PubMed Health⌕ Search

Biomedical subjects

B R Clark

Publications and source records attributed to B R Clark.

At least 37 records · Page 2Linked to original sources
PubMed1993

Lower thoracic upper lumbar spinocerebellar projections in rats: a complex topography revealed in computer reconstructions of the unfolded anterior lobe.

The topography of wheatgerm agglutinin-horseradish peroxidase/horseradish peroxidase-labeled mossy fiber terminals of lower thoracic-upper lumbar (T12-L3) spinal projections to the cerebellar anterior lobe was quantitatively analysed in adult rats. Computer-based image analysis mapped the orthogonal (parallel to the surface) distribution of labeled terminals in two-dimensional reconstructions of the unfoled anterior lobe cortex. The radial (perpendicular to the surface) distribution of terminals within the granule cell layer was mapped by computing whether the terminals were in either the outer- or inner-halves of this layer. The number of labeled terminals in each lobule was calculated. In the anterior lobe, lower thoracic-upper lumbar spinocerebellar projections terminate primarily in lobules II (mean 27.14%), III (mean 38.68%), and IV (mean 19.31%). Different-sized bilateral injections restricted to L1 were used to study the organization of intrasegmental spinocerebellar projections. Small injections into L1 labeled a limited number of terminals which were located either in clusters or were spatially isolated. Intermediate-sized intrasegmental injections resulted in additional clusters of labeled terminals. Many of the terminal clusters were spatially related and formed larger irregularly shaped patches. Large intrasegmental injections labeled terminal clusters and patches that were discontinuous but aligned parallel to the longitudinal (transverse) axis of lobules II-IV. Injections including segments rostral and caudal to L1 were used to study the topography of intersegmental lower thoracic-upper lumbar spinocerebellar projections. Multisegmental injections increased the number of labeled terminal clusters and patches which obscured the pattern of segmental input, but there was still a transversely oriented pattern of termination. Distinct transversely aligned terminal free areas remained apparent. Lower thoracic-upper lumbar spinocerebellar projections terminated in both the outer- and inner-halves of the granule cell layer, but overall were more numerous in the outer-half of this layer. In serially spaced sagittal sections, however, the majority of terminals alternated between the outer- and inner-halves of the granule cell layer. Outer- and inner-terminals were not spatially segregated in their orthogonal distribution. These results indicate lower thoracic-upper lumbar spinocerebellar projections have a complex three-dimensional topography in the anterior lobe. These findings are discussed in relation to previous findings for a sagittally oriented topography for lower thoracic-upper lumbar spinocerebellar projections and in the context of how cerebellar somatosensory afferent input may be organized.

Animals↗
PubMed1992

Antigen-specific stimulation of T cell extracellular acidification by MHC class II-peptide complexes.

A specific T cell response to a preformed complex of detergent-solubilized MHC class II molecule and cognate antigenic peptide was observed by monitoring the extracellular acidification. An increase in this rate was observed when the resting 4R3.9 T cell clone specific for the peptide fragment MBP(1-14) of myelin basic protein was exposed to preformed detergent-solubilized IAk-MBP(1-14)A4 complexes. MBP peptide alone, IAk alone, or complexes of IAs-proteolipid protein(139-151) and IAd-OVA(323-339), did not cause significant increases in the acidification rates of the MBP(1-14)-restricted 4R3.9 T cell clone. In addition, BW 5147 T lymphoma cells, which lack TCR, did not show any increase in rate when exposed to IAk-MBP(1-14)A4 complexes. Similar increases in acidification rate were observed in the presence of IL-2, anti-CD3 and anti-TCR antibodies. The enhanced acidification responses were blocked by genistein, a tyrosine kinase inhibitor.

Acids↗
PubMed1992

In vitro maximum binding of antigenic peptides to murine MHC class II molecules does not always take place at the acidic pH of the in vivo endosomal compartment.

