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Biomedical subjects

A Laslop

Publications and source records attributed to A Laslop.

At least 19 recordsLinked to original sources
PubMed2005

Total joint replacement of hip or knee as an outcome measure for structure modifying trials in osteoarthritis.

OBJECTIVE: The Group for the Respect of Ethics and Excellence in Science (GREES) organized a working group to assess the value of time to joint surgery as a potential therapeutic failure outcome criterion for osteoarthritis (OA) of the hip or knee in the assessment of potential structure modifying agents. METHODS: PubMed was searched for manuscripts from 1976 to 2004. Relevant studies were discussed at a 1-day meeting. RESULTS: There are no accepted guidelines for 'time to' and 'indications for' joint replacement surgery. A limited number of trials have examined joint replacement surgery within the study population. Several parameters, particularly joint space narrowing (interbone distance), correlate with surgical intervention. However, at the level of the knee, none of the parameters have positive predictive value for joint replacement surgery better than 30%. In contrast, lack of significant joint space narrowing has a strong negative predictive value for joint replacement surgery (>90%), that remains after controlling for OA pain severity. CONCLUSION: At this time, GREES cannot recommend time to joint surgery as a primary endpoint of failure for structure modifying trials of hip or knee OA-as the parameter has sensitivity but lacks specificity. In contrast, in existing trials, a lack of progression of joint space narrowing has predictive value of >90% for not having surgery. GREES suggests utilizing joint space narrowing (e.g., >0.3-0.7 mm) combined with a lack of clinically relevant improvement in symptoms (e.g., >/=20-25%) for 'failure' of a secondary outcome in structure modifying trials of the hip and knee.

Antirheumatic Agents↗
PubMed2002

The use of placebo-controlled and non-inferiority trials for the evaluation of new drugs in the treatment of postmenopausal osteoporosis.

Registration of new agents for the treatment of postmenopausal osteoporosis has been based over the past few years on placebo-controlled phase III trials with the incidence of patients with new vertebral/nonvertebral fractures as the most usual primary endpoint. The use of a placebo in diseases where an active treatment is available has been a matter of debate following the update of the Declaration of Helsinki by the World Medical Association which questioned this trial design. Current regulatory recommendations within the European Union suggest that placebo-controlled trials are still the best option when assessing the efficacy and safety of new drugs intended for the treatment of postmenopausal osteoporosis. This suggestion seems to be in apparent contradiction with the current content of the Declaration of Helsinki. This paper addresses the ethics and feasibility of placebo-controlled trials in the treatment of postmenopausal osteoporosis, in the light of available therapeutic options, and discusses possible alternative approaches in those patients where placebo treatment could be deemed to be unethical. It is concluded that placebo-controlled trials remain the most efficient design to establish the efficacy and safety of a new agent for the treatment of postmenopausal osteoporosis. Such trials are feasible and ethically acceptable in patients with osteoporosis but without prevalent vertebral fractures. Conversely, in patients with prevalent vertebral fractures, placebo-controlled trials are ethically questionable and non-inferiority trials are more appropriate. A relative margin of non-inferiority of 20-30% is suggested, to be discussed on a case by case basis.

Aged↗
PubMed2001

Regulation of chromogranin biosynthesis by neurotrophic growth factors in neuroblastoma cells.

