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R Paschke

Publications and source records attributed to R Paschke.

At least 19 recordsLinked to original sources
PubMed2001

Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase.

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.

Amino Acid Sequence↗
PubMed2001

Isoproterenol inhibits resistin gene expression through a G(S)-protein-coupled pathway in 3T3-L1 adipocytes.

Resistin was recently identified as a hormone secreted by adipocytes which leads to insulin resistance in vivo and in vitro and might therefore be an important link between obesity and diabetes. To clarify the regulation of resistin gene expression, 3T3-L1 adipocytes were treated with various agents known to modulate insulin sensitivity, and resistin mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction. Interestingly, isoproterenol treatment reduced the level of resistin mRNA to 20% of non-treated control cells. This effect was dose-dependent with significant inhibition occurring at concentrations as low as 10 nM isoproterenol. Moreover, pretreatment of adipocytes with the beta-adrenergic antagonist propranolol almost completely reversed the inhibitory effect of isoproterenol, whereas addition of the alpha-adrenergic antagonist phentolamine did not have any effect. Furthermore, the effect of isoproterenol could be mimicked by activation of G(S)-proteins and adenylyl cyclase. Thus, both cholera toxin and forskolin decreased resistin mRNA expression in a dose-dependent fashion by up to 90% of control levels. Taken together, these results suggest that resistin gene expression is regulated by a protein kinase A-dependent pathway in 3T3-L1 adipocytes.

3T3 Cells↗
PubMed2001

NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer.

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of approximately 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.

Amino Acid Substitution↗
PubMed2001

Detection of thyroid-stimulating hormone receptor and Gsalpha mutations: in 75 toxic thyroid nodules by denaturing gradient gel electrophoresis.

The actual frequency of constitutively activating thyrotropin receptor or Gsalpha mutations in toxic thyroid nodules (TTNs) remains controversial as considerable variation in the prevalence of these mutations has been reported. We studied a series of 75 consecutive TTNs and performed mutation screening by the more sensitive method of denaturing gradient gel electrophoresis (DGGE) in addition to direct sequencing. Furthermore, the likelihood of somatic mutations occurring in genes other than that for the thyroid-stimulating hormone receptor (TSHR) and exons 7-9 of the Gsalpha protein gene was determined by clonality analysis of TTNs, which did not harbor mutations in the investigated genes. In 43 of 75 TTNs (57%) constitutively active TSHR mutations were identified. Six TSHR mutations were detected only by DGGE, underlining the importance of a sensitive screening method. Novel, constitutively activating mutations were identified at positions 425 (Ser-->Leu) and 512 (Leu-->Glu/Arg). Furthermore, a new base substitution was detected at position Pro639Ala (CCA-->GCA). Ten of 20 TSHR or Gsalpha mutation negative cases (50%) showed nonrandom X-chromosome inactivation, indicating clonal origin. In conclusion, somatic, constitutively activating TSHR mutations appear to be a major cause of TTNs (57%), while mutations in Gsalpha play a minor role (3%). The mutation negative but clonal cases indicate a probable involvement of somatic mutations other than in the TSH receptor or Gsalpha genes as the molecular cause of these hot nodules.

Amino Acid Substitution↗
PubMed2001

Severe hypoglycemia due to insulin autoimmune syndrome with insulin autoantibodies crossreactive to proinsulin.

We report the case of a 69-year-old woman with insulin autoimmune syndrome first misdiagnosed as insulinoma. The case demonstrates the difficulties to correctly diagnose this rare disorder as both insulin and proinsulin levels were increased by crossreactive autoantibodies. No known triggering agent could be identified. We suggest that this diagnosis should be considered more often also in caucasian patients to avoid useless operations for such patients.

Aged↗
PubMed2001

Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity.

Constitutively activating mutations in the human thyroid-stimulating hormone (TSH) receptor (TSHr) have been identified as the most prevalent molecular cause of non-autoimmune hyperthyroidism. To investigate the feasibility of an animal model for non-autoimmune hyperthyroidism, we introduced two mutations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, basal cAMP and IP accumulation. In the human TSHr, the Ile 486 Phe mutation causes a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signaling. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2.4 kb PCR product from the mouse TSHr was cloned into the pGEM-T vector system. Ile was substituted with Phe at codon 486 and Val with Ala at codon 509. These mutated mouse TSHrs were subcloned in the pSVL expression vector. After transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined and directly compared to the human TSHr. Whereas constitutively activating mutations of the human parathyroid hormone (PTH)/PTH-related peptide receptor showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in constitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimmune hyperthyroidism by homologous recombination to study the pathogenetic mechanisms of non-autoimmune hyperthyroidism.

