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Biomedical subjects

A Woods

Publications and source records attributed to A Woods.

At least 19 recordsLinked to original sources
PubMed1992

Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl3 with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl2 moiety. The present study reveals that BrCCl3 also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCL2 or heme-CCl3 were bound to cysteine residue 93 of the beta-chain of hemoglobin. It appeared that the cysteine residue was bonded regiospecifically to the ring I vinyl group of the altered heme moiety, because the nonprotein-bound products of the reaction included the beta-carboxyvinyl and alpha-hydroxy-beta-trichloromethylethyl derivatives of the ring I vinyl moiety of heme. The absorption spectra of the protein-bound adducts in both the oxidized and reduced states were highly similar to those described for hemichromes, which are thought to be involved in the formation of Heinz bodies and subsequent red cell lysis.

Bromotrichloromethane↗
PubMed1992

Protein kinase C involvement in focal adhesion formation.

Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form. Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h and then treated with kinase inhibitors H7 and HA1004 for 2h, IRM indicated a reduction in focal adhesion formation at concentrations where protein kinase C (PKC) should be inhibited. In contrast, focal adhesions formed normally at concentrations of these inhibitors where cyclic AMP- or cyclic GMP-dependent kinases should be inactivated. Inhibition of PKC, but not that of cyclic AMP- or cyclic GMP-dependent kinases, also prevented the formation of stress fibers and induced a dispersal of talin and vinculin, but not integrin beta 1 subunits, from small condensations present at 1h. Consistent with the reduction in focal adhesion formation when PKC was inhibited, activation of PKC by 30 minutes of treatment with phorbol esters induced focal adhesion formation in cells spread for 3h on substrata composed of the cell-binding (RGD-containing) fragment of fibronectin, while untreated cells or those treated with inactive phorbol esters did not form these structures.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

A high molecular mass phosphoprotein defined by a novel monoclonal antibody is closely associated with the intermicrotubule cross bridges in the Trypanosoma brucei cytoskeleton.

The main component of the cell body cytoskeleton of Trypanosoma brucei is the highly organised array of stable, subpellicular microtubules on the cytoplasmic face of the plasma membrane. Although several microtubule associated proteins (MAPs) have been shown to be associated with this array, the mechanisms by which individual microtubules interact with one another and with the membrane are still largely undetermined. In this study we have used the T. brucei cytoskeleton as a complex immunogen for the production of monoclonal antibodies to define novel cytoskeletal antigens. Screening by immunofluorescence enabled the selection of an antibody, WCB-1, which detects an antigen associated specifically with the subpellicular microtubules and not with the flagellum microtubules. The antigen (WCB210) was shown to have a relative molecular mass of 210,000 by western blotting. Immunogold studies showed the epitope to be located on the membrane-facing side of the subpellicular cage; it appears to be closely associated with the cross-bridges lying between the microtubules. Unlike many MAPs this protein was shown not to be heat stable and is predicted to be a roughly globular monomer. Even though WCB210 is a very minor component of the cytoskeleton it is heavily phosphorylated. It is possible that this protein is involved in regulation of the subpellicular microtubule crossbridges by interaction with other proteins.

Animals↗
PubMed1992

Avascular necrosis of the femoral head post heart-transplantation and steroid dosage.

Avascular necrosis (avn) post heart-transplantation has been considered to be due to the high doses of steroids used to immunosuppress these patients in attempting to prevent transplant rejection. This study shows that avascular necrosis occurs even when low dose steroids regimes are used and demonstrates no significant correlation between steroid dosage and the development of avn. Patients with symptomatic avn benefit from early diagnosis and management of their condition in that the need for total joint arthroplasty can be prevented in many cases.

Adult↗
PubMed1991

p103 and A60: novel proteins of the neuronal membrane-associated cytoskeleton.

Associated with the neuronal plasma membrane are cytoskeletal proteins which probably control the specialization of the membrane into axonal and dendritic domains. Specialized isoforms of the proteins spectrin and ankyrin are located in each region and provide molecular mechanisms for locating specific transmembrane proteins at required points. However, spectrin and ankyrin were defined by extensions of the model for the erythrocyte membrane, an analogy unlikely to provide a complete account of the neuronal membrane skeleton. We have defined two new proteins of the neuronal membrane skeleton, designated p103 and A60. p103 is enriched in post-synaptic densities and binds with high affinity to integral membrane proteins--we suggest that it may have a role in linking the cytoskeleton to synaptic glycoproteins. A60 is a 60 kDa axonal protein, which appears to form a lining to the axolemma. It is almost exclusively axonal, although some neurons (such as Purkinje cells) appear to contain it in the cell body and initial dendrite segment. A60 binds both ankyrin and neurofilaments, and may have a role in transmitting information critical to axonal morphology to the membrane.