Presentation of Ag to the T cell requires binding of specific peptide fragments of the Ag to MHC II molecules. The ability of a peptide to bind to MHC class II appears to be pH dependent. Recent reports indicate that the binding of peptide to MHC class II molecules takes place primarily within an endosomal compartment of the cell at around pH 5. In this study, we have explored the in vitro pH dependence of peptide binding to different haplotypes of murine MHC class II molecules. The binding of peptides to MHC II was analyzed and quantitated by silica gel TLC, using radiolabeled peptides. The MBP peptide fragments, MBP(1-14)A4 and MBP(88-101)Y88, bound maximally at pH 8 to IAk and IAs, respectively. The binding of PLP peptide fragment, PLP(138-151)Y138, to IAs was maximal at around neutral pH. The maximum binding of an OVA peptide fragment, OVA(323-340)Y340, to IAd, was found to occur at pH 6. Results presented in this report thus suggest that the in vitro maximum binding of peptide is pH dependent and does not always occur at pH 5. The optimum pH range for maximum binding may depend on the nature and net charge of the peptide and its interaction with MHC class II molecules.

Amino Acid Sequence↗
PubMed1992

Cell adhesion in the stromal regulation of haemopoiesis.

Haemopoiesis occurs in close physical contact with elements of the bone marrow stroma. This review examines some adhesion molecules which are expressed in haemopoietic environments. These molecules may act to maintain cell contact between haemopoietic progenitors and the cellular and extracellular matrix elements of the marrow stroma. A review is undertaken of several families of adhesion molecules and their ligands, including integrins, CD44, LEC-CAMs, CAMs and variant glycosylation patterns expressed by haemopoietic cells. Both CD44 and integrin receptors have been implicated in mediating important cellular interactions within the haemopoietic system and the significance of these and other molecules in haemopoiesis is discussed.

Amino Acid Sequence↗
PubMed1991

Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex-peptide complexes.

Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) class II gene-linked autoimmune demyelinating disease of the central nervous system. To develop therapies that will specifically inactivate only the autoantigen-reactive T cells, mice were treated with soluble MHC class II molecules that had been complexed with encephalitogenic peptides. Intravenous injections of 300 micrograms of complexes consisting of encephalitogenic peptide 91-103 of myelin basic protein plus I-As protein on day 0, 4, and 7 were effective in preventing experimental allergic encephalomyelitis. Similarly, administration of 45 micrograms of I-As protein complexed to peptide 139-151 from proteolipoprotein on day 1, 4, and 7 prevented mortality and significantly reduced paralysis induced by immunization with the encephalitogenic proteolipoprotein peptide. Histological examination of sections of animal brains revealed that treatment with I-As protein plus myelin basic protein 91-103 peptide prevents the development of inflammatory lesions characteristic of experimental allergic encephalomyelitis. Thus, treatment with MHC-self-peptide complexes could serve as a highly specific therapeutic modality in treating autoimmune disease when the putative autoantigen and the MHC restricting elements are known.

Animals↗
PubMed1991

Binding of truncated peptides to the MHC molecule IA (d).

A peptide comprising amino acids 323-339 of chicken ovalbumin is known to bind to two heterodimeric conformations of the MHC molecule IA(d), and to each of its separate alpha- and beta-chains. We report that minor C- and N-terminal truncations of the parent peptide do not alter the binding pattern. A decrease in binding activity was observed upon deletion of the histidine residues of the already truncated peptides. Peptides as short as 4 amino acids associate weakly with all four proteins.

Amino Acid Sequence↗
PubMed1991

Energy transfer between two peptides bound to one MHC class II molecule.

The 17-amino acid peptide from chicken ovalbumin, Ova(323-339), was labeled at the amino terminus with fluorescein [FOva(323-339)] and near the carboxyl terminus with Texas Red [AcOva(323-338)KTR]. Fluorescence spectroscopy was carried out on resolved electrophoretic bands on nonreducing polyacrylamide gels derived from incubation mixtures containing major histocompatibility complex (MHC) class II molecules IAd and the FOva(323-339)- and AcOva(323-338)KTR-labeled peptides. Energy transfer between fluorescein and Texas Red was observed in the "floppy" alpha beta heterodimer band, but not in the "compact" alpha beta heterodimer band. Energy transfer was detected between the truncated peptides FOva(323-328)CONH2 and AcOva(331-338)KTR in both the compact alpha beta and floppy alpha beta gel bands. The energy-transfer data suggest that the two binding sites of floppy alpha beta arise from splitting apart a putative large, single binding site region in compact alpha beta.

