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Biomedical subjects

M Karl

Publications and source records attributed to M Karl.

At least 37 records · Page 2Linked to original sources
PubMed1994

Glucocorticosteroid resistance in humans. Elucidation of the molecular mechanisms and implications for pathophysiology.

Familial glucocorticoid resistance (FGR) is a rare hereditary disorder characterized by hypercortisolism and the absence of stigmata of Cushing's syndrome. The inability of glucocorticoids to exert their effects on target tissues is compensated for by increases in circulating corticotropin (ACTH) and cortisol, the former causing excess secretion of both adrenal androgens and adrenal steroid-biosynthesis intermediates with salt-retaining activity. There is considerable variability in the clinical presentations of FGR ranging from asymptomatic, to isolated chronic fatigue and to hypertension with or without hypokalemic alkalosis or to hyperandrogenism, or both. In women, hyperandrogenism can result in acne, hirsutism, menstrual irregularities, oligoanovulation, and infertility; in men it may lead to infertility and in children to precocious puberty. The reported molecular defects in FGR, such as point mutations and a microdeletion of the glucocorticoid receptor (GR) gene, cause partial resistance by, respectively, compromising the function of the GR or decreasing its intracellular concentration in glucocorticoid target tissues. Complete glucocorticoid resistance is believed to be incompatible with life in humans. Hence, the glucocorticoid resistance cases reported have been partial and of variable degree. The extreme variability in the clinical manifestations of the disorder can, additionally, be explained by differing sensitivity of target tissues to mineralocorticoids or androgens or both, and perhaps by different biochemical defects of the glucocorticoid receptor, causing selective resistance of certain glucocorticoid responses in specific tissues. Isolated tissue-resistance from a somatic mutation of the GR in a corticotropinoma from a patient with Nelson's syndrome was also found, suggesting that this may be a mechanism of tumorigenesis. There is additional evidence that defects of GR function can appear surreptitiously in a variety of clinical conditions, suggesting that glucocorticoid resistance in humans may be involved in the pathogenesis and/or clinical picture of a plethora of disease states, of which FGR is the archetype.

Adrenocorticotropic Hormone↗
PubMed1994

Clonal composition of human adrenocortical neoplasms.

The mechanisms of tumorigenesis of adrenocortical neoplasms are still not understood. Tumor formation may be the result of spontaneous transformation of adrenocortical cells by somatic mutations. Another factor stimulating adrenocortical cell growth and potentially associated with formation of adrenal adenomas and, less frequently, carcinomas is the chronic elevation of proopiomelanocortin-derived peptides in diseases like ACTH-dependent Cushing's syndrome and congenital adrenal hyperplasia. To further investigate the pathogenesis of adrenocortical neoplasms, we studied the clonal composition of such tumors using X-chromosome inactivation analysis of the highly polymorphic region Xcen-Xp11.4 with the hybridization probe M27 beta, which maps to a variable number of tandem repeats on the X-chromosome. In addition, polymerase chain reaction amplification of a phosphoglycerokinase gene polymorphism was performed. After DNA extraction from tumorous adrenal tissue and normal leukocytes in parallel, the active X-chromosome of each sample was digested with the methylation-sensitive restriction enzyme HpaII. A second digestion with an appropriate restriction enzyme revealed the polymorphism of the region Xcen-Xp11.4 and the phosphoglycerokinase locus. Whereas in normal polyclonal tissue both the paternal and maternal alleles are detected, a monoclonal tumor shows only one of the parental alleles. A total of 21 female patients with adrenal lesions were analyzed; 17 turned out to be heterozygous for at least one of the loci. Our results were as follows: diffuse (n = 4) and nodular (n = 1) adrenal hyperplasia in patients with ACTH-dependent Cushing's syndrome, polyclonal pattern; adrenocortical adenomas (n = 8), monoclonal (n = 7), as well as polyclonal (n = 1); adrenal carcinomas (n = 3), monoclonal pattern. One metastasis of an adrenocortical carcinoma showed a pattern most likely due to tumor-associated loss of methylation. In the special case of a patient with bilateral ACTH-independent macronodular hyperplasia, diffuse hyperplastic areas and a small nodule showed a polyclonal pattern, whereas a large nodule was monoclonal. We conclude that most adrenal adenomas and carcinomas are monoclonal, whereas diffuse and nodular adrenal hyperplasias are polyclonal. The clonal composition of ACTH-independent massive macronodular hyperplasia seems to be heterogeneous, consisting of polyclonal and monoclonal areas.

