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W Krone

Publications and source records attributed to W Krone.

At least 73 records · Page 4Linked to original sources
PubMed1997

Defects of insulin and IGF-1 action at receptor and postreceptor level in a patient with type A syndrome of insulin resistance.

The action of insulin and IGF-1 in comparison to non-diabetic controls was studied in cultured fibroblasts of a patient with an inherited syndrome of insulin resistance (Type A syndrome). Insulin binding was reduced due to decreased receptor affinity, but sequence analyses revealed no alterations of splicing or primary insulin receptor (IR) structure. Most likely due to the IR affinity defect analyses of signal transduction pathways showed an impairment of insulin action on glucose uptake, total RNA synthesis and phosphorylation as well as activity of MAP-kinase. In addition inducibility of c-fos mRNA level was strongly impaired by insulin and IGF-1, but comparable to controls by PDGF indicating a postreceptor defect. In conclusion, we provide evidence that genetic syndromes of insulin resistance can be associated with both, receptor and postreceptor defects.

Cells, Cultured↗
PubMed1997

Clonal origin of tumor cells in a plexiform neurofibroma with LOH in NF1 intron 38 and in dermal neurofibromas without LOH of the NF1 gene.

LOH at the NF1 locus was investigated in 38 neurofibromas of 26 NF1 patients. Only in one of these tumors LOH was observed. In this plexiform neurofibroma of a NF1 patient with a constitutional one base-pair insertion in NF1 exon 4b, a non-random X-inactivation pattern was found, strongly suggesting a clonal origin of the tumor cells. The analysis of X-inactivation patterns allowed the classification of some of the other neurofibromas with regard to the detectability of clonal LOH. In 3 of 6 neurofibromas without LOH amenable to this analysis, a comparable X-inactivation pattern was found in constitutional and neurofibroma derived DNA. A clonal LOH would not have been detected in these tumors. However, we observed a nonrandom pattern in 3 of the 6 neurofibromas, suggesting a clonal origin of the tumor cells. LOH was not detected in these tumors, but could, however, have occurred by mutational events below the level of large somatic deletions, loss of a whole chromosome 17 or somatic recombination.

Adolescent↗
PubMed1997

[Neurofibroma with contralateral linear hyperpigmentation along Blaschko lines].

We describe a patient with plexiform neurofibroma and contralateral circumscribed hyperpigmentation along the lines of Blaschko. Such findings represent a form of segmental neurofibromatosis. The origin and classification of segmental neurofibromatosis and its relationship to NFI (von Recklinghausen disease) are discussed.

Adult↗
PubMed1997

The second case of a t(17;22) in a family with neurofibromatosis type 1: sequence analysis of the breakpoint regions.

A reciprocal t(17;22)(q11.2;q11.2) was found in a female patient with neurofibromatosis type 1 (NF1) and in her affected daughter. Sequence analysis of cloned junction fragments traversing the breakpoints allowed the identification of the structures involved in the rearrangement. Aberrant bands in Southern hybridizations of restriction enzyme-digested DNA of the patient pointed to the disruption of the NF1 gene in intron 31. Semispecific polymerase chain reaction analysis of the genomic DNA of the patient with the specific primer anchored at NF1 exon 31 was used to obtain the breakpoint-spanning fragment of the derivative chromosome 17. The intron 31 sequence turned out to be interrupted within a large irregular (AT) repeat. The chromosome 22-derived sequence of the der(17) junction fragment allowed us to identify cosmids of the corresponding region from a chromosome 22 specific cosmid library. With the support of the breakpoint-spanning cosmids, the chromosome 22 region upstream of the fragment carried by the der(17) was characterized. Primers deduced from the sequence of this upstream region were used in combination with a primer in NF1 intron 31 distal to the breakpoint on chromosome 17 to amplify the der(22) junction fragment. The structure of the junction sequences suggested that the translocation had arisen by unequal homologous recombination between (AT)-rich repeats on chromosome 22 and on chromosome 17 in intron 31 of the NF1 gene. However, our data support the assumption of additional rearrangements prior to, or in the course of, the recombination event, leading to a loss of the sequences between the involved (AT) repeats on chromosome 22. In the direct vicinity of these (AT) repeats, two members of a previously undescribed low-copy repetitive sequence have been found, copies of which are also present on human chromosome 13.

Adult↗
PubMed1997

Mutational analysis and expression studies of the neurofibromatosis type 2 (NF2) gene in a patient with a ring chromosome 22 and NF2.