Amino Acid Sequence↗
PubMed1991

Novel methods to rapidly and sensitively analyze antigenic peptide binding to MHC class II molecules.

One of the important steps for antigen presentation by MHC class II molecules involves binding of a peptide fragment of the antigen to the class II molecule followed by recognition of the resulting complex by T cells. The most commonly used methods for studying binding of peptide to MHC II are: equilibrium dialysis, gel filtration chromatography, HPLC and polyacrylamide gel electrophoresis. Each of these methods has some limitations and is time consuming. In addition, each requires a considerable amount of native MHC class II, which is always difficult to obtain. In this report, we describe three different sensitive methods using radiolabeled peptide to study peptide binding to murine MHC class II molecules. These are: nitrocellulose filter binding, thin-layer chromatography (TLC) using plate-supported silica gel or PEI cellulose, and paper electrophoresis using Sepraphor cellulose polyacetate paper. All three methods are rapid, highly sensitive and require only ng quantities of affinity pure MHC class II molecules and peptides. These methods can be used to calculate the peptide occupancy of MHC class II molecules.

Animals↗
PubMed1991

In vitro peptide binding to the heavy chain of the class I molecule of the major histocompatibility complex molecule HLA-A2.

The heavy chain of class I molecules of the major histocompatibility complex forms the binding site for antigenic peptides. We describe the binding of a synthetic peptide to the purified heavy chain of the human major histocompatibility complex molecule HLA-A2. The peptide binding capacity is found to be markedly increased if the protein is first partly denatured by reduction of its disulfide bonds in detergent and subsequently renatured by reoxidation. In the presence of certain detergents, the heavy chain binds peptides even when the protein is partly unfolded.

Amino Acid Sequence↗
PubMed1991

Morphological evidence for calcium-dependent association of calgranulin with the epidermal cytoskeleton in inflammatory dermatoses.

The association of calgranulins, intracellular calcium-binding proteins, with the keratinocyte cytoskeleton has been studied. These molecules are expressed in various inflammatory dermatoses and in organ-culture explants. Triton X-100 extraction in the presence of calcium or EDTA suggested that calgranulins are detergent insoluble in the presence of calcium. The molecules were localized in a plaque-like structure at the cell periphery in lesional skin and in organ-culture explants. Following induction of calgranulins in vitro there was a redistribution of the intermediate filament cytoskeleton into a perinuclear halo, although desmosomes remained intact. These various features suggest that these members of the S-100 protein family have a role in cytoskeletal changes seen in various skin diseases.

Blotting, Western↗
PubMed1991

Kinetic studies on the biological activity of calmodulin in canine heart and liver during endotoxin shock.

Effects of endotoxin administration on the biological activity of calmodulin isolated from canine heart and liver were studied. Calmodulin was isolated and purified to homogeneity. The biological activity of calmodulin was determined by its ability to activate Ca(2+)-dependent phosphodiesterase. Results obtained 4 h after endotoxin administration show that the Vmax and A0.5 for calmodulin, the Vmax and Km for cAMP, and the Vmax and the Hill coefficient for Ca2+ were unchanged, while the S0.5 for Ca2+ for the activation of phosphodiesterase were significantly increased in the heart. The kinetic parameters as described above were not significantly altered in the liver. These data indicate that the biological activity of calmodulin is inhibited in the heart during endotoxin shock and that the nature of inhibition is associated with a mechanism involving a decrease in the affinity (1/S0.5) towards Ca2+ binding. Since calmodulin plays an important role in the regulation of cardiac function through calmodulin-dependent calcium transport systems, our findings may have a pathophysiological significance in contributing to the understanding of myocardial dysfunction in endotoxin shock.

Animals↗
PubMed1990

Calgranulin expression and association with the keratinocyte cytoskeleton.

