creatinine ratio. After adjustment for age, sex and body mass index, a SBP-Na regression coefficient of 0.091 mmHg/mmol Na (P = 0.02) was observed. On simple regression analysis, a significant association was also found between diastolic blood pressure and Na (P = 0.04). In the sub-group classified as 'complete' collectors by PABA excretion, BP-Na regression coefficients were larger than in analyses of the sample as a whole. BP was not significantly related to K or Na/K.

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Gastric absorption of D-xylose in the rat: its influence on the D-xylose absorption test.

Relatively high incidences of false-positive results of D-xylose absorption tests have been reported. Delayed gastric emptying is invariably listed as one important cause for such tests. However, this conclusion assumes that gastric absorption from the relatively concentrated D-xylose solutions used clinically is negligible. In our study, D-xylose was injected (0.5 gm/kg) into either the stomach or duodenum of rats that had undergone pyloric ligation. Blood xylose levels 30 minutes later were almost as high after intragastric administration (0.61 +/- 0.22 mmol/L) as they were after intraduodenal injection (0.65 +/- 0.16 mmol/L). A chymotrypsin-labile peptide (N-benzoyl-L-tyrosyl-p-aminobenzoate), given at the same time, was poorly absorbed from the stomach, as shown by the low plasma p-aminobenzoic acid levels (14 +/- 2 mumol/L vs. 158 +/- 22 mumol/L after intraduodenal injection). Intragastric absorption of xylose, therefore, did not appear to result from the surgical trauma of pyloric ligation. In rats given doses orally of the D-xylose, the peptide, and a nonabsorbable marker (phenol red), epinephrine and atropine both showed gastric emptying without any surgical trauma, decreased digestion and absorption of the peptide as expected, but did not significantly decrease xylose absorption. These results indicate that, at least in rats, D-xylose is absorbed from relatively concentrated solutions within the stomach. Consequently, circumstances that delay gastric emptying should not markedly decrease xylose absorption. Bacterial overgrowth and altered blood flow seem more likely causes for false-positive D-xylose absorption test results.

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N-acetylation of aromatic amines: genetic polymorphism in inbred rat strains.

The inheritance of rat liver N-acetyltransferase polymorphism was investigated with reciprocal genetic crosses between slow (NSD/N) and rapid (Peth/N) acetylator strains. Rat liver N-acetyltransferase activity was determined using a spectrophotometric assay which measured the amount of arylamine substrate present after incubation with N-acetyltransferase in vitro. Male N-acetyltransferase activities assayed in liver preparations using p-aminobenzoic acid and p-toluidine as substrates indicate bimodality of the parental strains and unimodality of the F-1 generation; limited data suggest trimodality (not significantly different from a 1:2:1 ratio) of the F-2 generation. Reciprocal crosses of WKY/N, another slow acetylator strain, and the Peth/N strain gave results similar to those of the NSD/N x Peth/N cross. Female N-acetyltransferase activities in all strains studied were lower than male N-acetyltransferase activities, but were similarly distributed in the parental and F-1 generations. The male/female N-acetyltransferase activity ratio was substrate- and genotype-dependent. Results show that regulation of the variation of rat liver N-acetyltransferase activity is consistent with autosomal Mendelian inheritance of two major alleles at a single gene locus.

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Ornithine decarboxylase induction in psoralen-treated mouse epidermis used as a test of UV-A sunscreen potency.

Inhibition by sunscreens of UV-A-induced epidermal ornithine decarboxylase (ODC) activity in 8-methoxypsoralen-treated mice was used to examine the UV-A sunscreen activity of two commercial preparations, Uval (para-aminobenzoic acid) and PreSun (a benzophenone derivative). Both gave significant protection against UV-A doses of up to 3 J/cm2 and Uval was more effective that PreSun in the UV-A range. At intermediate doses (5-15 J/cm2) neither Uval nor PreSun significantly altered the ODC response to UV-A. At doses of 20-40 J/cm2 UV-A, control and PreSun-treated animals showed a decline in ODC activity which histological studies suggested was due to severe epidermal damage. This decline in ODC activity and severe epidermal damage was not seen in Uval-treated animals. Topical zinc oxide was highly effective in preventing induction of ODC by UV-A irradiation at doses up to 40 J/cm2, and was significantly more effective than either PreSun or Uval.

