bluuebunny/crossref_metadata_2025_split huggingface dataset:
One‐ and two‐dimensional nmr spectral analysis of the consequences of single amino acid replacements in proteins
Authors: Markley John L. , Croll David H. , Krishnamoorthi R. , Ortiz‐Polo Gilberto , Westler William M. , Bogard W. C. , Laskowski M. 
Published: December 2004

AbstractThe traditional approach of using homologous sequences to elucidate the role of specific amino acid residues in protein structure and function becomes more meaningful as the number of differences is minimized, with the limit being alteration of a single residue. For small proteins in solution, NMR spectroscopy offers a means of obtaining detailed information about each residue and its response to a given change in the protein sequence. Extraction of this information has been aided by recent progress in spectrometer technology (higher magnetic fields, more sensitive signal detection, more sophisticated computers) and experimental strategies (new NMR pulse sequences including multiple‐quantum and two‐dimensional NMR methods). The set of avian ovomucoid third domains, which consists of the third domain proper plus a short leader (connecting peptide) and has a maximum of 56 amino acid residues, offers an attractive system for developing experimental methods for investigating sequence‐structure and structure‐function relationships in proteins. Our NMR results provide examples of sequence effects on pKa′ values, average conformation, and internal motion of amino acid side chains.
Phenotypic differences in subclones and long‐term cultures of clonally derived rat bone cell lines
Authors: Bellows Carlton G. , Sodek Jaro , Yao Kam‐Ling , Aubin Jane E. 
Published: December 2004

AbstractPrevious studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982].Several of these clonal populations have been used to study the effects of long‐term culture and inter‐ and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively αl(I)‐trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1‐2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long‐term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter‐ and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.
3′,5′‐Cyclic adenosine monophosphate as an intracellular second messenger of luteinizing hormone: Application of the forskolin criteria
Authors: Adashi Eli Y. , Resnick Carol E. 
Published: December 2004

AbstractThe role of adenosine 3′,5′‐cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10−7–10−4 M) of forskolin produced dose‐dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP‐phosphodiesterase activity with 3‐isobutyl‐1‐methyl‐xanthine (10−4 M) resulted in significant (P < 0.05) enhancement of the forskolin effect for all but the 10−4 M forskolin dose. Potency evaluation as judged by half‐maximal stimulation of testosterone accumulation revealed median effective doses (mean ± SE) of 1.25 ± 0.2 × 10−5, 1.7 ± 0.5 × 10−5, and 2.5 ± 0.4 × 10−10 M for forskolin, N6, O2′‐dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time‐dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P < 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of ⩽ 12hr was noted for hCG‐ and choleragen‐stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24‐hr time point. Although 10−7 M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6‐fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10−11 or 3 × 10−11M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10−4 M) and one of several testicular cell agonists [forskolin (10−4 M), choleragen (10−9 M) or hCG (10−9 M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin‐stimulated and forskolin‐amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH.
Inactivation of human plasma serine proteinase inhibitors (serpins) by limited proteolysis of the reactive site loop with snake venom and bacterial metalloproteinases
Authors: Kress Lawrence F. 
Published: December 2004

AbstractHuman plasma serine proteinase inhibitors (serpins) gradually lost activity when incubated with catalytic amounts of snake venom or bacterial metalloproteinases. Electrophoretic analyses indicated that antithrombin III, C1‐inhibitor, and α2‐antiplasmin had been converted by limited proteolysis into modified species which retained inhibitory activity. Further proteolytic attack resulted in the formation of inactivated inhibitors; α1‐proteinase inhibitor (α1‐antitrypsin) and α1‐antichymotrypsin were also enzymatically inactivated, but active intermediates were not detected. Sequence analyses indicated that the initial, noninactivating cleavage occurred in the amino‐terminal region of the inhibitors. Inactivation resulted in all cases from the limited proteolysis of a single bond near, but not at, the reactive site bond in the carboxy‐terminal region of the inhibitors. The results indicate that the serpins have two regions which are susceptible to limited proteolysis—one near the amino‐terminal end and another in the exposed reactive site loop of the inhibitor.
Identification of the chick neural retina cell surface N‐acetylgalactosaminyltransferase using monoclonal antibodies
Authors: Balsamo Janne , Pratt Rodney S. , Emmerling Mark R. , Grunwald Gerald B. , Lilien Jack 
Published: December 2004