Animals↗
PubMed2001

Growth factor expression in cold and hot thyroid nodules.

Hot thyroid nodules (HTNs) are predominantly caused by constitutively activating thyrotropin receptor (TSHR) mutations leading to an activation of the cyclic adenosine monophosphate (cAMP)-cascade that stimulates growth and function of thyroid epithelial cells and confers growth advantage. In contrast to HTNs, the molecular etiology of szintigraphically cold thyroid nodules (CTNs) is largely unknown. An increased prevalence of toxic multinodular goiters in iodine-deficient regions has been reported. Growth factors increase during early stages of iodine deficiency in rats. These growth factors could modulate the proliferation of thyrocytes. In order to determine if and which growth factors could modulate the increase in thyroid epithelial cell proliferation in late stages of CTNs and HTNs we investigated epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and TGF-beta1 concentrations by enzyme-linked immunosorbant assay (ELISA) in CTNs (n = 7), HTNs (n = 9), and their normal surrounding tissue (ST). Insulin-like growth factor-1 (IGF-1) was determined in CTNs (n = 5) and HTNs (n = 10) and their surrounding tissues by radioimmunoassay (RIA). We found lower concentrations of all investigated growth factors and iodine in CTNs compared to surrounding normal tissues (ST). Only iodine showed a significant difference. Furthermore, we found significantly lower concentrations of EGF and TGF-beta1 concentration in HTNs compared to their STs. Differences of TGF-alpha and IGF-1 were not significant. In conclusion, low EGF, TGF-alpha, and IGF-1 concentrations in most CTNs in spite of low iodine concentrations argue against a pathophysiologic role of EGF, TGF-alpha, or IGF-1 in late stages of CTNs. The low EGF, TGF-alpha, and IGF-1 concentrations in HTNs irrespective of their clonal origin or the presence or absence of activating mutations argue for increased cAMP as the primary cause for thyroid epithelial cell proliferation in established HTNs. However, the pathophysiologic significance of low TGF-beta1 concentrations in CTNs and HTNs remains to be elucidated. It might be possible that growth factors like EGF, TGF-alpha, TGF-beta1, and IGF-1 play a more prominent role during early clonal expansion and that aberrant intrinsic signaling through a somatic mutation (e.g., TSHR for HTNs) confers the predominant selective growth advantage in later stages of HTNs or CTNs.

Epidermal Growth Factor↗
PubMed2001

A free carboxylate oxygen in the side chain of position 674 in transmembrane domain 7 is necessary for TSH receptor activation.

A specific H-bonding network formed between the central regions of transmembrane domain 6 and transmembrane domain 7 has been proposed to be critical for stabilizing the inactive state of glycoprotein hormone receptors. Many different constitutively activating TSH receptor point mutations have been identified in hyperfunctioning thyroid adenomas in the lower portion of transmembrane domain 6. Position D633 in transmembrane domain 6 of the human TSH receptor is the only one in which four different constitutively activating amino acid exchanges have been identified. Further in vitro substitutions led to constitutive activation of the TSH receptor (D633Y, F, C) as well as to the first inactivating TSH receptor mutation in transmembrane domain 6 without changes of membrane expression or TSH binding (D633R). Molecular modeling of this inactivating TSH receptor mutation revealed potential interaction partners of R633 in transmembrane domain 3 and/or transmembrane domain 7, presumably via hydrogen bonds that could be responsible for locking the TSH receptor in a completely inactive state. To further elucidate the H-bond network that most likely maintains the inactive state of the TSH receptor, we investigated these potential interactions by generating TSH receptor double mutants designed to break up possible H bonds. We excluded S508 in transmembrane domain 3 as a possible interaction partner of R633. In contrast, a partial response to TSH stimulation was rescued in a receptor construct with the double-substitution D633R/N674D. Our results therefore confirm the H bond between position 633 in transmembrane domain 6 and 674 in transmembrane domain 7 suggested by molecular modeling of the inactivating mutation D633R. Moreover, the mutagenesis results, together with a three-dimensional structure model, indicate that for TSH receptor activation and G protein-coupled signaling, at least one free available carboxylate oxygen is required as a hydrogen acceptor atom at position 674 in transmembrane domain 7.