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Photopatch test reactivity: effect of photoallergen concentration and UVA dosaging.

We have studied the influence of variations in allergen concentration and UVA dosaging on the results of photopatch testing with the Scandinavian standard photopatch series in 29 patients with photocontact and/or contact allergy to 1 or several of the allergens in that series. Photocontact test reactions were more sensitive to allergen dilution than plain contact test reactions. Even dilution from the standard 5% to 2.5% significantly reduced para-aminobenzoic acid photocontact test reactions. Reducing the UVA dose from the standard 5 J/cm2 to 2.5 or 1 J/cm2 in 2 out of 5 cases turned a significant (++) reaction into a doubtful one (+). Increasing the standard UVA dose of 5 J/ cm2 to 20-40 J/cm2 turned a single + photocontact reaction to trichlorcarbanilide and a single 1 + plain contact reaction to chlorhexidine into ++ reactions. In the majority of cases, however, neither photocontact nor plain contact test reactions were augemented by UVA doses up to 80 J/cm2. We conclude that a UVA dose of 5 J/cm2 is sufficient for eliciting photocontact allergic test reactions, and that a reduction of either the UVA dose level or the standard allergen concentrations of the Scandinavian photopatch test guidelines may cause loss of significant photocontact test reactions in a proportion of the cases.

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Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii.

Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.

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Exocrine pancreatic function in children and adolescents with insulin-dependent diabetes mellitus.

Exocrine pancreatic function was evaluated in 21 diabetic children on the basis of a p-aminobenzoic acid (PABA) test and a determination of fasting serum amylase, pancreatic isoamylase, lipase, trypsin and elastase levels. Fecal chymotrypsin was also measured. Compared to the controls, the diabetic children had significantly lower levels of trypsin (P less than 0.001) and elastase (P less than 0.02). Fecal chymotrypsin appeared to be significantly lower (P less than 0.01) in diabetic children than in controls but in all patients fecal chymotrypsin values registered above the limit considered to be normal. No significant correlation was observed between pancreatic enzyme concentrations, serum and urinary PABA values, and chronologic age, HbA1 and insulin requirement. Only for serum PABA a significant negative correlation with duration of disease (P less than 0.01) has been observed. These data show that exocrine pancreatic function may be abnormal in children with IDDM.

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Inheritance of liver N-acetyltransferase activity in the rapid and slow acetylator inbred hamster.

p-Aminobenzoic acid and p-aminosalicylic acid-N-acetyltransferase activities were determined in 105,000 X g liver cytosols from homozygous rapid acetylator (Bio. 4.24, Bio. 41.56 and Bio. 65.67) and homozygous slow acetylator (Bio. 1.5) inbred hamsters. Liver N-acetyltransferase activities were substantially higher in rapid as opposed to slow acetylator hamsters. Genetic crosses yielded F1 generation progeny with unimodal distributions of N-acetyltransferase activity intermediate between those of the rapid and slow acetylator parental strains, and F2 generation progeny with trimodal distributions (low/intermediate/high) of N-acetyltransferase activity not significantly different from a ratio of 1/2/1. Backcross matings yielded bimodal distributions of N-acetyltransferase activity in a ratio (intermediate/parental) not significantly different from 1/1. Each of these findings is consistent with simple autosomal Mendelian inheritance of two codominant alleles at a single gene locus.

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Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein.