The case of a seriously disabled and retarded female patient with neurofibromatosis type 2 (NF2) is reported. She suffered from bilateral vestibular schwannomas, multiple intracranial meningiomas and neurinomas. The constitutional karyotype of the patient was 46, XX, r(22)/45,XX,-22. A constitutional G to A transition in the proximal 3' untranslated region of isoforms 1 and 2 was identified in the patient's NF2 gene and shown not to affect differential splicing or mRNA stability. The instability of the ring chromosome 22 with the associated loss of tumor suppressor genes on chromosome 22, in particular the loss of the NF2 gene, are assumed to have caused multiple tumorigenesis in this patient.

Adult↗
PubMed1997

A third neurofibromatosis type 1 (NF1) pseudogene at chromosome 15q11.2.

Sequences related to the neurofibromatosis type 1 (NF1) gene have been identified on several human chromosomes. In the centromeric region of chromosomes 14 and 15, two NF1 pseudogenes have been described. Sequence comparison between NF1-related exons amplified from two yeast artificial chromosome clones hybridizing to chromosomal region 15q11.2 and published NF1-related sequences localized at 15q11.2 suggested that a third NF1 pseudogene resides in this chromosomal region. The previous localization of an NF1-related locus to the telomeric part of chromosome 15 could not be confirmed by us. Our findings further support pericentromeric spreading of partial NF1 gene copies at chromosome 15q11.2 during evolution.

Base Sequence↗
PubMed1997

Stabilization of atherosclerotic plaque during lipid lowering.

Lipid lowering therapy leads to a great reduction of cardiovascular complications, but has almost no effect on the degree of stenosis of coronary arteries. These findings have lead to a new paradigm of coronary artery disease, i.e. clinical prognosis is not only determined by the extent of a single stenosis, but mainly by the number and structure of atherosclerotic plaque. Rupture of an instable or vulnerable plaque, characterized by a large lipid-rich central core, inflammatory cells, and a thin fibrous cap, causes sudden thrombus formation and thereby acute coronary syndromes. There is accumulating evidence that cholesterol lowering can result in plaque stabilization and improvement of endothelial dysfunction, reviewed recently in this journal. Accordingly, this review focuses on new molecular mechanisms which provide evidence that reduction of cellular cholesterol activates novel transcription factors, called sterol regulatory element binding proteins, which can regulate not only the LDL receptor, thereby reducing plasma cholesterol levels, but also a diverse number of other genes. These gene regulatory events might link cholesterol lowering not only to cellular cholesterol metabolism, but also to triglyceride metabolism, cell differentiation and other events potentially stabilizing vulnerable plaque.

Anticholesteremic Agents↗
PubMed1997

Post-transcriptional regulation of neurofibromin level in cultured human melanocytes in response to growth factors.

Among the symptoms that characterize neurofibromatosis type 1 (NF1) are pigmentation anomalies such as cafe au lait spots. It has been suggested that the reduction of the neurofibromin level in the epidermis of NF1 patients is responsible for the observed signs such as altered melanogenesis and altered density of melanocytes. Our studies show that in cultured normal human melanocytes, the neurofibromin level can be varied in vitro over a wide range by using different culture conditions. The influence of factors that control differentiation and proliferation of melanocytes on neurofibromin levels was studied. Immunoprecipitation followed by western blotting showed a 3- to 4-fold increase of neurofibromin after stimulation by PMA or bFGF, respectively, and a 1.5-fold increase in cells stimulated with steel factor. The increase of neurofibromin was not paralleled by a higher NF1 mRNA level as proved by northern blotting. Pulse-chase experiments with 35S-labeled melanocytes revealed an approximately 3-fold increase in the half-life of neurofibromin in bFGF- or PMA-stimulated cells compared to controls. These results indicate that the neurofibromin level of cultured melanocytes can be regulated by a mechanism independent of NF1 gene transcription and translation, which might influence the degradation rate of the protein.

Cells, Cultured↗
PubMed1997

Monosomy 6 in human cultured fibroblast-like cells after long-term stimulation with acidic fibroblast growth factor (FGF1).