Polypeptide growth factors secreted from the target tissue determine the choice of transmitter synthesis in the innervating nerves. We have investigated whether they also influence the expression of chromogranins and neuropeptide Y, components co-stored with the neurotransmitters within large dense-core vesicles. IMR-32 and SH-SY5Y human neuroblastoma cells were treated for up to six days with various neurotrophic growth and differentiation factors. For chromogranins A and B, no significant changes at the mRNA level were observed and for chromogranin A this was confirmed at the protein level. The expression of secretogranin II/pro-secretoneurin mRNA, however, was considerably enhanced in both cell lines after basic fibroblast growth factor treatment. In IMR-32 cells we determined a fast and continuous induction, whereas the up-regulation in SH-SY5Y cells was more delayed. A transient elevation of secretogranin II/pro-secretoneurin mRNA levels was seen in SH-SY5Y cells in response to epidermal growth factor. In these cells we also measured the amounts of secretogranin II/pro-secretoneurin protein which were increased by both growth factors. In addition to the above described changes in secretogranin II/pro-secretoneurin biosynthesis we extended and confirmed data available on neuropeptide Y. We found a qualitatively similar pattern of biosynthesis regulation as for secretogranin II/pro-secretoneurin, indicating that the ultimately increased expression of the two proteins may be characteristic of the phenotypic differentiation after growth factor treatment. Moreover, this finding of a concomitant regulation further emphasizes the concept of secretogranin II/pro-secretoneurin being a neuropeptide precursor from which the functional peptide secretoneurin is proteolytically liberated.

Cell Differentiation↗
PubMed2001

Differential regulation of chromogranin A, chromogranin B and secretogranin II in rat brain by phencyclidine treatment.

Chromogranin A, chromogranin B and secretogranin II belong to the chromogranin family which consists of large protein molecules that are found in large dense core vesicles. Chromogranins are endoproteolytically processed to smaller peptides. This study was designed to elucidate the regulation of chromgranin expression by acute and subchronic phencyclidine administration. The behavioral syndrome produced by phencyclidine represents a pharmacological model for some aspects of schizophrenia [Jentsch and Roth (1999) Neuropsychopharmacology 20, 201-225]. Tissue concentrations of chromogranins were measured with specific radioimmunoassays. Alterations in secretogranin II gene expression were investigated by in situ hybridization. A single dose of phencyclidine (10mg/kg) led to a transient decrease in secretoneurin tissue levels in the prefrontal cortex after 4h followed by an increase in secretoneurin tissue levels after 12h. Repeated phencyclidine treatment (10mg/kg/day) for five days resulted in elevated secretoneurin levels in cortical areas whereas chromogranin A and chromogranin B tissue levels were unchanged. After the same treatment, a significant increase in the number of secretoneurin containing neurons was found in cortical layers II-III, and V-VI as revealed by immunocytochemistry. The increases in secretoneurin levels were paralleled by an increased number of secretogranin II messenger RNA containing neurons as well as by an increased expression of secretogranin II by individual neurons. The present study shows that secretoneurin II tissue concentration and secretogranin II messenger RNA expression is distinctly altered after acute and subchronic phencyclidine application. From these results we suggest that phencyclidine may induce synaptic alterations in specific brain areas and may contribute to a better understanding of synaptic dysfunction which may also occur in schizophrenia.

Animals↗
PubMed2000

Regulation of the biosynthesis and processing of chromogranins in organotypic slices: influence of depolarization, forskolin and differentiating factors.

Slices from rat hippocampus in organotypic culture were used to study the biosynthesis regulation of chromogranins A and B and secretogranin II. Additionally, we investigated the proteolytic conversion of secretogranin II and the levels of prohormone convertases putatively involved. Forskolin treatment and depolarization with potassium plus BayK 8644 led to significant increases in secretogranin II mRNA in the principal cells of the hippocampus. Enhanced expression of secretogranin II was also reflected by a rise in peptide levels. Despite this induction of biosynthesis the extensive processing to secretoneurin normally observed in brain was maintained. Both forskolin and depolarization upregulated the prohormone convertase (PC)1, but not PC2, indicating that PC1 levels are critical for secretoneurin production under stimulating conditions. Results obtained for chromogranins A and B were less consistent. For chromogranin A mRNA, changes were restricted to granule cells; for chromogranin B, a response in granule cells was observed to depolarization but not to forskolin, and effects in pyramidal neurons were weak. Accordingly, we were unable to detect alterations in chromogranin A and B protein levels. Furthermore, we tested several neurotrophic growth factors and found that only basic fibroblast growth factor raised secretogranin II expression without affecting chromogranins A and B. The hippocampal slice preparation allowed well controlled treatment with identification of neuronal subpopulations and yielded data largely matching experiments in vivo and in cell culture. The pronounced regulation of secretogranin II and its effective processing underlines the importance of the resulting peptide secretoneurin as an active neuropeptide in the nervous system.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2000

Levels and molecular properties of secretoneurin-immunoreactivity in the serum and urine of control and neuroendocrine tumor patients.

