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H J Monstein

Publications and source records attributed to H J Monstein.

At least 37 records · Page 2Linked to original sources
PubMed1996

Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes.

Broad range PCR amplification and genus-specific 16S ribosomal DNA hybridization was used to demonstrate that Chlamydia, Helicobacter and Mobiluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR product obtained from pure cultures of the three genera. The sensitivity of the assay was determined by analysis of C. trachomatis serially diluted in urine. The detection limit was 1-10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S rRNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of Chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specimens using the C. trachomatis-specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other bacterial pathogens from the same PCR product by varying the hybridization probes.

Bacterial Typing Techniques↗
PubMed1996

Depletion of enterochromaffin-like cell histamine increases histidine decarboxylase and chromogranin A mRNA levels in rat stomach by a gastrin-independent mechanism.

BACKGROUND: Gastrin activates histidine decarboxylase (HDC) and increases HDC and chromogranin A (CGA) mRNA levels in histamine-producing enterochromaffin-like (ECL) cells in the rat stomach. We have studied how histamine depletion by subcutaneous infusion of the HDC inhibitor alpha-fluoromethyl-histidine (alpha-FMH) affects how ECL cells respond to hypergastrinemia in terms of HDC and CGA mRNA levels. METHODS: In one experiment rats received alpha-FMH for 24 h. In another experiment rats received alpha-FMH, omeprazole (perorally), or a combination of the two drugs for 10 days. In a third experiment antrectomized rats were treated with alpha-FMH for 48 h. The circulating gastrin level, oxyntic mucosal histamine concentration, HDC activity, and HDC and CGA mRNA levels were determined. RESULTS: alpha-FMH for 24 h increased the HDC and CGA mRNA levels without increasing the serum gastrin concentration. alpha-FMH for 10 days increased the serum gastrin concentration twofold. alpha-FMH + omeprazole resulted in the same serum gastrin concentration as after omeprazole alone (eightfold increase). HDC mRNA levels were higher after alpha-FMH + omeprazole than after omeprazole alone. alpha-FMH alone induced an HDC mRNA level that was similar in magnitude to that observed after omeprazole, although the serum gastrin concentration after alpha-FMH was much lower. In antrectomized rats alpha-FMH increased the HDC and CGA mRNA levels without increasing the serum gastrin concentration. CONCLUSION: ECL-cell histamine depletion will increase mRNA levels for HDC and CGA by a gastrin-independent mechanism, possibly involving abolished histamine autofeedback inhibition.

Animals↗
PubMed1996

Cholecystokinin-A and cholecystokinin-B/gastrin receptor mRNA expression in the gastrointestinal tract and pancreas of the rat and man. A polymerase chain reaction study.

BACKGROUND: Gastrin and cholecystokinin (CCK) are thought to exert trophic effects on the gastrointestinal tract and pancreas. Two types of receptors have been cloned, CCK-A and CCK-B/ gastrin. We have examined the occurrence of CCK-A and CCK-B receptor mRNA in the brain, digestive tract, pancreas, and kidney of the rat and man by Northern blot and reverse transcribed polymerase chain reaction (RT-PCR). METHODS: Total RNA was isolated from rat tissues and reverse transcribed into cDNA. cDNA from brain, kidney, and pancreas of the rat and man and from human whole stomach were commercially available. Northern blot and a PCR technique based on Taq polymerase-antibody interaction and using CCK-A and CCK-B receptor-specific primers, followed by Southern blot analysis, were the methods used. RESULTS: By means of Northern blots, CCK-A receptor mRNA was detected in rat fundus mucosa and pancreas but not in the remaining GI tract or brain. By means of RT-PCR, CCK-A receptor mRNA was demonstrated in the brain and the mucosa of the fundus, antrum, duodenum, and colon, kidney, pancreas and pancreatic islets. CCK-B receptor mRNA was detected by Northern blot analysis in the brain and the fundus mucosa but not in the rest of the digestive tract and not in the pancreas, pancreatic islets, or kidney. By RT-PCR, expression of CCK-B receptor mRNA could also be detected in antrum mucosa. In man, CCK-A receptor mRNA was detected in the brain, stomach, pancreas, and kidney, whereas CCK-B receptor mRNA was found in the brain, stomach, and pancreas but not in the kidney. Cloning and DNA-sequence analysis of the PCR-amplified rat and human CCK-A and CCK-B receptor DNA fragments, which cover the protein-encoding regions of the intracellular loop C3, showed complete sequence homology as compared with published rat and human sequences. CONCLUSIONS: It appears unlikely that CCK will have effects in the ileum, at least not effects mediated by CCK-A receptors. It also appears unlikely that physiologic concentrations of gastrin in the circulation will promote growth (or exert other effects) in the pancreas, duodenum, ileum, and colon, since CCK-B receptor mRNA is not expressed or is poorly expressed in these tissues.