Adenoma↗
PubMed1994

Regulation of HSP90 and corticosteroid receptor mRNA by corticosterone levels in vivo.

The non-activated 9S forms of several steroid hormone receptors are heterooligomeric complexes consisting of the aporeceptor and three heat shock proteins, hsp90, hsp70 and hsp56. Hsp90 appears to play a facilitatory role in high-affinity steroid binding and to promote the efficacy of steroid actions on target tissues. Circulating glucocorticoid levels have a major regulatory impact on the binding capacity of hippocampal and hypothalamic corticosteroid receptors, a phenomenon that affects the activity of the hypothalamic-pituitary-adrenal axis and neuronal excitability in general. This study demonstrates that hsp90 mRNA is present in substantial amounts in hippocampal and hypothalamic areas characterized by high densities of corticosteroid receptors, and in the thymus. Steady-state levels of hsp90 mRNA in these regions were altered by chronic changes of circulating glucocorticoid concentrations in a site-specific fashion. In the hippocampus, mRNAs coding for hsp90 and both types of corticosteroid receptors (type I, MR and type II, GR) displayed a coordinate increase following adrenalectomy and castration (ADX/GX); in the hypothalamus only hsp90 mRNA levels were elevated, and none of the parameters studied was affected in the thymus by steroid hormone deprivation. Supplementation of ADX/GX rats with various doses of corticosterone in vivo elicited differential responses. Moderate elevation of circulating corticosterone levels normalized ADX/GX-increased hsp90 mRNA concentrations in the hippocampus and the hypothalamic paraventricular nucleus (PVN); this was associated with similar changes in GR and MR mRNA levels in the hippocampus, while GR mRNA concentrations in the PVN were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenalectomy↗
PubMed1994

p53 mutations in human adrenocortical neoplasms: immunohistochemical and molecular studies.

The mechanisms of tumorigenesis of adrenocortical neoplasms have not been elucidated as yet. However, loss of heterozygosity at chromosomal locus 17p has been consistently observed in adrenocortical cancer. p53 is a recessive tumor suppressor gene located on chromosome 17p. Mutations in the p53 gene play an important role in the tumorigenesis of diverse types of human neoplasms including breast and colon cancers. More than 90% of all mutations discovered in such tumors have been detected in 4 hot spot areas that lie between exons 5 and 8. In contrast to wild-type p53, mutant p53 accumulates intracellularly and can be easily detected by immunohistochemistry. We therefore investigated the frequency of p53 mutations in human adrenocortical neoplasms using molecular biology and immunohistochemistry techniques. Five patients with adrenocortical adenomas (5 female; ages 39-72 yr), 11 patients with adrenocortical carcinomas (8 female, 3 male; ages 15-50 yr), and two adrenocortical tumor cell lines were studied. After DNA extraction from frozen tumor tissue or paraffin-embedded material, exons 5 through 8 were amplified using the polymerase chain reaction and directly sequenced by the dideoxy termination method. Immunohistochemistry was performed on paraffin-embedded tumor specimens obtained during adrenalectomy using a monoclonal antibody reacting with both wild-type and mutant p53. Prevalence of mutations was adenomas, 0/5, carcinomas, 3/11, and adrenocortical cell lines, 2/2. Single point mutations were detected in 3 cases (exons 5, 6, and 7, respectively), and rearrangements of exon 7/8 and 8 were found in 2 cases. Immunohistochemistry detected strong nuclear and/or cytoplasmic p53 immunoreactivity in all adrenocortical carcinomas with point mutations of the p53 gene but not in adenomas and carcinomas with the wild-type sequence or with deletion/rearrangement of the p53 gene. We conclude that p53 plays a role in the tumorigenesis of adrenocortical carcinomas but is of less importance to benign adenomas.

Adolescent↗
PubMed1994

Physiological and molecular aspects of mineralocorticoid receptor action in pseudohypoaldosteronism: a responsiveness test and therapy.

