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PubMed2002

Interactions between inducible nitric oxide synthase and heme oxygenase-1 in glomerulonephritis.

BACKGROUND: In anti-glomerular basement membrane (GBM) nephritis, inducible nitric oxide synthase (iNOS) and heme oxygenase (HO-1) are co-induced. Moreover, in glomerular mesangial cells iNOS-derived nitric oxide (NO) production stimulates HO-1 while HO-1 activation reduces iNOS expression/activity. These observations prompted us to explore regulatory interactions between iNOS and HO-1 in anti-GBM nephritis. METHODS: Rats with anti-GBM nephritis were pretreated with the iNOS inhibitor l-N6-(1-iminoethyl) lysine (L-NIL) or with the HO-1 inducer hemin. Glomerular HO-1 levels were assessed by Western blot analysis. iNOS activity was assessed by calculating conversion of l-arginine to l-citrulline. RESULTS: iNOS inhibition reduced glomerular HO-1 levels without altering the inflammatory response to anti-GBM antibody induced injury. Induction of HO-1 reduced glomerular iNOS activity. CONCLUSIONS: In anti-GBM nephritis iNOS up-regulates HO-1 presumably via high output NO production. Suprainduction of HO-1 attenuates iNOS activity. This negative feedback interaction points to HO-1 as a target for pharmacologic manipulation to reduce activity of prooxidant heme containing enzymes such as iNOS.

Animals↗
PubMed2001

The evolving technique of endovascular stenting of abdominal aortic aneurysm; time for reappraisal.

PURPOSE: to investigate the occurrence of intra-operative device-related complications during stent-grafting for abdominal aortic aneurysm. MATERIALS AND METHODS: data on patient characteristics, vascular morphology, operative technical details, procedural and device-related complications were obtained from the European collaborators on stent-graft techniques for abdominal aortic aneurysm repair (EUROSTAR) registry. Only intra-operative device-related complications were taken into account. Potential risk factors for device-related complications were examined by logistic regression analysis. The association between these complications and conversion to open surgery and death were determined. RESULTS: between January 1994 and July 2000, 2862 patients in 90 participating hospitals underwent endovascular abdominal aortic aneurysm repair. Device-related complications occurred in 238 (8.3%) patients. Complications were associated with the age of the patient (p=0.002), gender (p=0.05), smoking habit (p=0.001), pre-operative aneurysm diameter (p=0.005), type of device implanted (p=0.0001), fitness of the patient for open surgery (p=0.002), and year of operation (p=0.001). Adjusted for risk factors, the occurrence of complications decreased between 1994 to 2000 from 21.7% to 7.3%, respectively. Patients with device-related complications were 13.6 times (95% CI; 9.2-20.1) more likely to have conversion to an open procedure and 2.4 times (95% CI; 1.4-4.0) more likely to die within 30 days of the operation. CONCLUSIONS: intra-operative device-related complications were common, although appear to be decreasing in frequency, and were significantly related to conversion and post-operative death.

Aged↗
PubMed2001

Dynamic fatigue properties of the dental implant-abutment interface: joint opening in wide-diameter versus standard-diameter hex-type implants.

STATEMENT OF PROBLEM: The clinical long-term success of single-tooth implant restorations depends, in part, on a stable connection between the prosthetic restoration and the implant body. PURPOSE: The purpose of this experiment was to investigate the fatigue life of UCLA-style abutment screws in wide-diameter versus conventionally sized dental implant restorations. MATERIAL AND METHODS: Five 3.75 x 15-mm and five 6.0 x 15-mm hexed dental implants were used. Ten frameworks were fabricated, 5 with a single UCLA-style, 3.75-mm hexed gold alloy cylinder, and 5 with a single UCLA-style, 6.0-mm hexed gold alloy cylinder. To simulate a common laboratory procedure, 2 abutment interfaces were relieved with a one-quarter round bur for both diameters. The 3.75-mm implant used a Gold-Tite central abutment screw torqued to 32 Ncm, and the 6.0-mm implant used a titanium central abutment screw torqued to 25 Ncm. Frameworks were dynamically loaded ( approximately 10 Hz) with a 120 +/- 10-N, 4-mm off-axis force. Liquid metal strain gauges were used to measure joint opening. Measurements were made at intervals of 10(3), 10(4), 10(5), and 5x10(5) cycles. Gauge output data were converted to displacement with a conversion factor determined by calibration. Linear regression analysis then was performed. RESULTS: Two observations were made in this study. Two of three 3.75-mm nonadjusted specimens and all three 6.0-mm nonadjusted specimens maintained joint closure (range of opening 0-20 microm) while measured under dynamic loading. The median joint opening at 5x10(5) cycles for 3.75-mm nonadjusted specimens was 14 +/- 7 microm; for 6.0-mm specimens, it was 11 +/- 10 microm. Both 3.75-mm adjusted specimens and 1 nonadjusted specimen failed to maintain joint closure (excess joint opening >50 microm). One of the 3.75-mm adjusted specimens had abutment screw fracture. One of two 6.0-mm adjusted specimens failed to maintain joint closure because of screw fracture. CONCLUSION: The dental implant-abutment interface of 3.75-mm and 6.0-mm externally hexed implants experienced similar joint opening after periods of dynamic loading. Laboratory adjustment of the interface significantly decreased the service life of the abutment screw joint.