AbstractIntact embryonic chick neural retina cells have at their surface an N‐acetylgalactosaminyltransferase which catalyzes the incorporation of N‐acetylgalactosamine from UDP‐N‐acetylgalactosamine into endogenous macromolecular acceptors. The enzyme along with its endogenous acceptors can be isolated as a particulate complex following treatment of membrane‐enriched fractions with Triton X‐100. In this paper we report on two separate fusions generating monoclonal antibodies: one using as immunogen the particulate complex and the second using as immunogen a soluble N‐acetylgalactosaminyltransferase found in tissue‐culture‐conditioned medium which lacks endogenous acceptor activity. Antibodies from both fusions recognize an antigen which is tightly associated with the particulate transferase/acceptor complex and a soluble antigen having N‐acetyl‐galactosaminyltransferase activity toward exogenously added acceptors. The antibodies recognize a component of ca Mr 220,000, which shows N‐acetylgalactosaminyltransferase activity after SDS‐gel electrophoresis and transfer to nitrocellulose. This component comigrates on two‐dimensional gel electrophoresis with an iodinatable cell surface component whose presence at the cell surface correlates with endogenous transferase activity. We conclude that the antibodies recognize the transferase enzyme itself. Immunohistochemical analysis shows that the enzyme is initially localized throughout the embryonic neural retina in a pattern indicative of a cell surface disposition but becomes restricted to the outer plexiform layer and to outer segments in the adult.
A monoclonal antibody reactive with an activated ras protein expressing valine at position 12
Authors: Carney W. P. , Hamer P. , Petit D. , Rabin H. , Cooper G. , Lefebvre M. , Wolfe H. 
Published: December 2004

AbstractActivated ras transforming genes have been described in a variety of neoplasms and encode 21,000‐Dalton (p21) proteins with amino acid substitutions at positions 12, 13, and 61. In this report we describe a monoclonal antibody designated DWP that reacts. Specifically with synthetic dodecapeptides containing valine at position 12, to a lesser extent with peptides containing cysteine at position 12 and not with peptides containing glycine, arginine, serine, aspartic acid, glutamic acid or alanine at the same position. Western blot and immunoperoxidase studies showed that DWP specifically reacts with activated rasH or rasK proteins in NIH cells transformed by DNA from the human carcinoma cells that encode valine at position 12. DWP did not react with normal p21s encoding glycine at position 12, nor with activated p21s encoding aspartic acid, glutamic acid, arginine, serine, or cysteine at position 12. A survey of human tumor cell lines demonstrated that DWP reacted with the human bladder carcinoma cell line T24 but not with human tumor cell lines previously shown td contain other activating mutations at positions 12 or 61. DWP and perhaps additional antibodies that specifically react with alterations at positions 12 or 61 of the ras protein may be valuable in determining the presence and frequency of activated ras proteins in human malignancy.
Evolution in the structure and function of aspartic proteases
Authors: Tang Jordan , Wong Ricky N. S. 
Published: December 2004

AbstractAspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin. and chymosin). lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93–109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a “pro” enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens.It is the aim of this article to summarize the major models of structure‐function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.
Transformation by the v‐fms oncogene product: An analog of the CSF‐1 receptor
Authors: Rettenmier Carl W. , Jackowski Suzanne , Rock Charles O. , Roussel Marline F. , Sherr Charles J. 
Published: December 2004