Animals↗
PubMed1996

Rat stomach enterochromaffin-like cells are not stimulated by pylorus ligation. A biochemical and ultrastructural study.

BACKGROUND: The enterochromaffin-like (ECL) cells in the rat oxyntic mucosa produce histamine and contain cytoplasmic granules, microvesicles, and secretory vesicles. The cells respond to gastrin by the release of histamine (associated with loss of secretory vesicles), which is thought to mediate the gastrin-induced stimulation of the parietal cells. Gastric acid secretion is stimulated also by vagal excitation, which can be induced, for instance, by pylorus ligation. The present study addresses the question whether the ECL cells are involved in the acid response to pylorus ligation. METHODS: Rats were subjected to pylorus ligation and killed 4 or 16 h later. Other rats were subjected to sham operation (laparotomy). Some of the rats received human Leu15-gastrin-17 (5 nmol/kg/h) by intravenous infusion for 30 or 60 min before being killed. The serum gastrin concentration, the oxyntic mucosal histidine decarboxylase (HDC) activity, HDC mRNA concentration, histamine concentration, and gastric acid output were measured. Specimens from the oxyntic mucosa were processed for transmission electron microscopy. Electron micrographs of ECL cells were analyzed planimetrically. RESULTS: The gastric acid output was high, but the serum gastrin concentration was not affected by the pylorus ligation. The HDC activity and the level of HDC mRNA in the oxyntic mucosa were reduced, but the histamine concentration was unchanged. The secretory vesicles and granules of the ECL cells were unaffected, whereas the number and volume density of the microvesicles were reduced. Gastrin administration to sham-operated and pylorus-ligated rats lowered the oxyntic mucosal histamine concentration and increased the HDC activity in both groups. CONCLUSIONS: ECL cells in the rat stomach do not mediate the gastric acid response to pylorus ligation, and ECL cells in the pylorus-ligated stomach retain their ability to respond to gastrin with activation.

Animals↗
PubMed1995

Genomic structure of the gene encoding cionin--a cholecystokinin/gastrin like peptide.

Part of the gene encoding cionin from the protochordate Ciona intestinalis was amplified by polymerase chain reaction from genomic DNA employing synthetic oligonucleotides designed from the reported cDNA sequence. The nucleotide sequence of this gene revealed the presence of two introns of approximately 350 and 57 bp which interrupt the region that encodes pre-pro-cionin. A comparison of the positions of the exon/intron boundaries of the cionin and mammalian gastrin/cholecystokinin genes revealed a different exon/intron pattern. The 5'-donor and 3'-acceptor splice sites found in the cionin gene, however, are in agreement with the mammalian consensus sequence.

Animals↗
PubMed1995

Vitamin D3 effects on basal and cAMP modulated expression of cholecystokinin and somatostatin genes in a rat medullary thyroid carcinoma cell line [CA-77].