Asparagine-linked oligosaccharides present on hen egg-yolk immunoglobulin, termed IgY, were liberated from the protein by hydrazinolysis. After N-acetylation, the oligosaccharides were labelled with a UV-absorbing compound, p-aminobenzoic acid ethyl ester (ABEE). The ABEE-derivatized oligosaccharides were fractionated by anion exchange, normal phase and reversed phase HPLC, and their structures were determined by a combination of sugar composition analysis, methylation analysis, negative ion FAB-MS, 500 MHz 1H-NMR and sequential exoglycosidase digestions. IgY contained monoglucosylated oligomannose type oligosaccharides with structures of Glc alpha 1-3Man7-9-GlcNAc-GlcNAc, oligomannose type oligosaccharides with the size range of Man5-9GlcNAc-GlcNAc, and biantennary complex type oligosaccharides with core region structure of Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc. The glucosylated oligosaccharides, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, have not previously been reported in mature glycoproteins from any source.

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Dietary underreporting by obese individuals--is it specific or non-specific?

OBJECTIVE: To examine the distribution of patterns of macronutrient density in relation to obesity. DESIGN: Cross sectional. SETTING: Denmark. SUBJECTS: 323 men and women aged 35-65 years, selected randomly from a larger population sample of Danish adults. MAIN OUTCOME MEASURE: Bias in dietary reporting of energy and protein intake in relation to percentage body fat, assessed by comparison of data from an interview on dietary intake with data estimated from 24 hour nitrogen output, validated by administering p-aminobenzoic acid, and estimated 24 hour energy expenditure. RESULTS: Degree of obesity was positively associated with underreporting of total energy and protein, whereas compared with total energy reported, protein was overreported by the obese subjects. CONCLUSION: Errors in dietary reporting of protein seem to occur disproportionately with respect to total energy, suggesting a differential reporting pattern of different foods. Although, on average, all subjects showed a greater underreporting of energy than of protein, this was most common in the obese subjects. Snack-type foods may be preferentially forgotten when obese people omit food items in dietary reporting. These results seem to agree with the general assumption that obese people tend to underreport fatty foods and foods rich in carbohydrates rather than underreport their total dietary intake. These results may have implications for the interpretation of studies of diet and comorbidities related to obesity.

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Effects of model traumatic injury on hepatic drug metabolism in the rat. V. Sulfation and acetylation.

A previously validated small mammal trauma model, hind-limb ischemia secondary to infrarenal aortic ligation in the rat, was utilized to investigate the effects of traumatic injury on sulfation and N-acetylation, two of the major phase II conjugative reactions of hepatic drug metabolism. Hepatic cytosolic sulfotransferase activity, as measured by several aryl and hydroxysteroid sulfation assays, was depressed by 20-29% after model injury. N-Acetyltransferase activity towards both isoniazid and p-aminobenzoic acid showed a similar 20-22% decrease. Moreover, as was previously observed with hepatic oxidative drug metabolism and with glucuronidation, the in vivo reactions of sulfation and acetylation were found to be significantly decreased by model trauma. In both cases, this effect on in vivo pharmacokinetics appeared to be correlated closely with trauma's influence on the conjugating enzymes, and relatively independent of the post-traumatic response of the necessary co-substrates. In vivo acetaminophen sulfation was depressed (24%) in parallel with hepatic sulfotransferase activity, despite an increased availability of sulfate for conjugation. Similarly, in vivo acetylation of isoniazid was diminished (23%) in conjunction with hepatic N-acetyltransferase activity, though hepatic acetyl-CoA content was elevated after model trauma. Thus, traumatic injury appears to have wide-ranging effects on a variety of determinants of hepatic drug metabolism.

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Effect of black tea drinking on blood lipids, blood pressure and aspects of bowel habit.