Pseudohypoaldosteronism (PHA), characterized by congenital resistance to aldosterone and excessive salt loss, has been traditionally treated with salt replacement. Although the mineralocorticoid receptor (MR) has been suggested as a potential locus of the defect in this disease, no such abnormality has been identified as yet. We studied a 17-yr-old male patient with congenital multifocal target organ resistance to aldosterone. Both carbenoxolone, an 11 beta-hydroxysteroid dehydrogenase inhibitor, and a high dose of fludrocortisone normalized the patient's serum electrolyte concentrations and decreased his urinary excretion of sodium, suggesting that this patient's resistance was partial and could be overcome by high concentrations of endogenous or exogenous mineralocorticoids. We hypothesized that the beneficial effect of these treatments was mostly mediated by the MR, because the administration of dexamethasone, while this patient was receiving a therapeutic dose of carbenoxolone, caused its reversal. These findings convinced us that there was functional, albeit possibly defective, MR in this patient and led us to perform molecular studies. Both alleles of the MR gene were expressed in the patient and his clinically and biochemically normal father. A conservative heterozygous mutation (A760-->G760, Ileu180-->Val180) and a nonconservative homozygous mutation (C944-->T944, Ala241-->Val241) were identified in the complementary DNA of both the patient and his father. The first untranslated exon and 0.9 kilobase of the 5'-regulatory region were also identical in the two men. It appears that the mutations causing amino acid substitutions represent polymorphisms, as we found high frequencies of both in the general population. We conclude that carbenoxolone and fludrocortisone may help define the presence of functional MR in patients with PHA and that the former could be used in the long term therapy of this disease. We hypothesize that the defect causing PHA in this patient might be in a post-MR step of aldosterone action.

Adolescent↗
PubMed1993

Syndromes of glucocorticoid resistance.

Glucocorticoid resistance results from the partial, albeit apparently generalized, inability of glucocorticoids to exert their effects on target tissues. The condition is associated with compensatory increases in circulating pituitary corticotropin and cortisol, with the former causing excess secretion of both adrenal androgens and adrenal steroid biosynthesis intermediates with salt-retaining activity. The manifestations of glucocorticoid resistance vary from chronic fatigue (perhaps a result of glucocorticoid deficiency in the central nervous system) to various degrees of hypertension with or without hypokalemic alkalosis or hyperandrogenism, or both, caused by increased cortisol and other salt-retaining steroids and adrenal androgens, respectively. In women, hyperandrogenism can result in acne, hirsutism, menstrual irregularities, oligoanovulation, and infertility; in men, it may lead to infertility and in children, to precocious puberty. Different molecular defects, such as point mutations or a microdeletion of the highly conserved glucocorticoid receptor gene, alter the functional characteristics or concentrations of the intracellular receptor and appear to cause glucocorticoid resistance. The extreme variability in the clinical manifestations of glucocorticoid resistance and its mimicry of many common diseases can be explained by the overall degree of glucocorticoid resistance, differing sensitivity of target tissues to mineralocorticoids or androgens or both, and perhaps different biochemical defects of the glucocorticoid receptor, with selective resistance of certain glucocorticoid responses in specific tissues. The various different symptoms of classic glucocorticoid resistance and the theoretical potential of this condition to appear surreptitiously emphasize the importance of the glucocorticoid receptor in the pathogenesis of human disease.

Drug Resistance↗
PubMed1993

Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene.

The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. We studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene alpha, along with their 5'- and 3'-flanking regions, we identified a 4-base deletion at the 3'-boundary of exon 6 in one GR allele (delta 4), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G1220) in the proband, in one of her affected brothers and in her unaffected sister. The functional importance of this mutation was tested in a cotransfection study using the recombinant expression vector pRShGR-Ser363 and the glucocorticoid responsive vector mouse mammary tumor virus-chloramphenicol transferase. This amino acid substitution did not alter the function of the glucocorticoid receptor. Using reverse transcription-polymerase chain reaction we could only identify messenger RNA transcripts of the G1220-allele but not of the delta 4-allele in the affected members of this family who were heterozygous for the G1220 mutation. This deletion in the glucocorticoid receptor gene was, thus, associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism.

Adult↗
PubMed1993

No evidence for oncogenic mutations in guanine nucleotide-binding proteins of human adrenocortical neoplasms.