The regulation of cholecystokinin and somatostatin expression by vitamin D and cyclic AMP in the rat medullary thyroid carcinoma cell line CA-77 was investigated. Treatment with 100 nmol/l vitamin D did not affect cholecystokinin mRNA and peptide concentrations significantly; somatostatin mRNA level increased 6 times and the somatostatin peptide concentration increased 2-fold after 5 days of drug treatment. Under the same experimental conditions cyclic AMP increased cholecystokinin mRNA level 4.5 times and the cellular cholecystokinin-peptide concentration 2-fold; somatostatin mRNA and peptide concentrations were not significantly changed. Cyclic AMP stimulated peptide secretion from the cells were not affected by vitamin D, but cyclic AMP mediated increase in CCK peptide concentration was significantly inhibited by vitamin D (p < 0.05).

Amino Acid Sequence↗
PubMed1995

Broad-range PCR amplification and DNA sequence analysis reveals variable motifs in 16S rRNA genes of Mobiluncus species.

Using DNA primers based on highly conserved regions of bacterial 16S ribosomal RNA genes, a technique was established for detection of Mobiluncus species by polymerase chain reaction (PCR) and hybridization analysis. Part of the 16S rRNA genes of Mobiluncus mulieris, Mobiluncus curtisii and uncharacterized Mobiluncus strains were analyzed by broad-range PCR amplification and direct DNA sequencing analysis. Sequence comparison of the partial 16S rRNA genes of Mobiluncus curtisii, Mobiluncus mulieris and atypical Mobiluncus strains studied indicated genus and species-specific motifs within the variable regions V3, V4 and V9 of 16S ribosomal DNAs. A Mobiluncus curtisii-specific primer, located within the variable region V3 of the 16S rRNA gene, was designed for Southern blot hybridization analysis of broad-range PCR products. Broad-range amplification combined with a M. curtisii-specific hybridization probe, Mob V3, distinguished between Mobiluncus curtisii, Mobiluncus mulieris, and atypical Mobiluncus strains.

Base Sequence↗
PubMed1994

Acute responses of rat stomach enterochromaffinlike cells to gastrin: secretory activation and adaptation.

BACKGROUND/AIMS: Evidence for gastrin-induced histamine secretion from isolated rat enterochromaffinlike (ECL) cells was presented recently. We have investigated the gastrin-evoked secretory activation and adaptation of ECL cells in intact rats over a time span of a few minutes to several hours. METHODS: Fasted rats received a maximally effective dose of synthetic human Leu15-gastrin-17 by continuous intravenous infusion. ECL cell ultrastructure and ECL cell-related parameters (e.g., mucosal histamine and pancreastatin concentrations, histidine decarboxylase [HDC] activity, and messenger RNA [mRNA] concentration) were analyzed. RESULTS: Gastrin reduced the number of cytoplasmic vesicles in ECL cells while reducing the concentrations of histamine and pancreastatin in the oxyntic mucosa. The effects were maximal within a few hours after the start of gastrin infusion. The concentration of pancreastatin in serum was elevated for the duration of the study. The mucosal concentrations of histamine and pancreastatin returned to prestimulation values after 4-6 hours. The HDC activity and mRNA concentration increased progressively until after 6-8 hours of gastrin infusion. CONCLUSIONS: Gastrin promptly degranulates the ECL cells, releasing histamine and pancreastatin from the vesicles. Synthesis of histamine and pancreastatin is accelerated, a process associated with renewal of vesicles. The increase in HDC activity and mRNA concentration continues for several hours after restoration of the vesicles.

Adaptation, Physiological↗
PubMed1993

cDNA deduced procionin. Structure and expression in protochordates resemble that of procholecystokinin in mammals.