Ankyrins↗
PubMed1990

Congenital factor XIII deficiency associated with von Willebrand disease.

A boy with umbilical bleeding and severe hemorrhages after minor trauma, without family bleeding history, was studied. Coagulation tests showed abnormalities in FXIII subunits and FVIII/vWF complex. Both parents presented results compatible with a heterozygote state for FXIII deficiency and the father had abnormalities of FVIII/vWF. The propositus was diagnosed as congenital FXIII deficiency associated with vWD. No severe hemorrhagic complication was observed after a prophylactic regimen with cryoprecipitates.

Factor XIII Deficiency↗
PubMed1990

Molecular associations on the T cell surface correlate with immunological memory.

Different isoforms of CD45 are expressed on naive and memory CD4 T cells in the mouse, as revealed by an antibody to a set of isoforms of CD45 that utilize exon B, called CD45RB. Cloned TH1 and TH2 lines also differ for expression of isoforms detected by this antibody. Differential expression of CD45 isoforms correlates with different behavior of cell surface molecules involved in transmembrane signal transduction. On naive T cells, CD4, CD45 and the CD3/T cell receptor complex behave as independent entities. On memory T cells, these three molecules are stably associated on the T cell surface. Furthermore, on TH2 cells, which express intermediate levels of CD45RB, CD4 is stably associated with CD45 isoforms other than CD45RB, but this complex is not associated with the CD3/T cell receptor. These results lead us to propose that immunological memory in CD4 T cells consists of an altered structure of the T cell's specific signal transduction apparatus controlled by low-molecular weight CD45 isoforms. This altered receptor structure would allow the more sensitive triggering of the T cell characteristic of memory cells. The organization of multimolecular signal transduction systems may be a general means by which cells alter their physiological behavior, allowing the acquisition of new phenotypic characteristics.

Animals↗
PubMed1990

Fibronectin-cell interactions.

Fibronectins are widespread extracellular matrix and body fluid glycoproteins, capable of multiple interactions with cell surfaces and other matrix components. Their structure at a molecular level has been resolved, yet there are still many unanswered questions regarding their biologic activity in vivo. Much data suggests that fibronectins may promote extracellular matrix assembly, and cell adhesion to those matrices. However, one outstanding enigma is that fibronectins may, under different circumstances, promote both cell migration and anchorage. An analysis of the interaction of fibroblasts with proteolytically derived and purified domains of plasma fibronectin revealed that the type of adhesion and the correlated cytoskeletal organization depended on multiple interactions of fibronectin domains with the cell surface. Human dermal fibroblasts were capable of interacting with the integrin-binding domain and both heparin-binding domains of the plasma fibronectin molecule and their interactions determined the type of adhesion. The same principle was seen in a study of the ability of plasma fibronectin to promote basement membrane assembly in an endodermal cell line, PF-HR9. There also, interactions of both heparin- and integrin-binding domains combined to promote the deposition of a proteoglycan, laminin, and type IV collagen-containing basement membrane matrix. The underlying conclusion from our studies is, therefore, that fibronectins may, through their different isotypes, multiple receptors, and varying interaction of one or more domains with those receptors, result in a spectrum of responses in different cell types. The molecular details of this array of biologic activities is not resolved but is the target of much current research.

Animals↗
PubMed1989

Myosin regulation and calcium transients in fibroblast shape change, attachment, and patching.

Following our study in Balb/c 3T3 cells and other cultured fibroblasts of the changes in myosin light chain phosphorylation associated with alterations in cell shape, attachment, and receptor patching, we have now determined the corresponding changes in cytoskeletal myosin distribution, and in the cellular calcium concentration, since this might, in part, mediate such responses. Immunofluorescence microscopy showed that myosin assembly into ordered forms such as actomyosin bundles and myosin sheath almost always correlated with previously shown high phosphorylation levels of myosin regulatory light chain, whereas diffuse distributions usually correlated with low or undetectable levels. An exception was observed in treatment to alter cellular cAMP levels when, in a biphasic response, assembly was correlated inversely with the phosphorylation states shown previously. Fluorescent indicators for intracellular calcium concentration, [Ca++]i, showed that myosin disassembly by trypsin or EGTA acting externally on the cells was preceded by a transient increase in [Ca++]i. For EGTA this was associated with transient recruitment of myosin into dorsal sheath structure as well as the transient enhancement of phosphorylation shown earlier. Blockage of EGTA-induced disassembly could be achieved by azide, which also caused an immediate increase in [Ca++]i and inhibited its subsequent decline. Trypsin-induced dephosphorylation did not appear to involve an eventual reduction of [Ca++]i. Therefore, in many but not all of the systems studied, correlated changes were observed in myosin assembly, [Ca++]i, and the myosin phosphorylation levels shown earlier.