Adenylyl Cyclases↗
PubMed2001

Familial isolated primary hyperparathyroidism--a multiple endocrine neoplasia type 1 variant?

OBJECTIVE: Familial isolated primary hyperparathyroidism (FIHP) is defined as hereditary primary hyperparathyroidism without the association of other diseases or tumors. Linkage analyses suggest that different genotypes can lead to the same phenotype of primary hyperparathyroidism. Hereditary syndromes associated with primary hyperparathyroidism are multiple endocrine neoplasia type 1 and type 2 (MEN 1 and MEN 2). In MEN 1, multiple parathyroid adenomas occur in more than 90% of the patients. Therefore, it has been suggested that FIHP could represent a variant or partial expression of MEN 1. DESIGN: We report on a large FIHP kindred with a MEN1 gene mutation. Nineteen family members (aged 10 to 87 years) were screened. Furthermore, statistical comparison by Fisher's exact tests of FIHP families with MEN1 gene mutations and MEN 1 families with two or more endocrinopathies was carried out to investigate genotype-phenotype correlations. METHODS: Mutational analysis of leucocyte DNA was carried out by direct sequencing of the complete coding region of the MEN1 gene. Screening of MEN 1 manifestations was carried out by determination of serum calcium, phosphate, parathyroid hormone, prolactin, ACTH, cortisol, IGF-I, gastrin, glucose, insulin, glucagon, serum potassium, aldosterone, plasma renin and urinary hydroxyindoleacetic acid. RESULTS: We detected an in-frame deletion mutation in exon 8 of the MEN1 gene resulting in the deletion of one glutamine acid residue at position 363. It was found in eight individuals. Two of these family members (aged 42 and 60 years) were operated for primary hyperparathyroidism, and three (aged 13 to 40 years) showed mild hypercalcemia and parathyroid hormone levels within the upper normal range or slightly elevated, without any clinical symptoms. Two individuals (aged 12 and 19 years) were normocalcemic. One could not be tested. None of them had clinical evidence of other MEN 1 manifestations. Statistical comparison of the mutation types in families with FIHP and families with two or more MEN 1-associated endocrinopathies reported in other studies reveals a significant difference. In families with FIHP, missense/in-frame mutations have been found in 87.5% of cases whereas in families with tumors in various endocrine glands these mutation types occur much less frequently (21-34%, P<0.05). CONCLUSIONS: These studies indicate that FIHP can represent a partial MEN 1 variant and is often caused by missense/in-frame mutations.

Adolescent↗
PubMed2001

Plasma levels of tumor necrosis factor-alpha, angiotensin II, growth hormone, and IGF-I are not elevated in insulin-resistant obese individuals with impaired glucose tolerance.

OBJECTIVE: To investigate the relationship between insulin resistance and plasma concentrations of free fatty acids (FFAs), leptin, and potential agonists of the insulin receptor substrate (IRS) system, including tumor necrosis factor-alpha (TNF-alpha), IGF-I, growth hormone (GH), and angiotensin II in individuals with impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS: Because glucose toxicity per se leads to insulin resistance, the determination of the primary metabolic alterations leading to insulin resistance is best accomplished in individuals who are at an increased risk to develop type 2 diabetes. Therefore, 48 subjects with IGT and insulin resistance (IR), characterized by hyperinsulinemic-euglycemic clamps, were compared with 52 healthy insulin-sensitive (IS) control subjects with respect to the relationship between the plasma levels of TNF-alpha, IGF-I, GH, angiotensin II, FFA, leptin, and insulin resistance. RESULTS: Between the IR and the IS groups, there were no significant differences in the plasma concentrations of TNF-alpha, GH, angiotensin II, IGF-I, and leptin. However, plasma FFA levels were significantly elevated in the IR group compared with the IS group after matching for BMI. CONCLUSIONS: The plasma concentrations of FFA, but not TNF-alpha, IGF-I, GH, and angiotensin II, are elevated in patients at an early stage of insulin resistance, suggesting that FFAs, but not the other modulators of the IRS system, may be a primary metabolic abnormality leading to insulin resistance.

