bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insulin/IGF‐I receptor hybrids: A mechanism for increasing receptor diversity
Authors: Moxham Cary P. , Jacobs Steven 
Published: May 2005

AbstractInsulin and IGF‐l receptors are homologous disulfide linked α2β2 tetramers. These tetramers are formed biosynthetically when proreceptors containing α and β subunits in a single uninterrupted linear peptide from disulfide linked homodimers and are subsequently proteolytically cleaved at the α‐β junctions. Cells expressing both receptors also express hybrid receptors that contain one insulin receptor α and β subunit, and one IGF‐l receptor α and β subunit. These presumably from by the association of mixed proreceptors. Hybrid receptors greatly expand the possible repertoire of cellular responses to hormonal stimulation. Although not yet examined in detail, both the hormone binding and the signaling properties of the hybrid receptor appear to be different from that of either insulin or IGF‐l receptor. Regulatory mechanisms that involve either insulin or IGF‐l receptor, at the level of expression or subsequently, could alter the expression or function of the hybrid receptor or the other receptor. Similarly, pathology in one receptor could affect both the hybrid and other receptor, or perhaps be partially compensated for by a hybrid receptor. The magnitude of these effects could vary greatly in different tissues depending upon the relative level of expression of the different receptor forms. These postulated responses might explain some of the complex heterogeneity and linkage of these receptors that have been observed previously.
Heterotrimeric configuration is essential to the adhesive function of laminin
Authors: Lissitzky J. C. , Cantau P. , Martin P. M. 
Published: May 2005

AbstractMouse PFHR9 laminin, B1B2‐heterodimers, and free B1‐chains were separated from one another by gel filtration on superose 6. The cell attachment promoting activity of these species was measured after immunoprecipitation with monoclonal anti‐laminin antibodies coupled to Sepharose 6MB beads. These antibodies, Which did not react with the laminin E8 fragment, were directed against epitopes in the NH2‐terminus of the laminin B1‐chain and in the central region of laminin. After incubation with purified EHS laminin, the immunosorbents revealed efficient adhesion substrates for a rat rhabdomyosarcoma cell line which attached preferentially to the laminin E8 fragment. Although both were immunoprecipitated efficiently, B1B2‐heterodimers and B1‐chains, unlike PFHR9 laminin, did not support the attachment of RMS cells. On a molar basis B1B2‐heterodimers were 24 times less efficient than PFHR9 laminin or EHS laminin in supporting cell attachment. These data suggest that heterotrimeric configuration is essential to the adhesive function of the laminin E8 fragment.
The role of ion channels in insulin secretion
Authors: Boyd A. E. 
Published: May 2005

AbstractIon channels in beta cells regulate electrical and secretory activity in response to metabolic, pharmacologic, or neural signals by controlling the permeability to K+ and Ca2+. The ATP‐sensitive K+ channels act as a switch that responds to fuel secretagogues or sulfonylureas to initiate depolarization. This depolarization opens voltage‐dependent calcium channels (VDCC) to increase the amplitude of free cytosolic Ca2+ levels ([Ca2+]i), which triggers exocytosis. Acetyl choline and vasopressin (VP) both potentiate the acute effects of glucose on insulin secretion by generating inositol 1,4,5‐trisphosphate to release intracellular Ca2+; VP also potentiates sustained insulin secretion by effects on depolarization. In contrast, inhibitors of insulin secretion decrease [Ca2+]i by either hyperpolarizing the beta cell or by receptor‐mediated, G‐protein‐coupled effects to decrease VDCC activity. Repolarization is initiated by voltage‐ and Ca2+‐activated K+ channels. A human insulinoma voltage‐dependent K+ channel cDNA was recently cloned and two types of alpha1 subunits of the VDCC have been identified in insulin‐secreting cell lines. Determining how ion channels regulate insulin secretion in normal and diabetic beta cells should provide pathophysiologic insight into the beta cell signal transduction defect characteristic of non‐insulin dependent diabetes (NIDDM).
Monoclonal antibody to the human insulin receptor, but not insulin, stimulates S6 kinase via human insulin receptors mutated at three major tyrosine autophosphorylation sites
Authors: Sung Chin K. 
Published: May 2005