Thirty-one men (47 (SD 14) years) and thirty-four women (35 (SD 13) years) took part in a 4-week randomized cross-over trial to compare the effect of six mugs of black tea daily v. placebo (water, caffeine, milk and sugar) on blood lipids, bowel habit and blood pressure, measured during a run-in period and at the end of weeks 2, 3 and 4 of the test periods. Compliance was established by adding a known amount of p-aminobenzoic acid (PABA) to selected tea bags, and then measuring its excretion in urine. Mean serum cholesterol values during run-in, placebo and on tea drinking were 5.67 (SD 1.05), 5.76 (SD 1.11) and 5.69 (SD 1.09) mmol/l (P = 0.16). There were also no significant changes in diet, LDL-cholesterol, HDL-cholesterol, triacylglycerols, and blood pressure in the tea intervention period compared with placebo. Compared with placebo, stool consistency was softened with tea (P = 0.04), and no other differences were found in bowel habit. Results were unchanged when fifteen 'non-compliers', whose PABA excretion indicated that fewer than six tea bags had been used, were excluded from the analysis, and when differences between run-in and tea periods were considered separately for those who were given tea first or second.

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Acetylation of dapsone by human whole blood.

We studied the acetylation of dapsone (DDS) in vitro by whole blood taken from subjects with known acetylator phenotype. The acetylation of DDS by whole blood was both incubation time- and DDS concentration-dependent. Thus, it is highly recommended to separate plasma immediately after blood withdrawal during acetylation phenotyping using DDS. para-aminobenzoic acid (PABA) substantially inhibited the acetylation of DDS by whole blood taken from both slow and rapid acetylators, while procainamide (PAD) significantly inhibited DDS acetylation by whole blood taken from slow acetylators. At the highest PAD concentration used (208 microM), DDS acetylation by whole blood taken from rapid acetylators was also inhibited. In contrast, sulphanilamide (SAD) failed to produce any significant inhibition of the acetylation of DDS by whole blood taken from either slow or rapid acetylators. Furthermore, there was no correlation between DDS acetylation by whole blood in vitro and the acetylator status of the subject. It is therefore not possible to predict the acetylator phenotype by studying DDS acetylation by human whole blood. These results indicate that the DDS N-acetyltransferase of human whole blood is most probably of the monomorphic type.

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Intestinal changes in puppies infected with Toxocara canis.

Small intestinal histopathology and absorption were examined in Beagle puppies infected with either a moderate or a low burden of Toxocara canis. Infection with T. canis significantly reduced absorption of xylose, but only slightly delayed absorption of para-aminobenzoic acid. Fat assimilation was reduced and faecal proteolytic activity was increased. A significant reduction in villous height occurred and was inversely related to the extent of the infection. Villous goblet cell numbers, particularly those in the luminal third of the villus, were lowest and crypt goblet cell numbers were highest in the most heavily infected of the puppies. Villous goblet cell numbers increased rapidly after treatment of the puppies with piperazine or after the spontaneous elimination of the T. canis infection while crypt goblet cell numbers were less affected by elimination of the parasites. Intra-epithelial lymphocyte numbers were lowest in 33- to 37-day-old puppies infected with greater than 127 T. canis and highest in 44- to 46-day-old puppies losing their infection. Infection with T. canis had no apparent effect on mast cell numbers or pyroninophilic cell numbers in the lamina propria.

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Arylamine N-acetyltransferase in human red blood cells.

N-Acetyltransferase activities associated with erythrocytes from 20 individuals have been determined with p-aminobenzoic acid as substrate. A three-fold variation in Vmax is found. The N-acetyltransferase genotype of the individuals has been determined and there is no correlation between the extent of acetylation measured in the individuals' erythrocytes and the inheritance of alleles at the polymorphic NAT locus. Folate is confirmed to be an inhibitor of arylamine N-acetyltransferase activity measured in erythrocytes. The content of folate in erythrocytes of individuals also varies. The individual with the maximum folate content has the minimum N-acetyltransferase activity. The monomorphic N-acetyltransferase gene from individuals spanning the range of N-acetyltransferase activity have been amplified, using the polymerase chain reaction. The pattern of restriction enzyme digestion of the monomorphic N-acetyltransferase gene with a series of eight restriction enzymes is the same for individuals spanning the activity range of arylamine N-acetyltransferase in their erythrocytes.

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