AbstractThe product of the c‐fms proto‐oncogene is related to, and possibly identical with, the receptor for the macrophage colony‐stimulating factor, M‐CSF (CSF‐1). Unlike the product of the v‐erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v‐fms oncogene retains an intact extracellular ligand‐binding domain so that cells transformed by v‐fms express CSF‐1 receptors at their surface. Although fibroblasts susceptible to transformation by v‐fms generally produce CSF‐1, v‐fms‐mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF‐1 do not affect the transformed phenotype. An alteration of the v‐fms gene product at its extreme carboxyl‐terminus represents the major structural difference between it and the c‐fms‐coded glycoprotein and may affect the tyrosine kinase activity of the v‐fms‐coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v‐fms products in membranes was observed in the absence of CSF‐1 and was not enhanced by addition of the murine growth factor. Cells transformed by v‐fms have a constitutively elevated specific activity of a guanir.c nucleotide‐dependent, phosphatidylinositol‐4,5‐diphosphate‐specific phospholipase C. We speculate that the tyrosine kinase activity of the v‐fms/c‐fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphatc.
Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation
Authors: Wier Marjorie L. , Scott Robert E. 
Published: December 2004

AbstractThe differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two‐dimensional gel electrophoresis to evaluate changes in specific polypeptides that arc induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.
Oncogene amplification and chromosomal abnormalities in small cell lung cancer
Authors: Ibson J. M. , Waters J. J. , Twentyman P. R. , Bleehen N. M. , Rabbitts P. H. 
Published: December 2004

AbstractTwelve cell lines isolated from patients with small cell lung cancer have been studied for amplification of the three characterised members of the myc proto‐oncogene family (c‐myc, N‐myc, and L‐myc) and for abnormalities of chromosome 3. Ten of these lines were being studied for the first time. Ten of the 12 small cell lung cancer cell lines had amplification of one member of the myc proto‐oncogene family. Amplification of c‐myc was observed in only one small cell lung line—a “morphological variant.” One “classic” small cell lung cancer line expressed c‐myc but had no obvious amplification of the gene. N‐myc and L‐myc were more commonly amplified than c‐myc. Chromosomal abnormalities (mainly deletions) in chromosome 3 were observed in all small cell lung carcinoma cell lines examined. When the small cell lung carcinoma lines were grouped according to “classic” or “variant” characteristics, it was found that the “classics” had deletions of the short arm of chromosome 3, whereas the “biochemical variants” had deletions of the long arm of chromosome 3. The extent of the deletions varied between cell lines. For the deletion in the short arm of chromosome 3 the minimum common region of overlap was assigned to bands 3p23–3p24.
Identity of common phosphoprotein substrates stimulated by interleukin 2 and diacylglycerol suggests a role of protein kinase C for IL 2 signal transduction
Authors: Evans Stuart W. , Farrar William L. 
Published: December 2004

AbstractInterleukin 2 (IL 2) is a polypeptide growth factor essential for the proliferation and differentiation of T lymphocytes, large granulocytic lymphocytes, and, potentially, cells of the antibody‐producing lineage, B lymphocytes. Many of the biological properties of IL 2 may be mimicked or potentiated by a potent class of tumor promoters, phorbol esters. Phorbol esters have recently been shown to associate with and activate a unique phospholipid/Ca2+‐dependent phosphotransferase, protein kinase C (PK‐C). Utilizing two‐dimensional gel electrophoresis, we have compared the IL 2 and diacylglycerol‐induced protein phosphorylation patterns of several IL 2‐dependent murine cell lines. Both IL 2 and synthetic diacylglycerol, 1‐oleyl‐2‐acetyIglycerol (OAG), stimulated phosphorylation of a number of protein substrates in intact cells compared to unstimulated controls. Three groups of substrates were identified; the first showed increased phosphorylation following stimulation with either IL 2 or OAG, while the second and third groups showed increased phosphorylation following stimulation with IL 2 but not OAG, and with OAG but not IL 2, respectively. Here, we characterize the kinetics of phosphorylation of one cellular substrate, p68, which appears to be phosphorylated in response to direct activators of PK‐C or lymphoid or myeloid growth factors in their respective lineage cell lines. The observation that IL 2 also stimulates a unique series of phosphoproteins in addition to those induced by direct PK‐C activators suggests that IL 2 may initiate additional protein kinase activities, unrelated to PK‐C, which may also be critical for the ligand‐receptor signal transduction process regulating growth and gene expression.
Soluble 80‐kd fragment of cell‐CAM 120/80 disrupts cell‐cell adhesion
Authors: Wheelock Margaret J. , Buck Clayton A. , Bechtol Kathleen B. , Damsky Caroline H. 
Published: December 2004

