Search PubMed⌕ Search

Biomedical subjects

M Blum

Publications and source records attributed to M Blum.

At least 181 records · Page 10Linked to original sources
PubMed1991

Acetylation of serotonin in the rabbit pineal gland: an N-acetyltransferase with properties distinct from NAT1 and NAT2 is responsible.

Two rabbit arylamine N-acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, p-aminosalicylic acid, and p-aminobenzoic acid. In the pineal gland, serotonin is metabolized to N-acetylserotonin by an unknown N-acetyl-transferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N-acetyltransferase (AA-NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as p-phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N-acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross-hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA-NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N-acetylation of serotonin.

Acetylation↗
PubMed1991

Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain.

The rat preoptic area-anterior hypothalamic continuum (POA-AH) contains about 400-800 neurons that express the decapeptide GnRH and the 56-amino-acid GnRH-associated peptide. Originating from the olfactory placode, these neurons migrate and establish their final distribution and connections in the POA-AH several days before birth. The aim of the present study was to examine whether the biosynthesis of the mRNA encoding the precursor (proGnRH) common to GnRH and GnRH-associated peptide undergoes postnatal changes corresponding to the development of sexual maturation. The POA-AH content of proGnRH messenger RNA (mRNA) was followed from postnatal day 1 to day 90 in female and male Sprague-Dawley rats killed by decapitation between 1000-1200 h. Cytoplasmic RNA fractionated from individual POA-AH homogenates was purified using proteinase K digestion. Cytoplasmic proGnRH mRNA was quantitated simultaneously with cyclophilin mRNA (an internal standard control) using solution hybridization-RNase protection assay, with the protected fragments separated through polyacrylamide gel electrophoresis. In the POA-AH, the concentrations of proGnRH mRNA (femtograms mRNA per microgram total RNA) increased significantly with age in both sexes (P less than 0.001). In males, proGnRH mRNA levels increased by day 30 some 2-fold over the values of days 5 and 10, and the levels established on day 30 were maintained through adulthood. In females, the first rise in proGnRH mRNA levels occurred on day 30, followed by an additional increase on day 45 to levels seen in adulthood. Levels of proGnRH mRNA established in adulthood were significantly higher in females than in males (P less than 0.03). The concentrations of cyclophilin mRNA (picograms mRNA per microgram total RNA) remained essentially unchanged in both sexes during the same period of time when proGnRH mRNA levels were increasing. These results provide evidence for postnatal sex-related increases in the levels of proGnRH mRNA in the rat POA-AH, which are likely to reflect differential regulation by gonadal steroids.

Amino Acid Isomerases↗
PubMed1991

Adrenocorticotropin-releasing factor down-regulates glucocorticoid receptor expression in mouse corticotrope tumor cells via an adenylate cyclase-dependent mechanism.

Anterior pituitary POMC transcription and peptide release are negatively regulated by glucocorticoids and stimulated by CRF. Although pretreatment of corticotrope cells with CRF markedly inhibits subsequent glucocorticoid effects, the mechanism of this action is unclear. We have thus used a mouse corticotrope tumor (AtT20) cell line, to examine the effects of CRF on glucocorticoid receptor (GR) messenger RNA levels and on GR capacity/nuclear translocation. GR mRNA levels were measured by solution hybridization/S1 nuclease protection, and both total cell binding and nuclear binding were determined with [3H]dexamethasone ([3H]DEX). CRF treatment of AtT20 cells led to a rapid time-dependent decrease in GR mRNA levels which preceded a dose- and time-dependent decrease in GR binding capacity. Scatchard analysis showed a single class of high affinity binding sites (GR) in both control and CRF-treated cultures, and a decrease in the total number of GR after CRF treatment. The relative proportion of nuclear vs. cytoplasmic localized [3H]DEX-bound GR did not differ between control and CRF-treated cultures, indicating that CRF does not interfere with GR nuclear translocation. To investigate whether CRF regulates GR expression through the adenylate cyclase system, as it does POMC, AtT20 cells were treated with either forskolin or 8-bromo-cAMP, and specific nuclear GR binding was determined. Both drugs mimic the CRF-induced decrease in GR binding, and in addition forskolin decreased GR mRNA levels; in contrast, forskolin had no effect on GH3 cell GR levels. These results suggest that CRF can decrease the cellular concentration of GR, and thus potentially the response to glucocorticoids, through the same mechanism by which it stimulates anterior pituitary POMC expression.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism.

