Biomedical subjects

O Madsen

Publications and source records attributed to O Madsen.

27 records · Page 2Linked to original sources

PubMed1990

Effect of 5'-flanking sequence deletions on expression of the human insulin gene in transgenic mice.

Expression of the human insulin gene was examined in transgenic mouse lines carrying the gene with various lengths of DNA sequences 5' to the transcription start site (+1). Expression of the transgene was demonstrated by 1) the presence of human C-peptide in urine, 2) the presence of specific transcripts in pancreas, but not in other tissues, 3) the specific immunofluorescence staining of pancreatic islets for human C-peptide, and 4) the synthesis and accumulation of human (pro)insulin in isolated islets. Deletions in the injected DNA fragment of sequences upstream from positions -353, -258, and -168 allowed correct initiation of the transcripts and cell specificity of expression, while quantitative expression gradually decreased. Deletion to -58 completely abolished the expression of the gene. The amount of human product that in mice harboring the longest fragment contributes up to 50% of the total insulin does not alter the normal proportion of mice insulins I and II. These results suggest that expression of the human insulin gene in vivo results from the cooperation of several cis-regulatory elements present in the various deleted fragments. With none of the deletions used, expression of the transgene was observed in cell types other than beta-islet cells.

Animals↗

PubMed1989

B islet cells of pancreas are the site of expression of the human insulin gene in transgenic mice.

Transgenic mouse lines carrying the human insulin gene were previously shown to express it in pancreas but not in other tissues. The present study reports evidence that the expression of the transgene is restricted to a single category of cells. Immunofluorescence staining of frozen pancreas sections showed that the human C-peptide was present in pancreatic islets only, and more precisely in the B cells of the islets. Human insulin transcripts were initiated correctly in mouse pancreas at the same site as in human pancreas. Three different transgenic lines with different insertion sites and various copy numbers of the human insulin transgene had the same high levels of the transgene transcripts corresponding to a well-balanced contribution in insulin gene expression.

Animals↗

PubMed1989

Regulation of the phase switch controlling expression of type 1 fimbriae in Escherichia coli.

The expression of Escherichia coli type 1 fimbriae is phase-variable i.e. the bacterial cell is either fimbriated or non-fimbriated. The transition from one state to the other is caused by the change in configuration of an invertible DNA segment harbouring the promoter of the fimA gene. The position of this phase switch is controlled by two proteins, FimB and FimE, which mediate an 'on' or 'off' configuration of the switch, respectively. In this study, we have investigated how these proteins control the switch by means of fim-lac fusions on low-copy-number plasmids. It was found, by in trans and cis complementation, that the ratio of fimB to fimE and the total concentration of the gene products determine the configuration of the switch as well as the frequency of phase switching. It was also shown that transcription occurs from the promoter located at the phase switch when this is in the 'off' configuration. This suggests a regulatory mechanism, since the resulting transcript would be anti-sense to the fimE transcript.

Antigens, Bacterial↗

PubMed1989

A comparison of airway responsiveness in smokers with chronic bronchitis and in asthmatic subjects.

Fifty-two of 95 smokers with a forced expiratory volume in one second (FEV1) above 70% predicted and with chronic bronchitis were found to have increased bronchial responsiveness, expressed as PC20FEV1, upon challenge with inhaled histamine. The degree of responsiveness was significantly below that found in matched asthmatics, but substantially higher than that reported in normals. The degree of responsiveness was significantly correlated to prechallenge ventilatory capacity, age and tobacco consumption but not to sex. PC40MEF50 showed the same distribution as PC20FEV1, but did not add further information. The slope of the dose response curve expressing the maximum expiratory flow at 50% of vital capacity expired (MEF50) did not correlate with any of the parameters measured. The slope of the FEV1 dose-response curves showed significant correlation with tobacco consumption. The degree of bronchial responsiveness as an indication for future disability needs to be investigated.

Adult↗

PubMed1989

A trial of inhaled budesonide on airway responsiveness in smokers with chronic bronchitis.