Long-term exposure to fibroblast growth factor type 1 (FGF1) of fibroblast-like cells derived from neurofibromas of patients with neurofibromatosis type 1, from angiofibromas of patients with tuberous sclerosis, and from foreskin of unaffected donors resulted in the outgrowth of monosomy 6 in 7 out of 14 cell lines examined. After their initial detection by cytogenetic analysis, the proportion of cells which had lost one chromosome 6 was monitored by FISH using a-satellite probes specific for chromosome 6 and 7, and by PCR analysis of polymorphic microsatellite markers. Monosomy 6 exceeding baseline levels developed only in cultures exposed to FGF 1, and the emergence of monosomic cells could not be correlated with a given donor's genotype. During serial culture, the proportion of monosomic cells increased to over 90% in 5 of the 7 affected strains. A conspicuous change of cellular morphology from spindle-shaped to more epithelial-type cells was noted in monosomic cultures, even though none of them converted to a permanent cell line during the observation period. We conclude that long-term exposure of human fibroblast-like cell strains to FGF1 results in the emergence of monosomy 6 in 50% of the cultures so treated. A selective advantage for such monosomic cells is the most likely explanation for their steady increase during serial culture.

Adult↗
PubMed1997

Testosterone substitution normalizes elevated serum leptin levels in hypogonadal men.

The ob gene product leptin (OB) is a feedback signal from the adipocyte to the hypothalamus and is involved in regulation of food intake and energy expenditure in rodents. A major determinant of serum OB levels is fat mass. Several studies suggest that men have lower OB levels than women even after adjustment for percent body fat. We, therefore, investigated the influence of testosterone (T) substitution in hypogonadal men on serum OB levels. Hypogonadal men with T levels of 3.6 nmol/L or less and off substitution therapy for at least 3 months were assigned to two treatment groups: testosterone enanthate (TE; 250 mg, i.m., every 21 days; n = 10) or a single s.c. implantation of 1200 mg crystalline T (TPEL; n = 12). Blood samples for determination of T, 5 alpha-dihydrotestosterone (DHT), sex hormone-binding globulin, and 17 beta-estradiol were obtained before therapy and then every 21 days until day 189 and at follow-up visits on days 246 and 300. Serum OB levels were assessed on days 0, 42, 84, 126, 168, and 300. OB levels were referred to a normal range for men based on the analysis of OB levels in 393 adult men. Substitution with T led to a large rise in T and DHT in both groups compared to baseline values (average T, days 21-189: TE, 14.33 +/- 2.63 nmol/L; TPEL, 24.98 +/- 1.64; average DHT, days 21-189: TE, 4.20 +/- 0.57 nmol/L; TPEL, 5.11 +/- 0.56; P < or = 0.05). Concomitantly, 17 beta-estradiol increased in both groups, and sex hormone-binding globulin levels were significantly decreased. At baseline, serum OB levels in hypogonadal men were 3-fold elevated compared to those in normal men (12.39 +/- 2.93 micrograms/L vs. 4.28 +/- 0.52; P < 0.01) and not different between groups (TE, 13.7 +/- 5.6; TPEL, 11.3 +/- 2.9 micrograms/L). This elevation was retained after adjustment for body mass index in the normal control group [TE, 1.45 +/- 0.51 SD score (P < 0.0001); TPEL, 0.98 +/- 0.35 SD score (P < 0.0008)]. During T substitution serum OB was completely normalized (trough levels: TE, 4.6 +/- 1.0 micrograms/L; TPEL 4.3 +/- 0.9 micrograms/L). In multiple regression analysis, the androgen (T plus DHT)/estrogen ratio was the only significant determinant of OB levels (r = -0.32; P < 0.01). At baseline, OB levels did not correlate with body mass index, but during substitution, the correlation was considerably improved. We conclude that hypogonadal men exhibit elevated OB levels that are normalized by substitution with T. The only determinant of OB levels was the androgen/estrogen ratio, indicating a major influence of sex steroids on OB production. The interaction of T and OB might be part of a hypothalamic-pituitary-gonadal-adipose tissue axis that is involved in body weight maintenance and reproductive function.

Adult↗
PubMed1997

Occult eutopic Cushing's syndrome--failure of simultaneous bilateral petrosal sinus sampling to diagnose pituitary-dependent Cushing's syndrome.