AbstractCalcium‐dependent cell adhesion molecules (CAMs) mediate intercellular adhesion in epithelial cells and in preimplantation mammalian embryos. One of these molecules, cell‐CAM 120/80, is found on cells as a 120‐kd membrane glycoprotein and as a soluble 80‐kd species in conditioned culture medium [Damsky et al: Cell 34:455, 1983]. We have purified to homogeneity the soluble 80‐kd fragment of cell‐CAM 120/80 by using monoclonal antibody affinity chromatography. We have shown that the purified molecule can disrupt cell‐cell adhesion in cultured epithelial cells, thus indicating that it is directly involved in the adhesive process. In addition, we have further characterized both the 120‐kd cell‐associated molecule and its 80‐kd fragment, including N‐terminal sequence analysis.
Immunohistochemical demonstration of proteinase inhibitor alpha‐1‐antichymotrypsin in normal human central nervous system
Authors: Justice Deanna L. , Rhodes Roy H. , Tökés Zoltán A. 
Published: December 2004

AbstractThe presence of alpha‐1‐antichymotrypsin, a serine proteinase inhibitor with a high affinity for cathepsin G, is demonstrated in the normal human central nervous system (CNS) by immunohistochemical techniques. Paraffin‐embedded normal human CNS tissue from five adult, two fetal, one neonatal and three newborn autopsies were stained with monospecific rabbit antibodies to human alpha‐1‐antichymotrypsin using biotinylated goat anti‐rabbit antibodies and an avidin‐biotin‐ peroxidase complex. Positive immunostaining was seen in neurons and glial cells in the cerebral cortex, basal ganglia, hippocampus, cerebellum, brainstem, and spinal cord of the adults. The epithelium of the adult choroid plexus had the most intense staining in apical granular organelles corresponding in position to lysosomes or secretory granules. Ependymal cells, particularly those near the choroid plexus, were immunostained. The fetal CNS had no alpha‐1‐antichymotrypsin staining. Limited staining of choroid plexus, ependyma. and frontal lobe was found in the newborns. Immunostaining in the neonatal temporal lobe was only found in the choroid‐plexus epithelium. These observations establish a widespread distribution of this proteinase inhibitor in the normal human CNS. Developmental regulation of this inhibitor in the human CNS is also indicated.
Phosphatidylinositol cycle and its possible involvement in the regulation of cytoskeleton‐membrane interactions
Authors: Burn Paul 
Published: December 2004

AbstractReceptor‐mediated activation of many cells, including blood platelets, leads to changes at the cytoplasmic side of the membrane. In platelets, phospholipases, such as phospholipase C and phospholipase A2, have been shown to become activated. From phospholipids they generate the second messengers diacylglycerol and inositol phosphate(s) and fatty acids, respectively. At the same time, actin polymerization and reorganization of actin filaments into bundles and networks occurs. Here, the association of lipids, radiolabeled either with saturated (palmitic acid) or unsaturated (arachidonic acid) fatty acids, with the cytoskeletons of resting and activated human blood platelets was studied. The relative binding of lipid components to the cytoskeleton of activated platelets labeled with palmitic acid is six times higher than that of platelets labeled with arachidonic acid. Analysis of lipids associated with isolated cytoskeletons of resting and activated platelets (labeled with palmitic acid) showed a 30‐fold increase in the binding of labeled lipids to the cytoskeletal structures during activation. Both diacylglycerol and fatty acids were found to be associated with the cytoskeleton of activated platelets. Gel filtration, chromatofocusing, and immunoprecipitation studies demonstrated tight binding of these lipids to α‐actinin. α‐Actinin is one of the proteins that rapidly becomes associated with the cytoskeleton during platelet aggregation; it is also one of the molecules proposed to act as an actin‐membrane linker. The results, reported indicate a possible participation of α‐actinin, fatty acids, and the phosphoinositide‐derived second messenger diacylglycerol in the regulation of cytoskeleton‐membrane interactions. Together with the results of others they suggest a possible involvement of the phosphatidylinositol cycle in the assembly of actin filaments and their anchoring to membranes.