We have determined the levels of secretoneurin (SN), a novel 33-amino acid neuropeptide belonging to the class of chromogranins, in the serum and urine of healthy subjects and patients suffering from various tumors. SN serum levels averaged 22.1+/-1.1 fmol/mL. They were 5-fold higher in younger children and then declined continuously. SN levels were positively correlated with serum creatinine, suggesting an influence of renal function on the clearance of SN from the serum. In the urine 80.0 fmol/mL SN was present. In patients with endocrine tumors like gut carcinoids, endocrine pancreatic tumors, oat cell lung carcinomas, and pheochromocytomas, SN serum levels were elevated up to 45-fold. Patients suffering from neuroblastomas, insulinomas, pituitary adenomas including acromegaly, and solid nonendocrine tumors had concentrations in the normal range. In human serum, SN-immunoreactivity was confined to the free peptide SN; neither larger intermediate-sized forms nor the precursor secretogranin II were detected. An efficient removal of the small molecule SN from the serum by the kidney explains why SN serum levels are lower when compared to chromogranin A, which is present as large molecule in serum.

Adolescent↗
PubMed1999

The new chromogranin-like protein NESP55 is preferentially localized in adrenaline-synthesizing cells of the bovine and rat adrenal medulla.

The protein NESP55, a new member of the chromogranin family, is present in large dense-core secretory granules of neuroendocrine tissues. We investigated its cellular distribution in adrenal medulla with immunohistochemistry and in situ hybridization. A preferential co-localization of NESP55 with phenylethanolamine-N-methyltransferase in the adrenergic cell population was found by immunolabelling of consecutive sections. Noradrenergic cells also contained small amounts of NESP55, but the levels as measured by radioimmunoassay were five times lower. The distribution of NESP55 mRNA was similar to preproenkephalin mRNA which previously was shown to be confined to adrenaline-producing cells of the adrenal medulla. The present study indicates that stimulation of adrenergic cells will release significantly higher amounts of NESP55. The functional implications of this preferential secretion, however, have yet to be discovered.

Adrenal Medulla↗
PubMed1998

Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases.

Two experimental approaches were used to study the processing of chromogranin B and secretogranin II by prohormone convertases. In GH3 cells various prohormone convertases were overexpressed together with the substrate chromogranin B by use of a vaccinia virus infection system. PC1 appeared to be by far the most active enzyme and converted chromogranin B to several smaller molecules, including the peptide PE-11. In brain this peptide is cleaved physiologically from chromogranin B. Some processing of chromogranin B and formation of free PE-11 were also observed with PC2 and PACE4. Furin produced larger fragments, whereas PC5-A and PC5-B had negligible effects. As a second model, PC12 cells were stably transfected with PC1 or PC2 to investigate the processing of endogenous chromogranins. Both enzymes effectively cleaved chromogranin B and secretogranin II, liberating the peptides PE-11 and secretoneurin, respectively. However, in transfection experiments the ability to generate the free peptides was more pronounced with PC2 than with PC1. The extent of proprotein processing achieved by prohormone convertases apparently differed depending on the experimental system applied. This suggests that in vivo mechanisms to support and fine-tune the activity of the processing enzymes exist, which might be overlooked by using only one methodological approach.

Animals↗
PubMed1998

Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa.

The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.

Amino Acid Sequence↗
PubMed1997

Alpha1-antitrypsin Portland inhibits processing of precursors mediated by proprotein convertases primarily within the constitutive secretory pathway.