G-Proteins are membrane-bound heterotrimeric polypeptides that couple receptor signals to second messenger systems such as cAMP. Recently, point mutations at 2 codons of the highly preserved alpha-chain of Gs, the adenyl cyclase-stimulating G-protein, were found in GH-secreting pituitary tumors. These mutations resulted in constitutively activated Gs alpha and high intracellular cAMP levels. In addition, point mutations at similar codons of a different G-protein, G(i) alpha 2, were reported in adrenocortical neoplasms, suggesting a potential role of this isoform in the genesis of these tumors. We reevaluated the frequency of constitutively activating point mutations in the alpha-chain of the stimulatory (Gs alpha) and inhibitory (G(i) alpha 2) G-proteins in human adrenocortical tumors. Seven adrenocortical carcinomas, 2 human adrenocortical tumor cell lines, and 11 adrenocortical adenomas were studied. Genomic DNA was purified from either frozen tumor tissue or paraffin-embedded sections. Using specific primers and the polymerase chain reaction, DNA fragments surrounding codons 201 and 227 (Gs alpha) and 179 and 205 (G(i) alpha 2) were amplified and visualized on a 2% agarose gel. In a second asymmetric polymerase chain reaction, using nested primers, single stranded DNA was generated using 1-10 microL of the initial amplification mixture and directly sequenced using the dideoxy chain termination method of Sanger. We found no mutations at codons 201, 227 and 179, 205 of Gs alpha and G(i) alpha 2, respectively, in the tumors studied. We conclude that previously identified oncogenic point mutations in the stimulatory and inhibitory alpha-chain of G-proteins do not appear to be present at high frequency in adrenal neoplasms. Thus, the mechanism(s) of tumorigenesis in these tumors is different from that in GH-secreting adenomas and may involve oncogenic mutations of other cell constituents.

Adolescent↗
PubMed1991

Point mutation causing a single amino acid substitution in the hormone binding domain of the glucocorticoid receptor in familial glucocorticoid resistance.

Familial glucocorticoid resistance is a hypertensive, hyperandrogenic disorder characterized by increased serum cortisol concentrations in the absence of stigmata of Cushing's syndrome. Our previous studies of the first reported kindred showed a two- to threefold reduction in glucocorticoid receptor-ligand binding affinity in the propositus, and a lesser reduction in affinity in his mildly affected son and nephew. Glucocorticoid receptor cDNA from these three patients was amplified by polymerase chain reaction and sequenced. The cDNA nucleotide sequence was normal, except for nucleotide 2054, which substituted valine for aspartic acid at amino acid residue 641. The propositus was homozygous while the other relatives were heterozygous for the mutation. COS-7 monkey kidney cells were cotransfected with expression vectors for either wild type or Val 641-mutant receptors, together with the reporter plasmid pMMTV-CAT. Dexamethasone increased chloramphenicol acetyltransferase activity in cells expressing wild type receptor, but had no effect in cells expressing Val 641-mutant receptors, despite similar receptor concentrations, as indicated by Western blotting. The binding affinity for dexamethasone of the Val 641-mutant receptor was threefold lower than that of the wild type receptor. These results suggest that glucocorticoid resistance in this family is due to a point mutation in the steroid-binding domain of the glucocorticoid receptor.

Amino Acids↗
PubMed1990

Structural analysis of the regulatory region of the human corticotropin releasing hormone gene.

A DNA fragment containing the human corticotropin releasing hormone (CRH) gene, along with 9 kb of upstream and 4 kb of downstream sequences, was isolated from a human genomic DNA library. Nucleotide sequence analysis of the proximal 918 nucleotides 5' flanking the putative major mRNA start site of the human gene and comparison to the 866 nucleotide long homologous ovine sequence, revealed that this region of the CRH gene consists of two distinct areas with different degrees of homology, varying from 72% to 94%. The putative functional features of the human sequence were identified. Many, but not all, features were conserved in the ovine sequence. The highly conserved nature of the regulatory region of this gene makes it a good candidate for tracing possible related genetic defects of the hypothalamic-pituitary-adrenal (HPA) axis.

Animals↗
PubMed1990

Molecular analysis of duplicated esterase genes in Drosophila melanogaster.

Genomic clones containing sequences homologous to an esterase 6 (Est-6) cDNA clone were isolated from a library of Drosophila melanogaster DNA. Comparison of the genomic and cDNA sequences revealed that the Est-6 gene comprises two exons, one of 1,387 bp and one of 248 bp, separated by a short intron of 51 bp. Further sequencing revealed the presence of a tandem duplication of the Est-6 gene (denoted Est-P) which also has an exon of 1,387 bp and an exon of 248 bp, separated by a short intron of 56 bp. The two genes show similarities of 64% and 60% at the DNA and protein levels, respectively. The coding regions of the genes are 197 bases apart, and presumptive 5' regulatory sequences of Est-P overlap at least the 3' noncoding region of Est-6. Transcripts homologous to Est-P were detected in late larvae and adults of each sex, whereas Est-6 transcripts are present in all life stages but are predominant in adult males. This suggests different physiological functions for the products of the two genes. Southern and Northern blot hybridization analyses of the 20-kb region surrounding the Est-6/Est-P duplication failed to detect any other duplicated esterase genes, although this region is actively transcribed.