AbstractStudies were carried out to examine the role of the major insulin receptor tyrosine autophosphorylation sites in stimulation of S6 kinase activity. For these studies, we employed HTC rat hepatoma cells transfected with and expressing human insulin receptors. In cells transfected with and expressing a large number of normal human insulin receptors (HTC‐IR cells), the sensitivity of cells to insulin to stimulate S6 kinase was increased tenfold when compared to untransfected wild type HTC cells (HTC‐WT cells). However, in cells transfected with and expressing a large number of mutated human insulin receptors where the tyrosines at three major autophosphorylation sites (1158, 1162, and 1163) were mutated to phenylalanines (HTC‐F3 cells), there was no change in insulin sensitivity when compared to HTC‐WT cells. We next studied the effect of a human‐specific monoclonal antbody to the human insulin receptor, MA‐5, on S6 kinase activation. In HTC‐WT cells, MA‐5 did not interact with endogenous rat insulin receptors and thus did not stimulate S6 kinase. In HTC‐IR cells expressing normal human insulin receptors, MA‐5 stimulated S6 kinase. Interestingly, MA‐5, unlike insulin, was also able to stimulate S6 kinase in HTC‐F3 cells expressing mutated receptors. In order to further understand the signaling mechanisms by MA‐5 and insulin, two potential intermediate protein kinases were investigate. Neither insulin nor MA‐5 appears to activate either microtubule‐associated protein 2 (MAP‐2) kinase or protein kinase C in these cells.These studies suggest therefore that: 1) insulin and MA‐5 may signal S6 kinase activation by independent mechanisms that do not employ either MAP‐2 kinase or protein kinase C; and 2) under certain circumstances, S6 kinase appears to be activated by mechanisms that are independent of insulin receptor tyrosine autophosphorylation.
Transcriptional control of vitamin D‐regulated proteins
Authors: Lian Jane B. , Stein Gary S. 
Published: May 2005

AbstractVitamin D is a physiological regulator of gene transcription associated with control of a broad spectrum of biological processes that include but is not restricted to growth, differentiation and calcium‐mediated homeostatic control. Transcriptional regulation is mediated by sequence‐specific interactions of a 1,25(OH)2D3‐vitamin D receptor‐accessory factor complex with vitamin D responsive elements (VDRE) residing in the promoters of hormone responsive genes. Functioning primarily as a transcription enhancer, activity at the VDRE is controlled by diverse and integrated cellular signalling pathways acting synergistically and/or antagonistically with a series of basal regulatory elements and other hormone regulated sequences that are components of modularly organized vitamin D‐responsive gene promoters. Molecular mechanisms that integrate the activities at promoter elements contributing to vitamin D‐related transcriptional control include overlapping transcription factor binding domains within regulatory elements and cooperative activities at independent regulatory sequences that determine the level of vitamin D responsiveness.
Sharing of antigenic epitopes between synaptophysin and granulophysin
Authors: Shalev Abraham , Gerrard Jon M. , Robertson Catherine , Greenberg Arnold H. , Linial Michal 
Published: May 2005

AbstractThe immunological crossreactivity between the two granule‐specific membrane glycoproteins, synaptophysin and granulophysin, was studied using a series of site‐specific monoclonal and polyclonal antibodies. The epitope relatedness of six monoclonal antibodies against granulophysin was examined by competitive ELISA. The antibodies are shown to recognize distinct, but overlapping epitopes within a compact region that is constructed by the three‐dimensional configuration of the molecule. All these antibody clones also recognize rat neuronal synaptophysin. Two monoclonal antibodies against synaptophysin, of which one is the well‐characterized SY38 antibody, directed against the carboxy terminal of the molecule, are also shown to react with granulophysin. Characterized polyclonal antibodies against different peptide antigens of synaptophysin failed to recognize granulophysin. Synaptophysin and granulophysin are distinctly recognized in brain cell (white matter) and the pituitary both qualitatively and quantitatively. Based on these and other observations, it is suggested that the repeat motif in the cytoplasmic tail of synaptophysin represents an immunodominant construct that is the target for the observed crossreactive antibodies and that a similar tertiary construct has been preserved in granulophysin and in other transmembrane proteins.
Elevation of large‐T antigen production by sodium butyrate treatment of SV40‐transformed WI‐38 fibroblasts
Authors: Goldberg Y. Paul , Leaner Virna D. , Parker M. Iqbal 
Published: May 2005