The debrisoquine/sparteine polymorphism is associated with a clinically important genetic deficiency of oxidative drug metabolism. From 5% to 10% of Caucasians designated as poor metabolizers (PMs) of the debrisoquine/sparteine polymorphism have a severely impaired capacity to metabolize more than 25 therapeutically used drugs. The impaired drug metabolism in PMs is due to the absence of cytochrome P450IID6 protein. The gene controlling the P450IID6 protein, CYP2D6, is located on the long arm of chromosome 22. A pseudogene CYP2D8P and a related gene CYP2D7 are located upstream from CYP2D6. This gene locus is highly polymorphic. After digestion of genomic DNA with XbaI endonuclease, restriction fragments of 11.5 kb and 44 kb represent mutant alleles of the cytochrome CYP2D6 gene locus associated with the PM phenotype. In order to elucidate the molecular mechanism of the mutant allele reflected by the XbaI 11.5-kb fragment, a genomic library was constructed from leukocyte DNA of one individual homozygous for this fragment and screened with the human IID6 cDNA. The CYP2D genes were isolated and characterized by restriction mapping and partial sequencing. We demonstrate that the mutant 11.5-kb allele results from a deletion involving the entire functional CYP2D6 gene. This result provides an explanation for the total absence of P450IID6 protein in the liver of these PMs.

Base Sequence↗
PubMed1991

[Activities of a family planning clinic incorporated to a public medical service over a 4-year period].

Over a period of four years, 1769 women, all of whom were married with at least 2 children, agreed to use an intra-uterine contraceptive device (IUD), 1199 women (predominantly unmarried) preferred to use an oral contraceptive (in most cases, two- or three-phase, low-estrogen pills) and 62 a local contraceptive (spermicide or diaphragm). The period of use was 89.65% for the IUDs, with a single case of acute inflammation of the genital tract. As expected, we diagnosed a large number of cases of asymptomatic Chlamydia trachomatis infections in 24% of the women using oral contraceptives and in 15% of the women fitted with an IUD. Symptomatic cases took the form of abdominal discomfort, vaginal discharge and metrorrhagia. In the opinion of the authors, the successful results obtained were due to the cooperation between the GP and the gynecologist and to a clear and firm policy towards the choice of the optimum method of contraception. In this article, the authors describe the principles behind this policy.

Adolescent↗
PubMed1991

Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes.

A genetic polymorphism of human liver arylamine N-acetyltransferase (NAT; EC 2.3.1.5) enzyme activity divides populations into distinguishable "slow acetylator" and "rapid acetylator" phenotypes. Two human genes, NAT1 and NAT2, encoding NAT proteins [DNA Cell Biol. 9:193-203 (1990)] were transiently expressed in cultured monkey kidney COS-1 cells, and the resulting recombinant NAT1 and NAT2 proteins were compared with N-acetyltransferase activities in human liver cytosol with respect to their stability, chromatographic behavior on anion exchange columns, electrophoretic mobility, and arylamine acceptor substrate specificity. NAT1 was far less stable in vitro than NAT2. Under conditions designed to optimize enzyme stability, anion exchange chromatography experiments revealed that enzymes corresponding to both recombinant NAT1 and NAT2 were expressed in human liver. Recombinant and human liver NAT1 enzymes showed the same characteristic selectivity (low apparent Km, high Vmax) for the "monomorphic" substrates p-aminosalicylic acid and p-aminobenzoic acid. Such substrates fail to discriminate between the acetylator phenotypes in vivo. The same criteria established that recombinant NAT2 was indistinguishable from one of two previously observed N-acetyltransferases (NAT2A and NAT2B) whose liver contents correlate with acetylator phenotype in human populations. Recombinant NAT2 and the liver NAT2 isoforms NAT2A and NAT2B selectivity N-acetylated the "polymorphic" substrates sulfamethazine and procainamide, whose disposition in vivo is affected by the acetylation polymorphism. Interestingly, the carcinogen 2-aminofluorene was very efficiently metabolized by both NAT1 and NAT2. Independent regulation of NAT1 and NAT2 genes was suggested by a lack of correlation of NAT1 and NAT2 enzyme activities in cytosols from 39 human livers. The results provide strong evidence that the NAT2 locus is the site of the human acetylation polymorphism. In addition, the use of recombinant NAT1 and NAT2 will allow us to predict whether any given arylamine will be polymorphically acetylated in humans.