Amino Acid Sequence↗
PubMed1989

[Substitution of F XIII concentrate in ulcerative colitis].

A 33 years old female with ulcerative colitis was admitted with an acute exacerbation of the disease characterised by haematochezia, diarrhoea (10 stools/die) and anaemia (haemoglobin 6.5 g/dl). Therapy with 5-ASA and corticosteroids for six weeks failed to decrease the activity of the disease. Since deficiency of coagulation factor XIII (activity 60%, subunit A 56%) was present, in addition, concentrates of factor XIII (Fibrogammin HS, Behring, F.R.G.) 1250 U/die were given for ten days. The substitution resulted in an immediate increase of reduced F XIII activity (164%) and F XIII subunit A (333%) as well as in a marked improvement of symptoms.

Adult↗
PubMed1989

[HLA compatibility and susceptibility to habitual abortion. Results of histocompatibility testing of couples with frequent miscarriages].

We have performed class I HLA antigen testing in 42 women with recurrent habitual abortions and in their husbands. The main criterion for inclusion in this group was a frequency of more than two abortions without the known reasons for abortion and without a living child. 29 couples with a least two healthy children and no abortion in the clinical history served as a control group. The data were evaluated in respect to the association of HLA and disease as well as to the level of histocompatibility within the couples. In our results we did not find any significant association between HLA-A, B, C phenotypes and the habitual abortion. On the other hand, we observed a significantly higher level of histocompatibility in the couples with habitual abortions in comparison to the control couples. The frequency of identity in one and more histocompatibility antigens was in the patient group significantly higher than in the control group and as it could be expected by chance, calculated on the basis of antigen distribution within the loci A, B, C in the population in the southern part of the GDR. In no case we have revealed lymphocytotoxic antibodies as a possible cause for miscarriage. Our results are in favour of the hypothesis that a higher level of histocompatibility could be an immunological explanation for the habitual abortions. In addition, this hypothesis was supported by the successful immunotherapy with leukocytes derived from the husband or nonrelative donors.

Abortion, Habitual↗
PubMed1987

Muscarine receptors on the rat phrenic nerve, evidence for positive and negative muscarinic feedback mechanisms.

Neuronal transmitter stores of the rat phrenic nerve were labelled by incubation with [3H]choline. Release of [3H]acetylcholine was elicited by electrical nerve stimulation (100 or 1,500 pulses, 5 or 25 Hz) or by high potassium (27 mmol/l) and the effects of the muscarine receptor agonist oxotremorine and the antagonist scopolamine were investigated. Neither oxotremorine nor scopolamine affected the basal tritium efflux. A low concentration of oxotremorine (10 nmol/l) enhanced and a high concentration of oxotremorine (1 mumol/l) reduced the electrically evoked [3H]acetylcholine release. Likewise, the high potassium-evoked [3H]acetylcholine release was reduced by a high concentration of oxotremorine. Both effects of oxotremorine, increase and decrease, were abolished by a pretreatment (30 min before the first stimulation period) with 0.1 mumol/l scopolamine. Scopolamine (0.1 mumol/l) alone, enhanced [3H]acetylcholine release evoked by 100 pulses (5 Hz) or by high potassium. Scopolamine, however, reduced [3H]acetylcholine release evoked by 1,500 pulses (5 Hz or 25 Hz). The concentration-response curves obtained for scopolamine under these latter stimulation conditions were flat-running and biphasic which might indicate the involvement of two opposite effects (increase and decrease) of scopolamine under the present stimulation conditions. Both effects of scopolamine were reduced in the presence of 10 mumol/l neostigmine.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1987

Novel mRNA species from a Q-subregion class-I gene of the murine major histocompatibility complex.

A novel mRNA species is specified by the Q6/Q8 gene, a member of the Qa-2-determining family of class-I genes of the murine major histocompatibility complex. It is longer than all previously described class-I transcripts due to an extension at the 3' end of the mRNA past the predicted polyadenylation site. This mRNA was detected by Northern-blot analyses with single or low-copy-number probes to the 3' end of the Q6/Q8 gene. RNase protection experiments have confirmed this finding. The appearance of this mRNA in different strains of mice correlates with the synthesis of the Qa-2 molecule.

Animals↗