AbstractThe effects of sodium butyrate on simian virus 40 early gene expression were determined in SV40‐transformed human embryonic lung fibroblasts (SVWI‐38). Northern blot analysis and nuclear run‐off transcription studies revealed that treatment of cells with millimolar concentrations of sodium butyrate (2.5 to 10 mM) resulted in increased levels of SV40 early gene transcripts, with a concomitant increase in their corresponding proteins (large‐T and small‐t antigens). Although sodium butyrate treatment enhanced the expression of the early genes, it was associated with a reduction in cell growth and total protein synthesis, as measured by cell number and incorporation of 3H‐leucine into macromolecules, respectively. Immunoprecipitation of 35S‐labelled cellular proteins with anti‐p53 and anti‐T antibodies revealed that the level of the cellular protein, p53, declined markedly in the presence of sodium butyrate. Furthermore, in control cells only 30% of the p53 was complexed with large‐T antigen, whereas in butyrate‐treated cells all the p53 was complexed with large‐T antigen. The increased early gene expression was not due to altered methylation patterns, gene amplification, or rearrangement of the integrated SV40 genome. Sodium butyrate treatment did, however, result in the appearance of a new nuclear protein which bound specifically to a SV40 promoter fragment containing large‐T antigen binding sites I and II.
Cloning and expression of the human myeloid cell nuclear differentiation antigen: Regulation by interferon α
Authors: Briggs J. A. , Burrus G. R. , Stickney B. D. , Briggs Robert C. 
Published: May 2005

AbstractThe human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product wsa sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow λgt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5′ and 3′ untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon‐inducible genes: 204 and interferon regulatory factor 2. In addition, a 12‐base sequence matching the interferon‐stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5′ untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon α. The MNDA mRNA was not induced by interferon α in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell‐specific response to interferon.
Prospects for the genetic engineering of milk
Authors: Clark A. J. 
Published: May 2005

AbstractMilk and milk products comprise a substantial fraction of the protein intake of the industrialised West. The establishment of germline manipulation techniques in cows offers opportunities for directly manipulating milk composition to produce products with enhanced nutritional and processing properties. The major milk proteins are encoded by a small number of abundantly expressed single‐copy genes and a number of possible manipulations are described. Milk proteins exhibit complex interactions with each other and with other constituents of milk. It will, therefore, be necessary to utilise model systems to evaluate the consequences of these proposed changes before embarking upon the costly and time‐consuming process of manipulating the bovine genome.
Chromatin structure changes suggest a compensatory response to c‐myc gene amplification in malignant fibrous histiocytoma
Authors: Gibson Jane S. , Croker Byron P. 
Published: May 2005

AbstractChanges in chromatin structure as determined from DNAse I hypersensitive site analysis are associated with c‐myc amplification and increased transcript/protein levels in malignant fibrous histiocytoma (MFH) cell lines. A DNAse I hypersensitive site near the PO promoter region was observed in one MFH cell line (UR HCL 1), and in normal fibroblasts (HFF), but not in an MFH cell line with an amplified c‐myc gene (P3C). A DNAse I hypersensitive site exclusive to P3C amplified c‐myc was identified slightly 3′ of exon one. No alterations in c‐myc DNAse I hypersensitive site patterns were observed in HFF fibroblasts following serum release, when peak levels of c‐myc transcript were induced. DNAse I hypersensitive site patterns associated with gene amplification may reflect a compensatory response by P3C cells to an abundance of c‐myc transcript. Furthermore, elevated levels of protein in P3C cells provide additional evidence that amplified c‐myc is an oncogene in MFHs.
Monensin inhibits the binding of 3H‐flunitrazepam to and reveals the intracellular passage of GABAA/benzodiazepine receptor
Authors: Yin Hsiang Shu 
Published: May 2005