Acetylation↗
PubMed1991

[Indications, incidence of recurrence and late results after selective vagotomy].

At the Clinic and Polyclinic for General Surgery of the Westfälische Wilhelms Universität Münster a total of 125 patients were vagotomised because of chronic duodenal ulcers during the period from 1. 1. 1973-31. 12. 1988. The case histories were analysed retrospectively. Standardised questionnaires were sent to the patients in order to gain more information about the post-operative progress and about possible complications after vagotomy had been carried out. 12.2% of the patients questioned developed a recidive ulcer 1-16 years after the operation which resulted in 8 cases in a Billroth II gastrectomy with Y-anastomosis according to Roux. 7 of the 9 recidives occurred during the first two post-operative years. The average time before relapse occurred amounts to 1.7 years in spite of drug treatment. As the initial operation was performed more than 6 years ago in 88.1% of the patients and in this respect later occurring recidives could have been detected, it is to be assumed, that relapses manifest themselves, if at all, relatively soon after the initial operation. The surgical therapy of the recidive ulcer by means of B II gastrectomy was associated with a favourable prognosis; all patients were able to resume work. A comparative analysis of the later results in 67 patients of various age structures showed that with increasing age the rate of patients who considered the operation as successful decreased. Of those up to 25 years 66.7% considered themselves as recovered, of those between 41-45 years 22.2%. It can be concluded that a selective proximal vagotomy represents a curative method in the case of chronic relapsing uncomplicated duodenal ulcers. Satisfactory results could be obtained especially in young patients who develop temporary stress ulcers. Good long-term results can be obtained here, despite the high relapse rates described in our own collective of patients as well as in literature.

Adult↗
PubMed1990

A ligand-induced, temperature-dependent conformational Change in penicillopepsin. Evidence from nonlinear Arrhenius plots and from circular dichroism studies.

The effect of temperature on the rate constants of hydrolysis of various substrates by penicillopepsin is dependent on the length of the substrate. For the series Ac-(Ala)m-Lys-Nph-(Ala)n-amide (where Ac- is acetyl- and Nph- is p-nitrophenylalanyl-), where m and n = 0-2, substrates lacking both P'2 and P3 residues give linear Arrhenius plots with an energy of activation of about 55 kJ.mol-1. The Arrhenius plots of substrates in which an alanine residue occupies P'2 show a sharp break at an average transition temperature of 10.5 degrees C. The activation energies are approximately 90 kJ.mol-1 below and approximately 54 kJ.mol-1 above the transition temperature, respectively. For substrates in which P3 is occupied, the average transition temperature is 14.2 degrees C. In this case, the activation energies are 66 kJ.mol-1 below and from 26 to 39 kJ.mol-1 above the transition point. The most probable explanation of these phenomena is that substrate interaction at subsites S3 and/or S'2 of the enzyme induces a temperature-dependent conformational change. Physical evidence for this comes from the observation that the temperature dependence of a CD absorption band at 242 nm of a penicillopepsin-pepstatin complex shows a sharp break that corresponds to those observed in the Arrhenius plots of substrates with alanine at P'2 and P3, whereas the same CD band in the free enzyme is linearly dependent on temperature.

Amino Acid Sequence↗
PubMed1990

The Alzheimer's amyloid precursor protein is produced by type I astrocytes in primary cultures of rat neuroglia.

The production of beta-amyloid precursor protein (beta APP) by primary mixed glial cell cultures from cerebral cortex of 2-3 day postnatal rats was examined by Northern and Western blotting and by immunocytochemistry. A single 3.5 kb beta APP RNA transcript was detected using probes recognizing all forms of beta APP messenger RNA. No signal was detected with a probe specific for the alternately spliced Kunitz protease inhibitor (KPI) region. In Western blot analysis of protein extracts, antisera specific for either the cytoplasmic or extracellular parts of beta APP detected several proteins ranging from 105 to 140 kDa. None of these were recognized by an antiserum specific to the KPI insert of the beta APP. Multicolor immunofluorescence showed beta APP immunoreactivity in type I (GFAP+ A2B5-) astrocytes, distributed in a fibrillar pattern like that of glial fibrillary acidic protein (GFAP). No beta APP immunoreactivity was detected in oligodendrocytes (GC+) or in A2B5+ progenitor cells. Moreover, no cultured cells showed immunostaining with an antiserum specific for the KPI sequence of beta APP. We conclude that type I astrocytes in primary culture produce amyloid precursor protein, but that oligodendrocytes and their precursors do not. Moreover, type I astrocytes produce predominantly the beta APP subtypes which lacks the KPI sequence.