Simultaneous bilateral inferior petrosal sinus (IPS) sampling has been repeatedly proposed to be a highly specific approach for the diagnosis of Cushing's disease and 100% sensitivity in detecting autonomous pituitary ACTH secretion by an adenoma has been reported in a large series. We now report on a patient suffering from ACTH-dependent Cushing's syndrome in whom repeated bilateral IPS sampling failed to detect a central/peripheral gradient diagnostic for autonomous pituitary ACTH secretion during initial evaluation. Applying lysine vasopressin as the corticotroph secretatogue, the maximum central/peripheral gradient was 1.0 before and 1.1 following stimulation. Moreover, results of high dose dexamethasone and corticotrophin releasing hormone administration suggested ectopic ACTH secretion. Since thorough diagnostic procedures failed to localise a suspected carcinoid tumour, occult ectopic Cushing's syndrome was diagnosed. Eight years later, a pituitary macroadenoma was detected by magnetic resonance imaging (MRI), IPS catheterisation then revealed a maximal central/ peripheral gradient of 9.3 before and 20.4 after the intravenous administration of lysine vasopressin. Resected tumour tissue was classified as a typical densely granulated ACTH cell adenoma. We conclude that repeated MRI scans should be included in the follow-up of patients with a diagnosis of occult ectopic Cushing's syndrome to avoid the risk of overlooking 'occult eutopic Cushing's syndrome'.

Adenoma↗
PubMed1996

SREBP-1 mediates activation of the low density lipoprotein receptor promoter by insulin and insulin-like growth factor-I.

Transcription of the low density lipoprotein (LDL) receptor gene is regulated by intracellular cholesterol concentration, hormones, and growth factors. We studied the mechanisms by which insulin and estradiol stimulate promoter activity of the LDL receptor gene. Hormonal effects were analyzed in HepG2 cells after transient transfection with promotor reporter gene constructs. Successive 5' deletions of the LDL receptor promoter fragment from -537 to +88 revealed the sterol regulatory element 1 (SRE-1) between -65 and -56 as an insulin- and estradiol-sensitive cis-element. If the SRE-1 is point mutated at position -59 (C to G), which abolishes the binding of the SRE binding proteins (SREBP-1 and SREBP-2), no insulin or estradiol stimulatory effect on reporter gene expression was observed, indicating a role of SRE binding proteins in this regulatory mechanism. The concentration of the 125-kDa membrane-integrated SREBP-1 precursor protein in LDL repressed HepG2 cells is not altered by hormone treatment. Concentrations of SREBP-1 mRNA and precursor protein are reduced significantly by high and stable expression of an SREBP-1 antisense cDNA fragment in HepG2 cells (SREBP1(-) cells). Transfection of SREBP1(-) cells with promoter construct phLDL4 (-105 to +88) reduces induction of reporter gene activity by insulin and insulin-like growth factor-I to 35 and 17%, respectively, compared with HepG2 cells. The stimulatory effect of estradiol remains unchanged, and the inductions by pravastatin are enlarged. We conclude that different regulatory effects converge at SRE-1, but that SREBP-1 is selectively involved in the signal transduction pathway of insulin and insulin-like growth factor-I leading to LDL receptor gene activation.

Base Sequence↗
PubMed1996

Structure of the human apolipoprotein C-II gene promoter.

To localize transcriptional active cis elements and to study the effect of a single base transition at -86 derived from a patient (PS) with an inherited severe form of hypertriglyceridemia a reporter gene transfection assay with apo C-II promoter fragments was performed. Sequences from -170 to -140, -140 to -59 and -59 to -39 were transcriptional active. The A to G transition at -86 reduced promoter activity to 25% (pC2PS-13). A nuclear protein bound sequence specific to a DNA fragment from -102 to -70, but disappeared when the point mutation at -86 was introduced. We found that one of these cis elements enhances promoter activity as a response of cAMP elevation. Apo CII mRNA level increases after incubation of HepG2 cells with 10 microM forskolin within 2 hours as measured by northern blot analyses. Incubation of transfected cells (pC2W) with forskolin [10 microM] or a cAMP analogue resulted in an 6-fold increase of luciferase activity within 6 hours and a subsequent decrease. The results suggest that several positive acting elements are located between -170 and -36 and that one of them may be responsible for promoter activation by cAMP. The reduction of promoter activity by the point mutation may be a consequence of impaired protein-DNA interaction.

Apolipoprotein C-II↗
PubMed1996

A single nucleotide substitution in the promoter region of the apolipoprotein C-II gene identified in individuals with chylomicronemia.