The molecules calgranulin A and B are two intracellular calcium-binding proteins which are expressed by the lesional keratinocytes of inflammatory dermatoses. We have investigated the induction of the calgranulin proteins in an in vitro system and characterized the epidermal form of calgranulin. Using calgranulin-specific monoclonal antibodies, we have shown that these proteins are expressed within the epidermis of skin explants after 12-24 h culture in vitro. The induction of calgranulin-specific staining on culture was prevented, however, by the inclusion of cycloheximide in the culture medium, in sufficient quantities to prevent de novo protein synthesis. Indirect immunofluorescence staining was used to analyse the subcellular localization of the calgranulin proteins. The specific staining pattern with antibodies which recognize the individual calgranulin proteins was retained in detergent insoluble cytoskeletal preparations of epidermis. In Western blotting experiments epidermal calgranulins could be solubilized only by using a urea-based protein extraction buffer. After sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the epidermal extracts a single antigen, with a molecular weight of 13.0 kD was detected with the calgranulin-specific antibody MAC 387. The expression of calgranulins, similar to other members of the same protein family, may regulate cytoskeletal changes in skin disease.

Blotting, Western↗
PubMed1990

The invariant chain forms complexes with class II major histocompatibility complex molecules and antigenic peptides "in vivo".

The binding of a chicken ovalbumin peptide (residues 323-339), Ova-(323-339), to I-Ad molecules was investigated in vitro and in vivo. By using antigenic peptides labeled either with a hapten or with fluorescein, complexes formed in vitro between I-Ad and antigenic peptides were detected by Western blot analysis with an antibody recognizing the hapten 7-nitrobenzo-2-oxa-1,3-diazole and by scanning gels for fluorescence emitted by fluoresceinated peptide. Both techniques reveal that Ova-(323-339) binds not only to I-Ad alpha/beta heterodimers and separated alpha and beta chains but also to complexes of higher molecular mass. Additional analysis shows that one of these additional complexes contains I-Ad heterodimers, antigenic peptides, and also invariant chain. To explore the physiological role of these complexes, cells were incubated with haptenated peptide and the I-Ad-peptide complexes formed in vivo were purified by affinity chromatography using hapten-specific antibody. The complexes formed migrate with a significantly higher apparent molecular mass than the alpha/beta heterodimers. A band at 180 kDa contained the alpha/beta heterodimer, the antigenic peptide, and the invariant chain. These results show that in vivo high molecular mass complexes formed by the I-Ad heterodimer and the invariant chain bind antigenic peptides.

Animals↗
PubMed1988

Genomic localization of the gene encoding a 32-kDa capsid protein of human cytomegalovirus.

We have determined the map position of a viral gene encoding a 32-kDa late structural protein of human cytomegalovirus (HCMV) using a murine monoclonal antibody. This monoclonal antibody was reactive with two protein bands of 32 and 27 kDa in HCMV-infected cell lysates and with a single 32-kDa protein band in HCMV virions as detected by immunoblot analysis. When purified HCMV envelope preparation was used for immunoblotting, the monoclonal antibody did not display a detectable band. We used this monoclonal antibody to screen a cDNA library that was constructed from poly(A)+ RNA of late HCMV-infected cells and cloned into the expression vector lambda gt11. A cDNA clone that expressed an immunoreactive epitope of the late HCMV protein fused to beta-galactosidase was identified. Probing the restriction digests of HCMV (Towne and AD169) DNA with insert DNA from the immunoreactive lambda gt11 clone permitted us to localize the coding sequence within the long unique region between map coordinates of 0.62 and 0.64 of HCMV Towne and AD169 genomes. Using the same probe, a single transcript of 1.4 kb was detected in total RNA from HCMV-infected cells at late times after infection.

Animals↗
PubMed1985

A 64,000 dalton matrix protein of human cytomegalovirus induces in vitro immune responses similar to those of whole viral antigen.

Human cytomegalovirus contains approximately 30 to 35 structural polypeptides. Although antibodies to several of these proteins are made during natural infection, their relationship to T cell recognition of this virus and subsequent control of infection is poorly understood. We have purified one of these proteins (HCMVgp64) that is found in abundance in infected cell lysates in order to delineate the relationship of single viral proteins to the immune response caused by the virus. HCMVgp64 induced T cell reactivity only in individuals with serologic evidence of past infection. In addition, HCMVgp64 elicited similar in vitro immune reactions as the whole virus including T cell proliferation, interleukin 2 production, and receptor expression as well as interferon production. These studies suggest that single proteins of HCMV such as HCMVgp64 are capable of inducing T cell responses and may be important in the development of immune reactivity to HCMV.

Antigens, Viral↗