Adult↗
PubMed2000

Expression of beta-arrestins in toxic and cold thyroid nodules.

beta-Arrestins mediate agonist dependent desensitization of G protein-coupled receptors. Somatic TSH receptor mutations were identified in the majority of hot thyroid nodules. When transiently overexpressed in COS 7 cells these mutations resulted in constitutive activation of the cAMP pathway. However, the in vivo mechanisms and the in vivo desensitization of these TSH receptor mutations are unknown. Moreover, constitutively activated beta-adrenergic receptors are known to be constitutively desensitized. Therefore, we investigated the expression of beta-arrestins in toxic thyroid nodules (TTNs) with and without somatic TSH receptor mutation and in cold thyroid nodules (CTNs) by Western blotting and ELISA. Expression of beta-arrestin 2 was increased in all TTNs while beta-arrestin 2 expression was decreased in CTNs compared to their corresponding surrounding tissue. The mean beta-arrestin 1 expression was unchanged in the cytosol of TTNs, in membranes and cytosol of CTNs and decreased in the membranes of TTNs compared to their surrounding tissue. Transient coexpression of beta-arrestins 1 or 2 with the TSH receptor in HEK 293 cells and subsequent determination of cAMP showed that in vitro both beta-arrestins interact with the TSH receptor and are able to desensitize the receptor. The increased beta-arrestin 2 expression in TTNs and the desensitization of the TSH receptor by beta-arrestin 2 in vitro suggest that the beta-arrestin 2 expression is cAMP dependent and that beta-arrestin 2 very likely desensitizes the constitutively activated TSH receptor in toxic thyroid nodules.

Arrestins↗
PubMed2000

[Possible genetic causes for late complications of diabetes mellitus].

BACKGROUND: Hyperglycemia occurs in every patient with diabetes mellitus. It is the most important factor in the development of diabetic complications. However, the onset, intensity and the progression of complications show large interindividual variations. Manifestation in families and the lack of complications in some diabetics with poor metabolic control indicate a genetic predisposition to develop diabetic complications like nephropathy, neuropathy and angiopathy. NEPHROPATHY: Diabetic nephropathy occurs only in 25 to 40% of the diabetic patients. Therefore a genetic risk factor for this complication is very likely. Various variations in genes like ACE-gene and angiotensinogen-gene have been described, which could be associated with the development of diabetic nephropathy. NEUROPATHY: Peripheral diabetic neuropathy occurs in up to 66% of all diabetics. Therefore and because of the possible pathological mechanisms genetic risk factors like variations in the Na/K-ATPase-gene and in the aldose reductase-gene are discussed. RETINOPATHY: An association between diabetic retinopathy and polymorphisms in the ACE-gene and the aldose reductase-gene seems very unlikely, because up to 75% of the diabetic patients suffer from retinopathy after 15 years of diabetes. MACROANGIOPATHY: A large number of studies show an association between diabetic macroangiopathy and genetic variations in the ACE-gene (I/D-variant) and the paraoxonase-gene (2 isoforms). CONCLUSION: Based on the current evidence for associations of genetic markers with diabetic complications, the generation of an individual risk profile based on genetic markers seems to be possible. In addition to near euglycemia genetic markers could direct therapeutic strategies and lead to new therapeutic approaches.

Cardiovascular Diseases↗
PubMed2000

Hot microscopic areas of iodine-deficient euthyroid goitres contain constitutively activating TSH receptor mutations.