Analysis of Variance↗
PubMed2005

Conversion of nicotine to nornicotine in Nicotiana tabacum is mediated by CYP82E4, a cytochrome P450 monooxygenase.

Nornicotine is a secondary tobacco alkaloid that is produced by the N-demethylation of nicotine. Nornicotine production and accumulation in tobacco are undesirable because nornicotine serves as the precursor in the synthesis of the well characterized carcinogen N'-nitrosonornicotine during the curing and processing of tobacco. Although nornicotine is typically a minor alkaloid in tobacco plants, in many tobacco populations a high percentage of individuals can be found that convert a substantial proportion of the nicotine to nornicotine during leaf senescence and curing. We used a microarray-based strategy to identify genes that are differentially regulated between closely related tobacco lines that accumulate either nicotine (nonconverters) or nornicotine (converters) as the predominant alkaloid in the cured leaf. These experiments led to the identification of a small number of closely related cytochrome P450 genes, designated the CYP82E2 family, whose collective transcript levels were consistently higher in converter versus nonconverter tobacco lines. RNA interference-induced silencing of the CYP82E2 gene family suppressed the synthesis of nornicotine in strong converter plants to levels similar to that observed in nonconverter individuals. Although each of the six identified members of the P450 family share >90% nucleotide sequence identity, sense expression of three selected isoforms revealed that only one (CYP82E4v1) was involved in the conversion of nicotine to nornicotine. Yeast expression analysis revealed that CYP82E4v1 functions as a nicotine demethylase. Identification of the gene(s) responsible for nicotine demethylation provides a potentially powerful tool toward efforts to minimize nornicotine levels, and thereby N'-nitrosonornicotine formation, in tobacco products.

Base Sequence↗
PubMed1971

A bovine pancreatic enzyme catalyzing the conversion of proinsulin to insulin.

An enzyme that catalyzes the conversion of bovine proinsulin to insulin has been purified from a bovine pancreatic extract. The product of conversion was identified as insulin by aminoacid analysis and determination of carboxyl terminal aminoacid residues. The purified enzyme preparation showed one major protein band on polyacrylamide gel disc electrophoresis; it had a molecular weight of about 70,000 and an isoelectric point (pI) at a pH of 4.82.

Amino Acids↗
PubMed1989

Targeted homologous recombination at the endogenous adenine phosphoribosyltransferase locus in Chinese hamster cells.

We have developed a system that permits analysis of targeted homologous recombination at an endogenous, chromosomal gene locus in cultured mammalian cells. Using a hemizygous, adenine phosphoribosyltransferase (APRT)-deficient, Chinese hamster ovary (CHO) cell mutant as a transfection recipient, we have demonstrated correction of a nonrevertible deletion mutation by targeted homologous recombination. Transfection with a plasmid carrying a fragment of the APRT gene yielded APRT+ recombinants at a frequency of approximately 4.1 x 10(-7). The ratio of targeted recombination to nontargeted integrations of plasmid sequences was approximately 1:4000. Analysis of 31 independent APRT+ recombinants revealed conversions of the endogenous APRT gene, targeted integration at the APRT locus, and a third class of events in which the plasmid donor APRT fragment was converted to a full-length, functional gene.

Adenine Phosphoribosyltransferase↗
PubMed1993

On the directional specificity of ribosome frameshifting at a "hungry" codon.

Limitation for aminoacyl-tRNA promotes ribosome frameshifting at certain sites. We have previously demonstrated ribosome frameshifting to the right (3') at an AAG site in one context, and to the left (5') at an AAG site in a different context. Here, we demonstrate that the "rightwing" context is largely specific for frameshifting to the right, and the "leftwing" context is largely specific for frameshifting to the left. Analysis of these context rules, and the conversion of a sequence that promotes leftward frameshifting to one that promotes rightward frameshifting, demonstrated here, permits us to define a minimal heptanucleotide sequence sufficient for shiftiness in each direction at an AAG codon whose lysyl-tRNA is in short supply.

