bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phospholipid and Ca++ dependency of phorbol ester receptors
Authors: König Bernhard , Di Nitto Patricia A. , Blumberg Peter M. 
Published: December 2004

AbstractThe phospholipid and Ca++ dependency of a partially purified phorbol ester apo‐receptor from the soluble fraction of mouse brain homogenates was studied. This apo‐receptor is believed to be identical with the Ca++ and phospholipid‐dependent protein kinase C. Binding of phorbol esters to the receptor/kinase C was shown to be entirely dependent on phospholipids. The negatively charged phospholipids phosphatidylserine, phosphatidylinositol, and phosphatidic acid all fully reconstituted binding. The neutral phospholipids were inactive. Among active phospholipids and mixtures of phospholipids, substantial differences ( > 100‐fold) were observed in the amounts required to achieve reconstitution. Although Ca++ was not required for reconstitution of binding activity, it dramatically (up to 100‐fold) increased the potency of phospholipids for reconstitution. The phospholipids not only permitted reconstitution of the apo‐receptor but also played a major role in determining the binding characteristics of the complex. The KD values of [3H]phorbol 12,13–dibutyrate were in the range of 0.8 nM for the complex with phosphatidylserine to 30 nM for the complex with diolcoyl‐phosphatidic acid. Like the binding affinity, the stimulation of protein kinase C activity by phorbol esters was dependent on the phospholipid into which the receptor/kinase C was reconstituted. The importance of the lipid domain for controlling the receptor/kinase C activity and for modulation of cellular sensitivity to phorbol esters is discussed.
Basement membrane component changes in skeletal muscle transplants undergoing regeneration or rejection
Authors: Gulati Adarsh K. 
Published: December 2004

AbstractThe basement membrane of myofibers plays an important role during orderly regeneration of skeletal muscle after injury. In this report, changes in various basement membrane components were analyzed in skeletal muscle grafts undergoing regeneration (autografts) or immune rejection (allografts). The immunofluorescence technique using specific antibodies against laminin, types IV and V collagen, heparan sulfate proteoglycan, fibronectin, in combination with binding of concanavalin A (ConA) was used to monitor basement membranes. In normal muscle, these components were localized in the pericellular region of myofiber corresponding to its basement membrane, After transplantation, the majority of myofibers underwent degeneration as a result of is chemic injury, followed by regeneration from precursor myosatellite cells. Various components of basement membrane zone disappeared from the degenerating myofibers, leaving behind some unidentifiable component that still bound ConA. A new basement membrane appeared around the regenerated myotubes which persisted during maturation of the regenerating muscle, In rejected skeletal muscles, the immunoreactivity of various components persisted even after the disappearance of myotubes and myofiber cytoplasm. In addition, an accumulation of fibronectin was seen throughout the rejected muscle with the onset of immune rejection. These results demonstrate that the major basement membrane components disappear and reappear sequentially during myofiber degeneration and regeneration. Such a turnover is not seen in rejected skeletal muscles. Thus, the myofiber basement membrane is not a static structure as previously thought but one which changes chemically during degeneration and regeneration. This feature of basement membrane may be important in the orderly regeneration of skeletal muscle after injury.
Enzymes converting procollagens to collagens
Authors: Peltonen L. , Halila R. , Ryhänen L. 
Published: December 2004

AbstractConversion from procollagen to collagen is a specific process that is a requirement for proper alignment of collagen molecules to form functional fibers. This process is catalyzed by at least three structurally and functionally distinct enzymes cleaving collagen types I–III. The cleavage processes possibly taking place in the more recently discovered collagen types are not known to any extent at this time.Two amino‐terminal proteinases, one cleaving type I and type II procollagens and the other cleaving type III procollagen, have been purified close to homogeneity, and the more unspecific activity of carboxy‐terminal proteinase has been isolated from several tissues. In our experimental model, however, cleavage of the carboxy‐terminal propeptides of types I and III procollagen is differently affected by lysine. This suggests the presence of at least two distinct enzymes for the removal of carboxyl‐terminal propeptides.The regulation of the reaction process from procollagen to collagen is not well known at present. The importance of the phenomenon in terms of fibril formation, however, is demonstrated by several elegant studies in vitro; and certain genetic disorders in which this process is defective demonstrate the significance in vivo. Moreover, the factors shown to effect the cleavage process may be potentially beneficial in the treatment of the pathological processes with abnormal collagen accumulation such as fibrosis.In this paper we briefly review the current knowledge of the converting enzymes, including some very recent findings of our laboratory as well as the evidence presented for the biological significance of the conversion process.
Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix
Authors: Clark Richard A. F. , Nielsen Larry D. , Howell Samuel E. , Folkvord Joy M. 
Published: December 2004