Using an improved 3' RACE (PCR) amplification system containing oligonucleotide primer with an inosine at ambiguous codon positions and inverse PCR to amplify the 5' ends, we have isolated and characterized cDNA clones which encode cionin, a protochordean homologue of the mammalian hormones, cholecystokinin (CCK) and gastrin. The full-length cloned cDNA of 510 bp encoded a 128 amino acid preprocionin. Reverse transcription-PCR and subsequent cDNA cloning revealed that cionin mRNA is expressed in both the neuronal ganglion and the gut of the protochordate Ciona intestinalis. The primary structure of procionin resembles that of proCCK more than that of progastrin. Sequence-specific immunochemical analysis showed that the cionin gene is expressed also at peptide level in both the gut and the neural ganglion. The neuronal processing of procionin is, however, more complete both with respect to carboxyamidation and tyrosine O-sulfation. Hence, the tissue-specific expression of the cionin gene in Ciona intestinalis resembles that of the CCK gene in mammals.

Amino Acid Sequence↗
PubMed1993

Modulated cholecystokinin (CCK) mRNA expression is dependent on de novo protein synthesis.

The human neuroblastoma cell line SK-N-MC expresses cholecystokinin (CCK) and c-fos mRNA. Levels of CCK and c-fos mRNA expression were studied by nuclear run-off transcription and Northern blot analysis. Addition of foetal calf serum or forskolin to the medium stimulated basal CCK and c-fos mRNA expression. Addition of cycloheximide suppressed the stimulation of CCK mRNA expression, whereas expression of c-fos mRNA was super-induced by the simultaneous addition of cycloheximide and forskolin or foetal calf serum. Western blot analysis revealed that c-fos mRNA is translated into Fos. Expression of c-fos in SK-N-MC cells suggests that Fos may be part of a functional transcription complex and that it may be involved in the regulation of basal and stimulated CCK mRNA expression in SK-N-MC cells.

Base Sequence↗
PubMed1993

Identification of an AP-1 transcription factor binding site within the human cholecystokinin (CCK) promoter.

Neuropeptide gene expression, i.e. proenkephalin A, may be modulated by activating protein (AP-1) transcription factor complexes which associate with specific DNA sequence motifs, known as the AP-1 binding sites. Gel mobility shift assays revealed that nuclear extracts prepared from a human neuroblastoma cell-line, SK-N-MC, bind to the 5'-CTGCGTCAGCG-3' motif, present within the human cholecystokinin (CCK) promoter. In contrast, the mutated 5'-CTGCAACAGCG-3' motif did not bind any specific protein complex. In vitro run-off transcription and Western blot analysis revealed the expression of the protooncogenes Fos and Jun. This suggests that Fos/Jun homo- or heterodimeric complexes may interact with the 5'-CTGCGTCAGCG-3' motif, present within the human CCK promoter.

Actins↗
PubMed1993

Peptide hormone processing in tumours: biogenetic and diagnostic implications.

Insight into the biogenesis of peptide hormones has grown explosively by elucidation of gene, mRNA and prohormone structures. In addition, information about prohormone processing enzymes is rapidly accumulating. Prohormones vary in size and organization from poly- to monoprotein structures. According to their structural organization and sequence homology, hormones are grouped in families. Prohormones are processed to bioactive peptides by multiple modifications during the transport from the endoplasmic reticulum to secretory granules. The modifications comprise different proteolytic cleavages and amino acid derivatizations. By constitutive secretion, the processing is less pronounced. The same prohormone may be expressed in several cell types that process the precursor in different ways. Awareness of cell-specific processing patterns is important for understanding the tumour synthesis of peptides and for appropriate diagnosis of peptide-producing tumours. These tumours comprise not only well-known neuroendocrine neoplasias. An increasing number of common carcinomas also expresses peptide hormone genes. However, the translation and post-translational processing in tumours are generally attenuated. Consequently, the expression is often functionally and clinically silent. A new diagnostic tool, processing-independent analysis (PIA), seems promising in quantitation of hormone gene expression at peptide level irrespective of the degree of processing. Studies of progastrin expression and processing in tumours illustrate the diagnostic superiority of PIA.

DNA, Complementary↗
PubMed1992

Expression, but failing maturation of procholecystokinin in cerebellum.