Apolipoprotein (apo) C-II plays a major role as a cofactor for lipoprotein lipase, the enzyme involved in the hydrolysis of triglyceride-rich particles. We identified in two relatives of a family (mother and son) massive hypertriglyceridemia with chylomicronemia. In these individuals apoC-II was not measurable in plasma by radial immunodiffusion. On isoelectric focusing of very low density apolipoproteins, trace amounts of apoC-II became obvious in the regular position. By sequencing, no abnormalities in the exons or neighboring intron sequences were detected. However, three alterations in the DNA sequence were found upstream from the transcription initiation site. Two variations could be explained by differences in previously published DNA sequences. The third variation (A-->G; position -86; Das et al. 1987. J. Biol. Chem. 262: 4787-4793) was present only in the homozygous form in the two hypertriglyceridemic probands. In 46 hypertriglyceridemic individuals outside the family, this mutation was not found. In electrophoretic mobility shift experiments with nuclear extracts from HepG2 cells, the 31 bp DNA fragment carrying the A-->G substitution resulted in a markedly diminished protein binding compared with the wildtype DNA fragment. In promoter reporter gene assays, the activity of the basal promoter was reduced in the case of the A-->G substitution and the deletion of the bases -91 to -58. The pedigree analysis and the experimental results are evidence that this is the first mutation in the apolipoprotein C-II gene where a single nucleotide substitution diminishes the binding of a transcription factor to a positive cis-acting clement in the promoter resulting in a depletion of apolipoprotein C-II in plasma.

Adolescent↗
PubMed1995

Reduced neurofibromin content but normal GAP activity in a patient with neurofibromatosis type 1 caused by a five base pair duplication in exon 12b of the NF1 gene.

We screened a total of 87 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exons 11, 12a, and 12b of the NF1 gene using temperature gradient gel electrophoresis (TGGE). A novel mutation (1998insCCTCT) was found in exon 12b. The 5-bp duplication comprising nucleotides 1994 to 1998 is predicted to lead to a truncated protein product lacking three quarters of its C-terminal sequence including the entire GTPase-activating protein-(GAP)-related domain. This mutation is associated with a reduction by 50% of the detectable amount of neurofibromin found in this patient. Despite the reduced level of neurofibromin cellular GAP activity was normal, which suggests that defects in other functions of the neurofibromin molecule may be important in the pathogenesis of NF1.

Base Composition↗
PubMed1995

Recombinant human insulin receptor substrate-1 protein. Tyrosine phosphorylation and in vitro binding of insulin receptor kinase.

Insulin receptor substrate-1 (IRS-1) is a major endogenous substrate of the insulin receptor. To study the interaction of the insulin receptor with IRS-1 in vitro, we expressed in Escherichia coli the amino acids 516-777 of human IRS-1 (hIRS-p30) covering five potential tyrosine phosphorylation sites within YXXM motifs. Kinetic data for tyrosine phosphorylation of hIRS-p30 by partially purified insulin receptor and insulin-like growth factor I receptor and by baculovirus-expressed insulin receptor kinase domain were determined. Native insulin receptor demonstrated the highest affinity to hIRS-p30 (Km = 6.8 +/- 0.6 microM), followed by the insulin-like growth factor I receptor (Km = 9.9 +/- 1.0 microM). We used the soluble recombinant insulin receptor kinase domain, which phosphorylated hIRS-p30 with high affinity (Km = 11.9 +/- 0.8 microM), and affinity columns prepared by coupling hIRS-p30 to NHS-activated Sepharose for binding assays. The insulin receptor kinase domain phosphorylated the hIRS-p30 on the column, was bound by the immobilized hIRS-p30, and was eluted with high salt buffer. Autophosphorylated and EDTA-inactivated insulin receptor kinase domain was bound only by immobilized hIRS-p30 protein that has been prephosphorylated. Our results indicate that the recombinant hIRS-p30 protein is a high affinity substrate for insulin receptor and insulin-like growth factor I receptor in vitro. Moreover, we show that only tyrosine-phosphorylated hIRS-p30 is able to bind to the insulin receptor.

Amino Acid Sequence↗
PubMed1995

Single-cell PCR performed with neurofibroma Schwann cells reveals the presence of both alleles of the neurofibromatosis type 1 (NF1) gene.

It is commonly held that Schwann cells (SC) are the progenitor cells of benign neurofibromas. To test for loss of heterozygosity (LOH) at the neurofibromatosis 1 (NF1) gene locus, three intragenic polymorphic markers were analyzed after polymerase chain reaction amplification, starting from 98 single SC isolated from primary cultures of neurofibromas, of five informative NF1 patients. The patterns obtained did not provide evidence for LOH at the NF1 gene. LOH by nondisjunction, large deletions, or somatic recombination in SC seems not to be the mechanism of generation of neurofibromas.

Alleles↗