AbstractFibronectin and laminin production by human keratinocytes cultured in serum‐free, low‐calcium medium without a fibroblast feeder layer were examined by several techniques. By indirect immunofluorescence, fibronectin but not laminin appeared as short radial fibrils between the cells and the substratum, and in the pericellular matrix. Synthesis of fibronectin and laminin by 7‐day keratinocyte cultures was determined by 18 hr 35S‐methionine metabolic labeling followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Fibronectin accounted for 2.9% of total synthesized protein, 26.5% of fluid phase protein secretion, and 4.3% of deposited ECM protein. In contrast, only 0.1% of the total synthesized protein was laminin, little (6.3%) of this product was secreted, and none of this product was deposited in the ECM. Our results indicate that human keratinocytes under culture conditions that prevent terminal differentiation in vitro can synthesize, secrete, and deposit fibronectin in the extracellular matrix. Although these cells synthesize laminin, they secrete very little and deposit no detectable laminin in the matrix under these culture conditions. From these data we believe that fibronectin may play an important role in the interaction of epidermal cells with connective tissue matrix during wound healing or morphogenesis in in vivo situations in which the epidermis is not terminally differentiated.
Immunochemical and biochemical characterization of a glioma‐associated extracellular matrix glycoprotein
Authors: Bourdon Mario A. , Matthews Thomas J. , Pizzo Salvatore V. , Bigner Darell D. 
Published: December 2004

AbstractA novel human glioma‐associated extracellular matrix (ECM) glycoprotein has been identified by murine monoclonal antibody 81C6. The glycoprotein, designated GMEM, is expressed in the ECM of glioma and mesenchymal cell cultures, in the perivascular matrix of endothelial proliferations of human gliomas, and in the stroma of human glioma xenografts in athyrnic mice, where it has been used as a target antigen for monoclonal antibody tumor localization and radioimaging. We report here on the immunochemical and biochemical characterization of GMEM. Polyacrylamide gel analysis of immunoprecipitated [3H]‐leucine‐ and [3H]‐glucosamine‐labeled ECM from the human glioma cell line U‐251MG has shown that GMEM is a high‐molecular‐weight macromolecule (Mr ∼ 1,000,000) composed of Mr ∼ 230,000 disulfide‐bonded glycoprotein subunits. Immunoprecipitation, immunoblot, and one‐dimensional peptide map analysis have shown that GMEM is distinct from human fibroblast and plasma fibronectin. These results support previous immunohistology and absorption analysis findings, indicating that GMEM is distinct from fibronectin, laminin, and glycosaminoglycans secreted by U‐251MG.
Pteridine formation during lectin‐induced lymphocyte activation
Authors: Ziegler Irmgard 
Published: December 2004

AbstractAfter iodine oxidation, biopterin, 6‐hydroxymethylpterin, and 6‐formylpterin were identified in mouse spleen lymphocytes by means of reverse‐phase HPLC, Crithidia assay, and oxidative degradation. Concanavalin A activation induces a 30‐fold increase in the pteridine amounts; biopterin as well as the sum of the carbinol and the aldehyde attain levels of 6–8 × 10−12 mol/106 cells. The most rapid increase occurs during the first 24 hr. Thus, pteridine accumulation precedes the period of lymphocyte proliferation; maximum DNA synthesis was found after 72 hr. Biopterin remains largely inside the cells, whereas 6‐hydroxymethylpterin and 6‐formylpterin were found in the supernatant if the stimulated cells were subsequently incubated in a phosphate buffered salt solution (PBS). Isoxanthopterin was found in the PBS supernatant of control cells that previously were kept in medium alone rather than subjected to lectin stimulation. Only minimal amounts were found inside these cells, and this pterin was absent from the stimulated lymphocytes. The early increase in cellular pteridines and their differential release may well provide the basis for their modulating effect on interleukin‐2 activity (Ziegler I, et al: Lymphokine Research 3:284, 1984).
Cell surface galactosyltransferase: Immunochemical localization
Authors: Shaper Nancy L. , Mann Paul L. , Shaper Joel H. 
Published: December 2004