Amino Acid Sequence↗
PubMed2000

Reaction mechanism and stereochemistry of gamma-hexachlorocyclohexane dehydrochlorinase LinA.

gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) catalyzes the initial steps in the biotransformation of the important insecticide gamma-hexachlorocyclohexane (gamma-HCH) by the soil bacterium Sphingomonas paucimobilis UT26. Stereochemical analysis of the reaction products formed during conversion of gamma-HCH by LinA was investigated by GC-MS, NMR, CD, and molecular modeling. The NMR spectra of 1,3,4,5,6-pentachlorocyclohexene (PCCH) produced from gamma-HCH using either enzymatic dehydrochlorination or alkaline dehydrochlorination were compared and found to be identical. Both enantiomers present in the racemate of synthetic gamma-PCCH were converted by LinA, each at a different rate. 1,2,4-trichlorobenzene (1,2,4-TCB) was detected as the only product of the biotransformation of biosynthetic gamma-PCCH. 1,2,4-TCB and 1,2,3-TCB were identified as the dehydrochlorination products of racemic gamma-PCCH. delta-PCCH was detected as the only product of dehydrochlorination of delta-HCH. LinA requires the presence of a 1,2-biaxial HCl pair on a substrate molecule. LinA enantiotopologically differentiates two 1,2-biaxial HCl pairs present on gamma-HCH and gives rise to a single PCCH enantiomer 1,3(R),4(S),5(S),6(R)-PCCH. Furthermore, LinA enantiomerically differentiates 1,3(S),4(R),5(R),6(S)-PCCH and 1,3(R),4(S),5(S),6(R)-PCCH. The proposed mechanism of enzymatic biotransformation of gamma-HCH to 1,2,4-TCB by LinA consists of two 1,2-anti conformationally dependent dehydrochlorinations followed by 1,4-anti dehydrochlorination.

Bacterial Proteins↗
PubMedDate not supplied

Human nutritional supplements in the horse. Dehydroepiandrosterone versus androstenedione: comparative effects on the androgen profile and consequences for doping analysis.

Dehydroepiandrosterone (DHEA) and androstenedione are weak androgens, which need conversion to more potent testosterone in order to enhance anabolic action. Consequences of oral dosing at 1 mg/kg on the urinary and plasma androgen profile of mare and gelding have been evaluated with an analytical method involving conjugate fractionation and selective hydrolysis, group separation, and quantitation by gas chromatography-mass spectrometry with selected ion monitoring of trimethylsilyl ethers. Peak levels of testosterone total conjugates in urine (range 300-6000 microg/L) were attained a few hours after dosing. Renal clearance was fast, so the testosterone detection period lasted only 20 to 33 h, the longest time being generated by androstenedione. The urinary testosterone/epitestosterone ratio for detection of exogenous testosterone in the mare was inoperative after DHEA administration because there was a concomitant increase of epitestosterone, which thereby acted as a masking agent. Androstanediols and androstenediols, as well as some 17-ketosteroids, were additional markers. A transient increase of circulating free testosterone has been evidenced, and this would support possible anabolic/androgenic action by supplementation with DHEA and androstenedione along the oral route.

Adjuvants, Immunologic↗
PubMed2003

Release and interconversion of P2 receptor agonists by human osteoblast-like cells.

Nucleotides, acting as agonists at P2 receptors, are important extracellular signaling molecules in many tissues. In bone they affect both bone-forming osteoblast and bone-resorbing osteoclast cell activity. The presence of nucleotides in the extracellular microenvironment is largely determined by their release from cells and metabolism by ecto-enzymes, both of which have scarcely been studied in bone. We have investigated adenosine 5'-triphosphate (ATP) release from SaOS-2 osteoblastic cells and the activities of cell surface ecto-enzymes on ATP metabolism. ATP, but not LDH, was detected in SaOS-2 cell conditioned medium, suggesting these cells were actively releasing ATP. Introduction of ADP resulted in increased ATP concentrations in the medium, which was found not to be receptor mediated. Nucleotide inhibition and substrate specificity studies revealed an ecto-nucleoside diphosphokinase (ecto-NDPK) was responsible for the ADP-->ATP conversion; PCR and immunocytochemistry confirmed its presence. Analysis of ATP metabolism over time demonstrated overall ATP degradation was increased by inhibiting ecto-NDPK activity; confirming that the combined action of multiple osteoblast-expressed ecto-enzymes affected extracellular nucleotide concentration. The data establish the coexistence of ATP-consuming, and for the first time, ATP-generating activities on the osteoblast cell surface, the discovery of which has significant implications for studies involving P2 receptor subtypes in bone.