Amyloid↗
PubMed1990

The correlation between Chlamydia antigen, antibody, vaginal colonization and contraceptive method in young unmarried women.

In a group of 55 unmarried women, mean age 25 years, attending a family planning clinic and having minor gynecological complaints, the correlation between Chlamydia trachomatis (CT) antigen, CT antibodies, vaginal colonization by Candida or bacteria and the method of contraception was investigated. The correlation between CT antigen and CT antibodies (IgG) was significant in oral contraceptive users (p = 0.003), as was the correlation with vaginal colonization by Candida and potential pathologic bacteria. In the group using the natural family planning method, a statistically significant correlation was found between CT antigen, IgG (p = 0.002), IgA (p = 0.02) antibodies, and vaginal candidiasis (p = 0.002), but not with bacterial colonization (p = 0.90). The discrepancy between CT antigen and antibodies is discussed. Differences in the prevalence of Chlamydia trachomatis infection were found among groups using different birth control methods, indicating an association between Chlamydia infection and the contraceptive method used.

Adolescent↗
PubMed1990

Can children use a verb without exposure to its argument structure?

We hypothesize that canonical sentence schemas (e.g. Agent-verb-Patient) can sometimes assign argument structure to verbs. In particular, they provide a default argument structure early in learning when a verb's lexical entry may record the nature of the action but lack a specific argument structure. To test the theory and its application to causative verb errors (e.g. stay it there), novel action verbs were modelled, some as causative, some as intransitive, and some unmarked for transitivity. Spontaneous usage was recorded, along with responses to agent-questions ('What is the [Agent] doing?') and patient-questions ('What is the [Patient] doing?'). Comparable data were obtained for familiar English verbs, some of fixed and some of optional transitivity. Subjects were willing to use all novel verbs both transitively and intransitively, although adults respected assigned transitivity more than children. All subjects largely respected the transitivity of familiar verbs. The discourse pressure of the agent- and patient-questions greatly affected observed transitivity. No evidence was found for the intransitive-to-causative derivational process postulated by Bowerman. We propose that the kind of causativity error observed by Bowerman is due to assignment of argument structure from canonical sentence schemas, especially under pressure of a need to make a sentence with a particular argument (Agent or Patient) as subject. The theory has the advantage of explaining errors without postulating the acquisition of erroneous lexical entries that have to be unlearned, and it can be extended to other kinds of errors in the choice and placement of arguments.

Adult↗
PubMed1990

Purification of an inhibitor of erythroid progenitor cell cycling and antagonist to interleukin 3 from mouse marrow cell supernatants and its identification as cytosolic superoxide dismutase.

We have isolated a protein from media conditioned by a murine marrow-derived cell line (PB6) and from mouse marrow supernatants that antagonizes interleukin 3-dependent proliferation of cells in culture and reversibly inhibits DNA synthesis of erythroid progenitor cells (BFU-E) in vitro. This protein, p16 (monomer Mr = 16 kD on SDS-PAGE), was purified to homogeneity and amino acid sequencing of a polypeptide fragment yielded a sequence identical to that of murine cytosolic Cu,Zn-containing superoxide dismutase (SOD). The identification of p16 as SOD was confirmed by the detection of SOD enzymatic activity in pure p16 fractions, and when a commercial human erythrocytic SOD preparation was tested it showed the same cell inhibitory activities as p16. These observations show that superoxide dismutase is able to affect the cycling and growth factor responses of hematopoietic cells, activities that have not previously been associated with this enzyme.

Amino Acid Sequence↗
PubMed1990

Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression.