AbstractA cell surface UDP‐galactose:N‐acetylglucosamine galactosyltransferase (GT) has been directly localized on bovine cells in tissue culture by immunohistochemical techniques. A conventional rabbit heteroantiserum was prepared against an affinity‐purified soluble form of GT from bovine milk, and a monospecific IgG fraction was isolated by affinity chromatography on a GT adsorbent. As demonstrated by indirect immunofluorescence, antigen to this antibody is present on the surface of all three bovine cell lines tested. It was uniformly distributed over the exposed membrane surface of fixed cells. Exposure of living cells to the anti‐GT antibody resulted in its time‐dependent aggregation in the plane of the membrane. Antigen (GT) was released from the membrane surface by trypsin digestion, and its reappearance required protein synthesis, since cycloheximide effectively prevented repopulation of the cell surface.
Stoichiometric binding of diacylglycerol to the phorbol ester receptor
Authors: König Bernhard , DiNitto Patricia A. , Blumberg Peter M. 
Published: December 2004

AbstractThe major phorbol ester receptor is the Ca++‐activated, phospholipid‐dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2‐dioleoylglycerol interacts with the receptor with the predicted stoichiometry.
Ankyrin and synapsin: Spectrin‐binding proteins associated with brain membranes
Authors: Bennett Vann , Baines Anthony J. , Davis Jonathan Q. 
Published: December 2004

AbstractBrain membranes contain an actin‐binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purifed from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease‐resistant domains of Mr = 72,000 that contain spectrin‐binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the union channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrin beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin‐associated act in filaments.Another spectrin‐binding protein has been purified from brain membranes, and this protein cross‐reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP‐dependent and Ca/calmodulin‐dependent protein kinases with equivalent physical properties, immunological cross‐reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.
Comparative analysis of p21 proteins from various cell types by two‐dimensional gel electrophoresis
Authors: Feuerstein Nili , Ali Iqbal Unnisa 
Published: December 2004

AbstractThe products of the ras gene family arc related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two‐dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H‐labeled translation initiation factor [elF‐4D]) as a standard internal marker for isoelectric point (pI), we show that p2l proteins from various cells differ only slightly in molecular weight (21–24 kDa) but express a wide variety in charge (pl 4.8 to 7) that could only be detected by the use of two‐dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pl 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21‐associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus‐transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21‐associated proteins, one at 21 kDa (pl, 5.2 and 5.3) and the other at 23 kDa (pl, 5.1 and 5.2). In HL‐60 leukemic cells there was an additional, more acidic form (pl 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2‐D gel electrophoresis as an important tool in future studies involving p21 proteins.
Functional ion channel formation by mouse macrophage IgG Fc receptor triggered by specific ligands
Authors: Young John Ding‐E , Unkeless Jay C. , Cohn Zanvil A. 
Published: December 2004

AbstractThe mouse macrophage Fcγ 2b/γ 1R has previously been purified with the aid of the monoclonal antibody 2.4G2. That this FcγR functions as a ligand‐dependent ion channel is supported by the following evidence: (a) Employing [3H]tetraphenylphosphonium ([3H]Ph4P+) as a probe for membrane potential changes in intact cells, we found a biphasic change in membrane potential following treatment with immune complexes, monoclonal antibody 2.4G2 IgG and 2.4G2 Fab‐Sephadex particles. We observed an immediate depolarization followed by prolonged hyperpolarization. (b) [3H]Ph4P+ uptake experiments with plasma membrane vesicles prepared from J774 macrophages showed that binding of ligands to the FcR led to transmembrane monovalent cation flow. (c) Similar [3H]Ph4P+ uptake experiments were done with phospholipid vesicles containing purified and reconstituted Fcγ 2b/γR. Following challenge with specific ligands, transmembrane monovalent cation flow was observed. (d) Purified FcR was reconstituted into planar lipid bilayers; exposure to ligands led to transient bilayer conductance increase. The conductance change was resolved into single channel events.Quin‐2 measurements showed an increase of free cytosolic calcium levels in macrophages following exposure of cells to different ligands of the FcR. An optimal range of calcium was found to be required for phagocytosis, below and above which inhibition of ingestion occurred.
Nerve growth factor biosynthesis: Isolation and characterization of a guinea pig prostate kallikrein
Authors: Dunbar Joan C. , Bradshaw Ralph A. 
Published: December 2004

AbstractGuinea pig prostate contains one major soluble esteropeptidase activity. The protein has been purified and characterized and found to be a glycoprotein comprised of a single polypeptide chain. The molecular weight of the deglycosylated protein is approximately 26,000. The esteropeptidase has a similar Km for lysine and arginine synthetic substrates, although the Vmax for arginine is much greater than that for lysine. Amino‐terminal sequence analysis has also revealed a marked degree of homology to mouse γ‐nerve growth factor (NGF) and the kallikrein family of serine proteases. In contrast to γ‐NGF, however, the guinea pig enzyme does not appear to form stable complexes with β‐NGF.
Aluminum ions stimulate mitosis in murine cells in tissue culture
Authors: Jones Trevor R. , Antonetti Donna L. , Reid Ted W. 
Published: December 2004