Adenosine Diphosphate↗
PubMed1990

Articulatory impairment associated with tardive dyskinesia.

The speech of two patients with tardive dyskinesia was studied, and one neuroleptic-treated patient having no signs of overt tardive dyskinesia served as control. A structured interview, including reading, repetition of sentences, and spontaneous conversation, was performed. A phonetic transcription and analysis of abnormal phonemes was done by a linguist under blind conditions. Both patients with tardive dyskinesia had abnormal phonemes whereas the control patient had none. These differences could not be explained by age, direct neuroleptic effect, or neuroleptic exposure time. The abnormal phonemes were all consonants. The authors conclude that tardive dyskinesia may cause articulatory communication problems.

Adult↗
PubMed2002

Assimilatory detoxification of herbicides by Delftia acidovorans MC1: induction of two chlorocatechol 1,2-dioxygenases as a response to chemostress.

Proteome analysis of bacteria that can detoxify harmful organic compounds enables the discovery of enzymes involved in the biodegradation of these substances and proteins that protect the cell against poisoning. Exposure of Delftia acidovorans MC1 to 2,4-dichlorophenoxypropionic acid and its metabolites 2,4-dichlorophenol and 3,5-dichlorocatechol during growth on pyruvate as a source of carbon and energy induced several proteins. Contrary to the general hypothesis that lipophilic or reactive compounds induce heat shock or oxidative stress proteins, no induction of the GroEL, DnaK and AhpC proteins that were used as markers for the induction of heat shock and oxidative stress responses was observed. However, two chlorocatechol1,2-dioxygenases, identified by amino terminal sequence analysis, were induced. Both enzymes catalyse the conversion of 3,5-dichlorocatechol to 2,4-dichloro-cis,cis-muconate indicating that biodegradation is a major mechanism of resistance in the detoxifying bacterium D. acidovorans MC1.

Betaproteobacteria↗
PubMed1986

Establishment and growth kinetics of the mutant Ehrlich-Lettré ascites cell strain HD33 in permanent suspension culture.

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 X 10(-5) of the explanted cells continued to grow in vitro. The resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. The corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. The duration of the G1 phase was effectively zero. Both PLM data and [3H]/[14C] thymidine double-labelling measurements revealed an S-phase duration of between 11 and 12 hr. The G2 phase lasted 3-5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.

Animals↗
PubMed1985

The effects of age and body composition on circulating serum oestrogens and androstenedione after the menopause.

Circulating levels of oestrone, oestradiol and androstenedione were measured in two large groups of postmenopausal women, in one group the women were between 46 and 56 years of age and in the second, older group they were 70 years of age. In addition the fat mass was calculated from the height, weight and age of the women. Serum concentrations of both oestrogens did not change with age, whereas the serum androstenedione concentration decreased significantly. A change in body composition included decreased height and increased fat mass in the older group. Serum concentrations of both oestrogens correlated significantly with the fat mass and serum androstenedione as well as with each other. From the correlation analysis it may be concluded that the conversion rate of androstenedione to oestrone, and of oestrone to oestradiol, increases with age, which presumably explains the unchanged concentrations of the circulating oestrogens in relation to postmenopausal age, although the precursor decreases during the same period.

Age Factors↗
PubMed1980

A caution in the use of tritiated substrates for monoamine oxidase assays.

A monoamine oxidase assay utilizing generally labeled [3H]serotonin as substrate became nonlinear after only approximately 5% conversion of initial c.p.m. to product. Subsequent analysis showed that a significant proportion of the tritium label was readily exchangeable into water and that monoamine oxidase activity increased release of label as water. The use of generally labeled substrates for oxidase activities is not recommended.

Animals↗
PubMed1994

A morphometric study of the effect of bilateral cervical sympathetic ganglionectomy on the architecture of pial arteries in spontaneously hypertensive and normotensive rats.