The cerebellum is the only region of the central nervous system which has been found to be devoid of cholecystokinin (CCK). The assays used, however, have been directed against the alpha-amidated C-terminus of fully processed CCK peptides. Using Northern blot analysis and a library of radioimmunoassays specific for different sequences of proCCK in combination with chromatography and enzyme cleavage, we have now examined the expression and processing of proCCK in fetal, neonatal and adult cerebellar tissue from man, pig and rat. In rat cerebellum CCK mRNA was present already in the fetal state. Two weeks after birth the concentrations declined. Also proCCK was found in significant concentrations in the fetal human and rat cerebellum (approximately 20 pmol/g); but already before birth the expression began to decrease towards low concentrations in adults. The adult porcine cerebellum contained 3.2 pmol proCCK and glycine-extended processing intermediates per gram (range less than 0.1-10.4 pmol/g), and 0.8 pmol carboxyamidated CCK per gram (range 0.1-4.1 pmol/g) varying in size from CCK-58 to CCK-5. For comparison, the adult porcine cerebral cortex contained 757 pmol carboxyamidated CCK/g, 20 pmol glycine-extended CCK/g and no proCCK. We conclude that cerebellum expresses proCCK with the highest level of expression in fetal life. In comparison with other regions of the brain, the maturation to transmitter-active, carboxyamidated CCK peptides is, however, attenuated in both fetal and adult cerebellar tissue.

Aging↗
PubMed1992

Cloning and expression of two different genes from Streptococcus dysgalactiae encoding fibronectin receptors.

Binding of bacteria to fibronectin has been implicated as a mechanism of bacterial adhesion to the host tissue. In this report we have analyzed the binding of a strain of Streptococcus dysgalactiae to fibronectin. The cells bind to a site in the NH2-terminal domain of the protein via trypsin-sensitive cell surface components. Furthermore, a lysate prepared by sonication of streptococcal cells contained fibronectin-binding proteins that inhibit the binding of the ligand to intact bacteria. When the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to an Immobilon-P filter, and probed with 125I-labeled fibronectin, a 140-kDa fibronectin-binding protein was identified along with a number of smaller binding proteins. A genomic DNA library was constructed and screened for the expression of fibronectin-binding proteins. Two clones were isolated and shown to contain unrelated inserts by restriction mapping and cross-hybridization experiments. The two encoded proteins were also immunologically distinct although both bound to the same region of the fibronectin molecule, and both effectively inhibited the binding of 125I-fibronectin to bacterial cells. Immunological analyses showed that only one of the two proteins tentatively identified as fibronectin receptors was expressed in detectable quantities in the Streptococcus dysgalactiae strain under the culture conditions employed.

Bacterial Adhesion↗
PubMed1992

Modulation of cholecystokinin (CCK) gene-expression in a human neuroblastoma cell line: effects of serum on enhanced CCK and c-fos mRNA expression.

The effect of fetal-calf serum (FCS) and Forskolin (FKN) on cholecystokinin (CCK) and proto-oncogene c-fos mRNA expression in the human neuroblastoma cell line SK-N-MC, cultured in serum free medium was studied by Northern blot analysis and nuclear run-off transcription analysis. Addition of FCS or FKN gradually increased the basal CCK mRNA level approximately four to six-fold after 2-4 h. In contrast, a strong and transient increase of the c-fos mRNA-level was observed, approximately forty to fifty-fold after 50-60 min over unstimulated cells. Nuclear run-off transcription analysis indicates that c-fos mRNA is constitutively expressed and transcription may be further stimulated by FCS and FKN. Moreover, in SK-N-MC nuclei, transcription of the c-fos gene clearly precedes stimulated CCK-mRNA expression. This suggests that FOS, which is known to form a AP-1 heterodimer transcription complex with the proto-oncogen product, Jun, may bind to the tentative AP-1 binding site, found within the human CCK promoter and thereby modulates basal and enhanced CCK-mRNA expression in SK-N-MC cells.

Base Sequence↗