N-Acetylation by hepatic arylamine N-acetyltransferase (NAT, EC 2.3.1.5) is a major route in the metabolism and detoxification of numerous drugs and foreign chemicals. NAT is the target of a common genetic polymorphism of clinical relevance in human populations. We have used our recently isolated rabbit cDNA rnat to clone three human NAT genes from human leukocyte DNA. None of the three genomic coding sequences was interrupted by introns. Two genes, designated NAT1 and NAT2, each possessed open reading frames of 870 bp. Both genes have been assigned to human chromosome 8, pter-q11. Following transfection they were transiently expressed in monkey kidney COS-1 cells. NAT1 and NAT2 gave rise to functional NAT proteins, as judged by their NAT enzyme activity with the arylamine substrate sulfamethazine. Western blots with NAT-specific antisera detected proteins of apparent molecular weight of 33 and 31 kD in NAT1- and NAT2-transfected cultures, respectively. The product of NAT2 had an identical apparent molecular weight as that of NAT detected in human liver cytosol. The deduced amino acid sequence of NAT2 also contained 6 peptide sequences which had previously been determined from tryptic peptides of the polymorphic NAT purified from human liver. These data suggest that NAT2 encodes the polymorphic NAT protein. The third gene, NATP, had multiple deleterious mutations and did not encode a functional NAT protein; it most likely represents a pseudogene.

Acetyltransferases↗
PubMed1990

Corticotropin-releasing factor differentially regulates anterior and intermediate pituitary lobe proopiomelanocortin gene transcription, nuclear precursor RNA and mature mRNA in vivo.

The proopiomelanocortin (POMC) gene and its peptide products are under complex regulation in the pituitary by multiple hormonal, neurohormonal and neurotransmitter factors. Corticotropin-releasing factor (CRF) stimulates the release of POMC-derived peptides in both anterior and intermediate lobes of the pituitary, while having differential long-term effects on levels of POMC mRNA in the two pituitary lobes in vivo. In the present study, we have analyzed the release of POMC-derived peptides, as well as changes in POMC gene transcription, primary transcript, nuclear mRNA and cytoplasmic mRNA levels following acute and chronic in vivo CRF administration in an attempt to elucidate the mechanism whereby this tissue-specific differential regulation occurs. Subcutaneous injection of CRF led to a substantial increase in plasma adrenocorticotropin, but only a minor sustained rise in plasma alpha-melanocyte-stimulating hormone. CRF was shown to induce rapid, time-dependent increases in POMC gene transcription in both anterior and intermediate pituitary lobes, which were reflected by increases in the level of POMC primary transcript in the nucleus. POMC primary transcript remained 2-fold elevated in the anterior lobe for at least 4 h after a single injection of CRF; in contrast, CRF stimulation of POMC primary transcript in the intermediate lobe was short-lived, and had returned to control values by 60 min after injection. After 7 days of repetitive CRF administration, POMC primary transcript, mature nuclear mRNA and cytoplasmic mRNA were 2.5 to 3.0-fold elevated in the anterior pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1990

Biphasic effect of orchiectomy on pro-opiomelanocortin gene expression in the hypothalamus.

Previous studies have shown that the hypothalamic concentration of pro-opiomelanocortin (POMC) mRNA and several POMC-derived peptides increases in the rat 4 weeks after orchiectomy and that this increase can be prevented by testosterone replacement. In this study, we have examined the short-term effects of orchiectomy on POMC gene expression in the medial basal hypothalamus (MBH). Adult male rats were studied at various time points between 1 day and 4 weeks after orchiectomy and compared to sham-orchiectomized rats. The MBH was homogenized and, after an aliquot was removed for beta-endorphin (beta-EP) radioimmunoassay, the RNA was isolated and the amount of POMC mRNA was measured using a solution hybridization S1 nuclease protection assay. In the first experiment, POMC mRNA was significantly higher 4 weeks after orchiectomy compared to that of the intact controls: 1.34 +/- 0.14 vs. 0.86 +/- 0.04 pg/micrograms RNA (p less than 0.01). Three days after orchiectomy, POMC mRNA was somewhat lower, 0.71 +/- 0.06 pg/micrograms RNA, but not significantly different from the controls. In a second experiment, POMC mRNA levels were measured 1, 2, 3, 4 and 7 days after orchiectomy. At 1 and 2 days after orchiectomy, POMC mRNA was lower than the controls: 0.51 +/- 0.02 and 0.52 +/- 0.06 vs. 0.70 +/- 0.09 pg/micrograms RNA. Levels then steadily increased to 0.61 +/- 0.04, 0.70 +/- 0.09 and 0.78 +/- 0.11 pg/micrograms RNA at 3, 4 and 7 days after orchiectomy, respectively. The mean level at 1-2 days after orchiectomy was significantly less than the controls (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