Constitutively activating TSH receptor mutations have been established as the most common molecular basis for the pathogenesis of toxic thyroid nodules. These mutations result in uncontrolled signalling through the TSH receptor that is likely to cause hyperfunction and proliferation. The incidence of toxic multinodular goitres has been demonstrated to be related to iodine deficiency. Moreover, scintigraphically autonomous areas are found in 40% of euthyroid goitres from iodine-deficient areas. To investigate the molecular cause of these autonomous areas, small autoradiographically hot areas were examined for somatic TSH receptor mutations using archival tissue sections from 14 patients with euthyroid goitres, which had been originally prepared nearly 20 years ago. All patients had received (125)I 17 h preoperatively for the autoradiographic investigation of their thyroid. Areas with high and low (125)I-labelling on autoradiography sections were collected separately either from serial paraffin-embedded tissue sections, or Eukitt-embedded tissue sections containing the autoradiograph. After genomic DNA extraction, the transmembrane segment of the TSH receptor was PCR-amplified and directly sequenced. Somatic TSH receptor mutations were identified in areas with high (125)I-labelling in four patients: A623I, L629P, F631L, and T632I. This is the first evidence that TSH receptor mutations occur in microscopic areas with increased (125)I-labelling in euthyroid goiters and it suggests that TSH receptor mutations in these areas confer the potential to develop into toxic thyroid nodules. It is therefore very likely that toxic thyroid nodules originate from small autonomous areas in iodine-deficient euthyroid goitres that contain a TSH receptor mutation.

Female↗
PubMed2000

Functional characterization of five constitutively activating thyrotrophin receptor mutations.

OBJECTIVE: Gain of function mutations of the thyrotrophin receptor (TSHR) affect several functional characteristics, such as cAMP and inositol phosphate (IP) accumulation, cell surface expression and TSH affinity. In this study we compared five constitutively activating TSHR mutations, four receptors with a point mutation (S505N, L629F, I630L, V656F) and a nine amino acid (aa) deletion mutant (aa positions 613-621) for these functional parameters in parallel transfection experiments. METHODS: The wild-type TSHR (wt) and TSHRs containing the mutations S505N, L629F, I630L, V656F and the deletion 613-621 (all cloned in the expression vector pSVL) were transiently expressed in COS-7 cells in parallel experiments. Forty-eight hours after transfection the basal and stimulated cAMP and inositol phosphate accumulation as well as the cell surface expression (by FACS and ELISA), KD-values and TSHR down regulation by different stimuli were determined. RESULTS: In contrast to the very different values for specific constitutive activity (sca) (ranging from 7.5 to 100.3-fold wt) and very different levels of receptor cell surface expression (11-94% wt level) the basal cAMP accumulation determined in transfected COS-7 cells was surprisingly uniform (6.5-8.0 over wt basal). None of the point mutated receptors constitutively activates the phospholipase C cascade. In contrast the deletion 613-621 mutant showed constitutive activity for the IP pathway with a twofold increase in basal IP accumulation compared to the wild type TSHR. All investigated TSHR-mutants showed a TSH-stimulated receptor down-regulation, which seems to be independent of the phospholipase C pathway. CONCLUSIONS: The uniform basal cAMP values in spite of the large variation in specific constitutive activity values suggest that the COS-7 cell overexpression system used for the in vitro characterization is partly regulated. This regulation is most likely due to receptor down regulation. The TSHR deletion mutant (613-621) showed a constitutive activity for both the Galphas and the Galphaq/11 pathways. The TSH-mediated IP-stimulation by this mutant contrasts with its unresponsiveness to TSH for cAMP accumulation and therefore supports the model of different active conformations of the TSHR.

Animals↗
PubMed2000

Novel TSHR germline mutation (Met463Val) masquerading as Graves' disease in a large Welsh kindred with hyperthyroidism.

Hereditary nonautoimmune hyperthyroidism is caused by activating germline mutations in the thyrotropin (TSH) receptor (TSHR) gene. We describe an extended Welsh kindred with toxic thyroid hyperplasia affecting 8 family members in three generations and a history consistent with thyrotoxicosis in a further three generations now deceased. A novel heterozygous germline mutation (ATG --> GTG; Met463Val) was identified in the second membrane spanning TSHR region in 6 relatives with thyrotoxicosis and goiter and absence of TSHR antibodies. Screening of 5 additional family members led to the identification of 2 siblings with the mutation, who were asymptomatic at the time, although subsequent thyroid function tests in these children showed suppressed serum TSH and increased serum free triiodothyronine (FT3) and free thyroxine (FT4) concentrations. Functional studies of the novel TSHR germline mutation demonstrated a constitutive activation of the cAMP pathway, which in the thyroid controls both thyroid hormone production and stimulation of thyroid growth. The molecular diagnosis in this family has clinical implications: genetic counseling is required, and primary thyroid ablation should be advocated as the preferred treatment in the patients with the constitutively activating TSHR germline mutation.

Animals↗