Animals↗
PubMed1989

Differential activation of cytokine genes in normal CD4-bearing T cells is stimulus dependent.

Studies of cloned CD4+ T cell lines have shown that they can be separated into two distinct subsets with distinctions in their functional capabilities and by the differential release of either interleukin 2 (IL 2) (TH1/inflammatory type) or IL 4 (TH2/helper type) upon activation. To establish if in vivo-derived CD4+ T cells can exhibit distinct subsets we have investigated whether normal CD4+ T cells demonstrate differential expression of IL 2 and IL 4 mRNA, and secretion of IL 2 and IL 4 after primary stimulation in vitro. Utilizing the technique of in situ hybridization IL 2 and IL 4 gene expression in individual CD4+ T cells was readily detectable after concanavalin A (Con A) phytohemagglutinin (PHA) or pokeweed mitogen (PWM)-mediated activation. The frequencies of activated T cells producing IL 2 and IL 4 mRNA after Con A or PHA activation were approximately equivalent (30-40% of cells); however, after PWM activation the number of CD4+ T cells expressing IL 4 mRNA (78%) was more than twofold greater than the number of cells producing IL 2 mRNA (30%). Maximal levels of IL 2 gene expression occurred 24 h after mitogen activation whereas the highest levels of IL 4 mRNA were not detected until 48 h after mitogen activation. Similar distinctions in the kinetics of IL 2 and IL 4 secretion after mitogen activation were also found demonstrating good concordance in the observed expression of IL 2 and IL 4 mRNA and the levels of secreted lymphokines detected by bioassay. Most importantly, we have shown by in situ hybridization analysis that the majority of individual CD4+ T cells produce only IL 2 or IL 4 mRNA, and not both, after primary activation in vitro. By contrast, most CD4+ T cells activated in the presence of PMA and ionophore express both IL 2 and IL 4 mRNA. Our studies demonstrate that in normal, non-clonal populations of CD4+ T cells, the production of IL 2 and IL 4 is independently regulated in the majority of cells and appears to be stimulus dependent.

Animals↗
PubMed1989

Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies.

The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.

Animals↗
PubMed1989

Evidence for a Mr 88,000 glycoprotein with a transmembrane association to a unique flagellum attachment region in Trypanosoma brucei.

We have examined the relationship of externally accessible proteins associated with the internal cytoskeleton of procyclic Trypanosoma brucei. Two approaches were taken. First, externally disposed glycoproteins were identified with lectins and examined for their persistence and location in isolated cytoskeletons. Second, proteins containing tyrosine residues available for chemical modification on the outer surface were identified in isolated cytoskeletons and probed for glycosylation. The procyclic form of T. brucei that was employed does not express the variable surface glycoprotein. The lectin concanavalin A (ConA) bound to the outer surface of T. brucei in two discrete locations; one a narrow line close to the flagellum attachment zone on the cell body, the other at the distal tip of the flagellum itself. Of these, only the cell body labelling was detected when isolated cytoskeletons were probed with fluorescein isothiocyanate-labelled ConA. When cytoskeletons were prepared from cells labelled with gold-conjugated ConA, a narrow line of label was detected parallel to the flagellum attachment zone but was distinct from it. Only one cytoskeletal protein, of Mr 88,000, could be labelled at the cell surface by the 125I/iodogen procedure. This protein could be precipitated from SDS-solubilized cytoskeletons with ConA-agarose. These data indicate the existence of a previously undetected cytoskeletal structure, situated in the cell body, close to the point of flagellum attachment, which has a transmembrane association with an external Mr 88,000 glycoprotein.

Animals↗
PubMed1988

Autocrine growth of CD4+ T cells. Differential effects of IL-1 on helper and inflammatory T cells.

The growth factor requirements of cloned lines representing two major subsets of CD4+ T cells were examined. The helper subset, which produces IL-4 as its autocrine growth factor, proliferates in response to IL-2 or to IL-4 in the presence of IL-1. The inflammatory subset, which produces IL-2 as its autocrine growth factor, proliferates in response to IL-2 and, in the presence of limiting amounts of IL-2, shows increased proliferation in the presence of IL-4. The inflammatory subset does not proliferate in response to IL-1 plus IL-4. This ability to respond to the combination of IL-1 plus IL-4 correlates with the presence of IL-1R on the cloned lines tested. These data suggest that IL-1 may play a controlling role in the clonal expansion of CD4+ T cells of different functional types. This, in turn, suggests means by which the immune response could be directed into humoral or cell-mediated responses.

Animals↗