We studied the extent of cellular inhibitory activity of alpha1-antitrypsin Portland (alpha1-PDX), a potent inhibitor of proprotein convertases of the subtilisin/kexin type. We compared the inhibitory effects of alpha1-PDX on the intracellular processing of two model precursors (pro-7B2 and POMC) mediated by six of the seven known mammalian convertases, namely furin, PC1, PC2, PACE4, PC5-A, PC5-B, and PC7. The substrates selected were pro7B2, a precursor cleaved within the trans-Golgi network (TGN), and pro-opiomelanocortin, which is processed in the TGN and secretory granules. Biosynthetic analyses were performed using either vaccinia virus expression in BSC40, GH4C1, and AtT20 cells, or stable transfectants of alpha1-PDX in AtT20 cells. Results revealed that alpha1-PDX inhibits processing of these precursors primarily within the constitutive secretory pathway and that alpha1-PDX is cleaved into a shorter form by some convertases. Evidence is presented demonstrating that in contrast to the full-length alpha1-PDX (64 kDa), the cleaved (56 kDa) secreted product does not significantly inhibit furin activity in vitro. Cellular expression of alpha1-PDX results in modified contents of mature secretory granules with increased levels of partially processed products. Biosynthetic and immunocytochemical analyses of AtT20/alpha1-PDX cells demonstrated that alpha1-PDX is primarily localized within the TGN, and that a small proportion enters secretory granules where it is mostly stored as the cleaved product.

Animals↗
PubMed1996

Chromaffin granule membrane glycoprotein IV is identical with Ac45, a membrane-integral subunit of the granule's H(+)-ATPase.

Glycoprotein IV of bovine adrenal chromaffin granule membranes was purified by membrane fractionation with Triton X-114 and lectin affinity chromatography. An antiserum raised against this protein recognized the same component as one directed against subunit Ac45 of the proton-translocating adenosine triphosphatase in the granule membrane. Amino acid sequencing confirmed that glycoprotein IV and Ac45 are identical proteins, and also showed that they are derived from a larger precursor by removal of a 246-amino acid N-terminal sequence. Enzymatic deglycosylation indicated an apparent polypeptide molecular mass of 29 kDa for the mature Ac45/glycoprotein IV. Blue Native electrophoresis confirmed that this protein is a component of the membrane sector of the V-ATPase.

Adrenal Medulla↗
PubMed1996

Processing of chromogranins in chromaffin cell culture: effects of reserpine and alpha-methyl-p-tyrosine.

Bovine chromaffin cell cultures were treated with either reserpine or alpha-methyl-p-tyrosine for up to 10 days. Afterwards the cells were harvested and the degree of proteolytic processing of secretogranin II, chromogranin A and chromogranin B was determined by immunoblotting and HPLC followed by RIA. There was a significant increase in the proteolysis of all three chromogranins after 4-6 days in the presence of reserpine. The small peptides formed in the presence of reserpine in vitro are also produced in vivo. A similar effect was observed with alpha-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, but the response took up to 10 days to develop. Both drugs decreased catecholamine levels but reserpine was more effective, reaching a high degree of depletion after 4 days. In addition, experiments in vitro indicate that low millimolar amounts of either adrenaline (IC50 5.2 mM) or noradrenaline (IC50 2.4 mM) can significantly impair the proteolytic activity of recombinant murine prohormone convertase 1 when assayed with synthetic fluorogenic and/or peptidyl substrates. We conclude that a lowering of catecholamine levels in chromaffin granules leads to a concomitant increase in proteolytic processing of all secretory peptides. Apparently within chromaffin granules the endoproteases are inhibited by catecholamines and thus their removal leads to increased proteolysis.

Adrenal Medulla↗
PubMed1996

Differences in the composition of chromaffin granules in adrenaline and noradrenaline containing cells of bovine adrenal medulla.