AbstractEffects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H‐flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 μM monensin for 3 h blocked the binding of 3H‐flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 μM monensin, as detected by assaying the incorporation of 3H‐leucine and 3H‐galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down‐regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.
Extraction and partial characterization of non‐histone nuclear proteins of Schistosoma mansoni
Authors: Rabelo Elida M. L. , Campos Elida G. , Fantappié Marcelo R. , Rumjanek Franklin D. 
Published: May 2005

AbstractA pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one‐dimensional and two‐dimensional PAGE. Stage‐specific differences in the HM‐like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P‐F‐10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3′untranslated end of the F‐10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.
Inhibition of renal Na+‐Pi cotransporter by mercuric chloride: Role of sulfhydryl groups
Authors: Loghman‐Adham Mahmoud 
Published: May 2005

AbstractWe studied the role of sulfhydryl groups in Na+‐Pi contransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na+‐dependent 32Pi transport in a dose‐dependent manner (IC50 = 54 μM). Na+‐independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium–exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D‐glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (> 100 μM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 ± 0.13 to 0.75 ± 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected agaisnt inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na+‐Pi cotransporter of renal BBM.
Expression and partial characterization of rat protein kinase C‐δ and protein kinase C‐ξ in insect cells using recombinant baculovirus
Authors: McGlynn E. , Liebetanz J. , Reutener S. , Wood J. , Lydon N. B. , Hofstetter H. , Vanek M. , Meyer T. , Fabbro D. 
Published: May 2005

AbstractExpression of rat protein kinase C‐δ (PKC‐δ ) and PKC‐ξ in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype‐specific antisera. Although the PKC‐ξ cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC‐δ were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC‐α. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC‐ξ. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC‐δ or PKC‐ξ when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC‐δ and PKC‐ξ. In contrast to PKC‐ξ, the PKC‐δ enzyme activity phosphorylated MBP or histone in a phosphatidylserine‐(PS)/diacylglycerol(DG)‐dependent manner, albeit not to the same extent as PKC‐α. Lack of stimulation of the enzyme activity of PKC‐ξ by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC‐ξ, whereas several insect cell proteins were phosphorylated by PKC‐δ in a PS/DG‐dependent manner, including a protein of 78 kD.Our data demonstrate that the 76 kD PKC‐ξ, in contrast to PKC‐δ, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS of PS/DG. In addition, staurosporine was about 2–4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC‐δ and PKC‐δ when compared to PKC‐ξ.
SPARC antagonizes the effect of basic fibroblast growth factor on the igration of bovine aortic endothelial cells
Authors: Hasselaar Paula , Sage E. Helene 
Published: May 2005

AbstractMigration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose‐dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N‐ and C‐terminal Ca2+‐binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell‐associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high‐affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]‐thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum‐free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.
Isolation of recombinant proteins from milk
Authors: Wilkins Tracy D. , Velander William 
Published: May 2005

AbstractMilk is a complex bio‐colloid which presents some unique problems for the protein isolation chemist, but the majority of the processing criteria for purifying recombinant proteins are the same as with any complex biological mixture. The casein micelles and fat globules behave as separate phases; they prevent filtration of the milk and interfere with the usual separation methods. The usual first step is to centrifuge the milk to remove the fat and precipitate the casein micelles with low pH or precipitating agents. Some recombinant proteins may associate to some degree with the micelles which may necessitate solubilizing them with chelating agents. If the majority of the product protein associates with either the fat or micelles, this can be used to advantage. Once the casein micelles have been removed or disrupted, the clarified milk can be processed by the usual separation methods. There also are proteases in milk which can degrade recombinant proteins. The greatest advantage of producing recombinant proteins in milk is the high concentration which can be obtained. The high levels of product protein can alleviate many problems associated with the application of classical purification strategies to transgenic milk proteins.