AbstractAddition of aluminum to the culture medium of Nakano mouse lens epithelial (NMLE) cells and Swiss 3T3K cells induced both 3H‐thymidine incorporation and mitosis. This is in contrast to other metal ions such as vanadium, which, at concentrations high enough to increase 3H‐thymidine incorporation, actually inhibits mitosis (Jones and Reid, J Cell Physiol 121:199, 1984 [1]). Aluminum concentrations between 20 μM and 50 μM were most effective. The 3T3 cells respond to aluminum with a 7.6‐fold increase, and NMLE cells respond with a 21‐fold increase in 3H‐thymidine incorporation. DNA synthesis in NMLE cells was also found to be synergistically stimulated by aluminum and low concentrations of insulin (4.5 × 10−8 M). A 3.25‐hr incubation with 50 μM aluminum was sufficient to induce 50% of maximum 3H‐thymidine incorporation during the 40‐hr assay. Aluminum‐stimulated 3H‐thymidine incorporation is inhibited by hydroxyurea, and aluminum causes an increase in cell number. Also, by sedimentation equilibrium analysis of the product of aluminum‐stimulated DNA synthesis it was found that a single copy of DNA was synthesized following addition of aluminum to quiescent cells. These facts indicate that aluminum induces both S‐phase DNA synthesis and mitosis. However, only 48% of the NMLE cells found to be labeled with DNA went on to divide. In contrast, although only a small percentage of 3T3 cells were found to be labeled after aluminum treatment, all of these cells appeared to go through mitosis.
Mechanisms of cytoskeletal regulation: Functional and antigenic diversity in human erythrocyte and brain beta spectrin
Authors: Harris Alan S. , Anderson John P. , Yurchenco Peter D. , Green L. A. David , Ainger Kevin J. , Morrow Jon S. 
Published: December 2004

AbstractA study of human erythrocyte and brain spectrin with particular emphasis on the beta subunits revealed a structural homology but functional dissimilarity between these two molecules. Six monoclonal antibodies raised to human erythrocyte beta spectrin identify three of the four proteolytically defined domains of erythrocyte beta spectrin. Five of these monoclonal antibodies cross‐react with human brain spectrin. None of a previously identified set of alpha erythrocyte spectrin monoclonal antibodies [Yurchenco et al: J Biol Chem 257:9102, 1982] reacted with brain spectrin. A domain map generated by limited tryptic digestion shows that brain spectrin is composed of proteolytically resistant domains analogous to erythrocyte spectrin, but the brain protein is more basic. The binding of brain spectrin to erythrocyte ankyrin, both in solution and on erythrocyte IOVs, yielded an association constant approximately 100 times weaker than for erythrocyte spectrin. The binding of azido‐calmodulin under native conditions was specific for the erythrocyte beta subunit but was not calcium dependent. In contrast, azido‐calmodulin bound only to the alpha subunit of brain spectrin in a calcium‐dependent manner. The similarity of structure but modified functional character‐istics of the brain and erythrocyte beta spectrins suggest that these proteins serve different cellular roles.
Association of spectrin with desmin intermediate filaments
Authors: Langley Robert C. , Cohen Carl M. 
Published: December 2004

AbstractThe association of erythrocyte spectrin with desmin filaments was investigated using two in vitro assays. The ability of spectrin to promote the interaction of desmin filaments with membranes was investigated by electron microscopy of desmin filament‐erythrocyte inside‐out vesicle preparations. Desmin filaments bound to erythrocyte inside‐out vesicles in a spectrin‐dependent manner, demonstrating that spectrin is capable of mediating the association of desmin filaments with plasma membranes. A quantitative sedimentation assay was used to demonstrate the direct association of spectrin with desmin filaments in vitro. When increasing concentrations of spectrin were incubated with desmin filaments, spectrin cosedimented with desmin filaments in a concentration‐dependent manner. At near saturation the spectrin:desmin molar ratio in the sedimented complex was 1:230. Our results suggest that, in addition to its well characterized associations with actin, spectrin functions to mediate the association of intermediate filaments with plasma membranes. It might be that nonerythrocyte spectrins share erythrocyte spectrin's ability to bind to intermediate filaments and function in nonerythroid cells to promote the interaction of intermediate filaments with actin filaments and/or the plasma membrane.