Several constituents of chromaffin granules were quantitatively determined in noradrenaline and adrenaline cells purified from bovine adrenal medulla. As far as secretory peptides are concerned noradrenaline granules contained slightly more secretogranin II, but much less chromogranin A than adrenaline granules. This can be explained by the dependence of the biosynthesis of chromogranin A on corticosteroids. Proteolytic processing of chromogranin A and secretogranin II was higher in noradrenaline cells which was paralleled by a higher content of the prohormone convertase PC2. Noradrenaline granules also contained a higher concentration of the vesicular monoamine transporter (vMAT2). No differences were found for dopamine beta-hydroxylase, prohormone convertase PC1, carboxypeptidase H and synaptophysin. These results indicate that the secretory cocktail of peptides released from these cells differs significantly between adrenaline and noradrenaline storing cells.

Adrenal Cortex Hormones↗
PubMed1996

Nicotine and prostaglandin E induce secretogranin II levels in bovine chromaffin cells.

The synthesis regulation of secretogranin II was investigated in bovine chromaffin cells by treatment with various first messengers. Nicotine and prostaglandin E2 elevated secretogranin II mRNA and protein up to three-fold. Angiotensin II, atrial natriuretic peptide, apomorphine, bradykinin and clonidine on the other hand had no effect. The prostaglandin E induced elevation of secretogranin II mRNA was transduced via the calcium/calmodulin pathway but not via the protein kinase A or C pathways as shown by using specific inhibitors. Exposure of chromaffin cells to drugs specifically activating second messenger pathways both elevated and decreased secretogranin II mRNA. The calcium channel agonist Bay K, forskolin and phorbol esters increased secretogranin II mRNA whereas 8-Br-cGMP repressed the secretogranin II message. Thus, although secretogranin II expression can be altered by all major second messenger transduction systems, regulation of secretogranin II in vivo occurs mainly via the calcium/calmodulin pathway. Chromogranin A and B mRNA were not changed by any of the first messengers investigated indicating a differential synthesis regulation of components co-stored in bovine chromaffin granules.

Animals↗
PubMed1995

Processing of secretogranin II by prohormone convertases: importance of PC1 in generation of secretoneurin.

Secretoneurin is a recently characterized neuropeptide present in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.

Adrenal Medulla↗
PubMed1995

Biosynthesis of large dense-core vesicles in PC12 cells: effects of depolarization and second messengers on the mRNA levels of their constituents.

mRNA levels of various constituents of large dense-core vesicles were determined in PC12 cells during depolarization and/or in the presence of BayK 8644, forskolin or phorbolester. For the soluble (secretory) proteins of the vesicles the mRNAs of chromogranin A and B, secretogranin II, neuropeptide Y and VGF were analyzed. Depolarization in the presence of BayK induced a strong up-regulation of the messages for chromogranin B, neuropeptide Y and VGF. Addition of forskolin enhanced this response for neuropeptide Y and VGF, phorbolester did the same only for VGF. Partly membrane-bound and membrane-spanning components analyzed were carboxypeptidase H, dopamine beta-hydroxylase and glycoprotein III (clusterin), peptidylglycine alpha-amidating mono-oxygenase and cytochrome b-561, respectively. Changes of mRNAs for these components were in general smaller and delayed. Six days of depolarization caused an up-regulation of glycoprotein III, peptidylglycine alpha-amidating mono-oxygenase and carboxypeptidase H mRNA levels which were not further increased by cyclic AMP and phorbolester. The dopamine beta-hydroxylase message increased after 6 days of depolarization, however, addition of phorbolester reduced this effect. For cytochrome b-561 there was no change after any of the conditions employed. These in vitro results are compared with those obtained for the biosynthesis regulation of large dense-core vesicles under in vivo conditions. It is suggested that in vivo acetylcholine and vasoactive intestinal polypeptide released from splanchnic nerve induce a differential change in the biosynthesis of large dense-core vesicles by acting via calcium and protein kinase A and C.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