The aim of the present randomized, double-blind study was to evaluate the effect of inhaled budesonide on daily symptoms, ventilatory capacity, and airway responsiveness in smokers with chronic bronchitis. Twenty-five subjects with a provocative concentration producing a 20% fall in forced expiratory volume in one second PC20(FEV1) less than 2.0 mg.ml-1, by bronchial histamine challenge, were included. Eighteen subjects accomplished the entire 12 week study, eight receiving inhaled budesonide 400 micrograms b.i.d. and ten receiving placebo. Cough decreased significantly in the actively treated group during the treatment period, but no change could be demonstrated in expectoration, dyspnoea, or sleep disturbances. No changes in any of these symptoms were found in the placebo group, and no differences in symptoms scores were found between the groups. No significant differences in ventilatory capacity or bronchial responsiveness could be demonstrated. In conclusion, a moderately high dose of inhaled steroid in eight subjects with chronic bronchitis did not improve the symptom scores, ventilatory capacity, or airway responsiveness to any clinically relevant degree.

Administration, Inhalation↗

PubMed1988

Gene probes to detect cross-culture contamination in hormone producing cell lines.

Cross-culture contamination of cell lines propagated in continuous culture is a frequent event and particularly difficult to resolve in cells expressing similar phenotypes. We demonstrate that DNA-DNA hybridization to blotted endonuclease-digested cell DNA effectively detects cross-culture contamination to monitor inter-species as well as intra-species cross contamination. An insulin-producing cell-line, Clone-16, originally cloned from a human fetal endocrine pancreatic cell line did not produce human c-peptide as anticipated. DNA from these cells showed no hybridization to the human ALU sequence probe, BLUR, and lacked restriction fragment length polymorphism typical for the human HLA-DQ beta-chain gene. Although a human insulin gene probe showed a weak, nonhuman hybridization pattern, a cDNA probe for the Syrian hamster insulin gene hybridized strongly consistent with a single copy hamster insulin gene. Karyotyping confirmed the absence of human chromosomes in the Clone-16 cells while sizes, centromere indices, and banding patterns were identical to Syrian hamster fibroblasts. We conclude that the insulin-producing Clone-16 cells are of Syrian hamster origin and demonstrate the effective use of gene probes to control the origin of cell cultures.

Adenoma, Islet Cell↗

PubMed1986

Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

Animals↗

PubMed1985

Direct identification of prohormone conversion site in insulin-secreting cells.

We have localized proinsulin in B cells of human and rat pancreatic islets, using a proinsulin-specific monoclonal antibody revealed by immunocytochemistry. Proinsulin is abundant in Golgi stacks and clathrin-coated secretory granules. It rapidly disappears from these compartments when protein synthesis is inhibited. Depletion of ATP stores prevents movement of proinsulin from the Golgi stacks to the secretory granules; under these conditions, the prohormone in preformed coated granules is converted to insulin, whereas that bound to the Golgi complex is not. Non-coated granules show a low level of proinsulin reactivity under all incubation protocols. These findings provide direct evidence that coated secretory granules are the major, if not the only, cellular site of proinsulin to insulin conversion. They also suggest that the Golgi stack is not involved in conversion, and that intercisternal transport and coated granule formation are hitherto unrecognized energy-requiring steps that precede conversion.

Animals↗

PubMed1985

Relationship between swimming velocity and lactic concentration during continuous and intermittent training exercises.

The present study examined the relationship between lactic acid concentration in capillary blood and swimming velocity during 11 typical endurance exercises (continuous swimming for 30 and 60 min, interval swimming with distances between 50 and 400 m, and with rest periods of 10 and 30 s) and during the "two-speed test" recently described by Mader. It was expected that a better understanding of these relationships could provide evidence how to adjust training intensities from results obtained during the two-speed test. Fifty-nine male swimmers of the German national level participated in this study. After a 30-min maximal swimming test, a mean lactic acid concentration of 4.01 +/- 0.75 mmol/l was found. The corresponding mean velocity was similar to the speed (V4) calculated for the 4 mmol/l level on the basis of the results obtained during the two-speed test (2 X 400). During 30 min continuous swimming at 95% to 105% of the velocity V4, there was a significant correlation (r = 0.82, P less than 0.001) between the swimming speed and the lactic acid concentration. In the 30-min maximal test, the velocity V4 correlated significantly with both the lactic acid concentration (r = -0.58, P less than 0.005) and the swimming speed (r = 0.97, P less than 0.001). During the interval exercises with rest periods of 10 s, the swimming velocities corresponding to the same lactic acid level as during continuous swimming, increased for the 50, 100, 200, and 400 m by 11.23%, 4.21%, 2.95%, and 2.02% of V4, respectively. With rest periods of 30 s, the swimming velocity for the 100, 200, and 400 m increased by 7.34%, 4.22%, and 3.01% of V4, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