The influence of the cranial sympathetic nerves on the architecture of pial arteries in normo- and hypertension was examined. For this purpose the effect of bilateral superior cervical ganglionectomy was evaluated in normotensive rats (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP). The operations were performed at the age of 1 wk, which is just prior to the onset of ganglionic transmission. The length of the inner media contour was measured and the media cross-sectional area was determined planimetrically, with computerized digitalization of projected photographic images of transversely sectioned pial arteries. Four wk after sympathectomy there was a 20% reduction in media cross-sectional area and a consequent reduction in the ratio between media area and calculated luminal radius in the major pial arteries at the base of the brain in WKY but not in SHRSP. Conversely, in small pial arteries linear regression analysis showed that in WKY subjected to ganglionectomy the relationship between media cross-sectional area and luminal radius was significantly larger in arteries with a radius less than 21 microns compared to untreated WKY. No such effect was seen in the corresponding SHRSP vessels. In addition, the cross-sectional area of the internal elastic membrane (IEM) in the basilar arteries of WKY was measured by means of a computerized image-analysing system. Mean cross-sectional area of the IEM was approximately 45% larger following SE than in control animals. The present findings propose a 'trophic' role for the sympathetic perivascular nerves in large pial arteries of the rat. The increased media-radius ratio in the small pial arteries of the WKY following sympathectomy might reflect a compensatory hypertrophy due to reduced protection from the larger arteries against the pressure load. The inability to detect any morphometrically measurable effect of the sympathectomy in the cerebral arteries of SHRSP is probably explained by a marked growth-stimulating effect of the high pressure load in these animals.

Animals↗
PubMed2006

Selective metabolism of vincristine in vitro by CYP3A5.

Clinical outcomes of vincristine therapy, both neurotoxicity and efficacy, are unpredictable, and the reported pharmacokinetics of vincristine have considerable interindividual variability. In vitro and in vivo data support a dominant role for CYP3A enzymes in the elimination of vincristine. Consequently, genetic polymorphisms in cytochrome P450 (P450) expression may contribute to the interindividual variability in clinical response, but the contributions of individual P450s and the primary pathways of vincristine metabolism have not been defined. In the present study, vincristine was incubated with a library of cDNA-expressed P450s, and the major oxidative metabolites were identified. CYP3A4 and CYP3A5 were the only P450s to support substantial loss of parent drug and formation of the previously unidentified, major metabolite (M1). The structure of M1, arising as a result of an oxidative cleavage of the piperidine ring of the dihydro-hydroxycatharanthine unit of vincristine, was conclusively established after conversion to suitable derivatives followed by spectroscopic analysis, and a new pathway for vincristine metabolism is proposed. CYP3A5 was more efficient in catalyzing the formation of M1 compared with CYP3A4 (9- to 14-fold higher intrinsic clearance for CYP3A5). The formation of M1 was stimulated (3-fold) by the presence of coexpressed cytochrome b5, but the relative efficiencies of M1 formation by CYP3A4 and CYP3A5 were unaffected. Our findings demonstrate that in contrast to most CYP3A biotransformations, the oxidation of vincristine is considerably more efficient with CYP3A5 than with CYP3A4. We conclude that common genetic polymorphisms in CYP3A5 expression may contribute to the interindividual variability in the systemic elimination of vincristine.

Animals↗
PubMed1989

Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes.

Biochemical analysis of the enzymatic activity catalyzing the conversion of chorismate to isochorismate in the enterobactin biosynthetic pathway attributed the reaction to the isochorismate synthetase enzyme, designated EntC. However, the lack of mutations defining this activity has hampered the precise identification of the entC structural gene. In this study, we engineered a stable insertion mutation into the chromosomal region between the enterobactin genes fepB and entE. This mutation disrupted the structural gene for a previously identified 44-kilodalton protein and eliminated production of 2,3-dihydroxybenzoic acid, the catechol precursor of enterobactin. The complete nucleotide sequence of this gene was determined and compared with the sequences of other genes encoding chorismate-utilizing proteins. The similarities observed in these comparisons not only indicated that the locus is entC but also supported the premise that these enzymes constitute a family of related proteins sharing a common evolutionary origin. In addition, in this and the accompanying paper (M. S. Nahlik, T. J. Brickman, B. A. Ozenberger, and M. A. McIntosh, J. Bacteriol. 171:784-790, 1989), evidence is presented indicating that the entA product is potentially a secondary factor in the chorismate-to-isochorismate conversion and that the prototypic entC lesion (entC401) resides in the structural gene for the EntA protein. Finally, polarity effects from the insertion mutation in entC on downstream biosynthetic genes indicated that this locus is the promoter-proximal cistron in an ent operon comprising at least five genes. Appropriate regulatory signals upstream of entC suggest that this operon is regulated by iron through interaction with the Fur repressor protein.

Amino Acid Sequence↗