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Biomedical subjects

C A Smith

Publications and source records attributed to C A Smith.

At least 271 records · Page 15Linked to original sources
PubMed1995

Structural studies of myosin:nucleotide complexes: a revised model for the molecular basis of muscle contraction.

The structures of the MgADP-beryllium fluoride and MgADP-aluminum fluoride complexes of the truncated myosin head from Dictyostelium myosin II are reported. These reveal the location of the nucleotide complex and define the amino acid residues that form the active site. The tertiary structure of the beryllium fluoride complex is essentially identical to that seen previously in the three-dimensional structure of chicken skeletal muscle myosin. By contrast, significant domain movements are observed in the aluminum fluoride complex. These structural findings form the basis of a revised model for the structural basis of the contractile cycle. It is now suggested that the narrow cleft that splits the central 50-kDa segment of the heavy chain provides not only the communication route between the nucleotide-binding pocket and actin but also transmits the conformational change necessary for movement.

Adenosine Diphosphate↗
PubMed1994

Characterization of the activation-associated isoform of CD43 on murine T lymphocytes.

A rat mAb termed 1B11 recognizes a 130-kDa cell surface glycoprotein expressed on T lymphocytes. Transfection studies using the Cd43 gene transfected into murine L cells, and immunoblots using anti-peptide Abs specific for the CD43 polypeptide identified the 1B11 Ag as the 130-kDa isoform of murine CD43. mAb 1B11 fails to recognize the other major CD43 isoform, 115-kDa CD43, either by Western blotting or by FACS analysis, thus differing from the previously characterized anti-CD43 mAb S7 that recognizes only the CD43 115-kDa isoform and not the CD43 130-kDa isoform. CD43 130-kDa recognized by mAb 1B11 is differentially expressed on T lymphocytes. Whereas most CD4-8-, CD4+8+, and CD4-8+ thymocytes express 130-kDa CD43 constitutively, the Ag is expressed by less than 20% of CD4+ T cells in immature and mature populations. On activation, expression of 130-kDa CD43 is up-regulated dramatically on CD4+ T lymphocytes, and to a lesser extent on CD8+ T lymphocytes. In contrast, T cell activation resulted in only minor up-regulation of 115-kDa CD43. CD43 130-kDa contains sialylated O-linked carbohydrate; however, recognition by mAb 1B11 is not dependent on the presence of sialic acid. Interestingly, removal of sialic acid by neuraminidase treatment of 1B11-negative CD4+ T lymphocytes or 1B11-negative EL4 cells confers 1B11 reactivity, suggesting that the 1B11 epitope is masked by sialic acid residues on the CD43 115-kDa isoform. The isoelectric point (pl) of 130-kDa CD43 was determined to be 6.0, which is higher than the pl reported for 115-kDa CD43. Different molecular properties of 115-kDa and 130-kDa CD43 and their differential expression in T cell subsets may indicate specific roles for these CD43 isoforms in T cell ontogeny and/or T cell function.

Animals↗
PubMed1994

Regulation of intercellular adhesion molecule-1 gene expression involves multiple mRNA stabilization mechanisms: effects of interferon-gamma and phorbol myristate acetate.

Although the intercellular adhesion molecule-1 (ICAM-1) is constitutively expressed at a low level on a subpopulation of hematopoietic cells, on vascular endothelium, on fibroblasts, and on certain epithelial cells, it is dramatically increased at sites of inflammation. Interferon-gamma (IFN-gamma) and phorbol myristate acetate (PMA) are known to increase the expression of ICAM-1 on many cell types. Because both human and murine ICAM-1 mRNAs contain putative destabilizing AUUUA sequences in their 3' untranslated regions (UTRs), we examined the role of mRNA stability in the regulation of ICAM-1 gene expression. The treatment of the murine monocytic cell line P388D1, which constitutively expresses ICAM-1 mRNA at a low level, with IFN-gamma or PMA rapidly enhanced the level of ICAM-1 mRNA and dramatically prolonged its half-life. To determine whether the putative destabilizing sequences are responsible for this effect of IFN-gamma and PMA, fibroblast L cells were transfected with either the full-length ICAM-1 cDNA or a truncated form (ICAM-1 delta 3) lacking the putative destabilizing AUUUA sequences. Although ICAM-1 delta 3 mRNA was more stable than the full-length ICAM-1 mRNA, IFN-gamma treatment induced the accumulation of both mRNA species and prolongation of their half-lives. The transplantation of the ICAM-1 delta 3' UTR into a stable ICAM-2 mRNA rendered it unstable, and it was unresponsive to IFN-gamma. Therefore, the treatment with IFN-gamma stabilizes the otherwise labile ICAM-1 mRNA, but the IFN-gamma-responsive sequence may at least in part reside within the protein coding region. PMA also upregulated ICAM-1 gene expression by mRNA stabilization. However, unlike IFN-gamma, PMA treatment only increased the level of the full-length, but not of the truncated, ICAM-1 mRNA. This shows that the PMA-responsive element is located within the 3'UTR. Furthermore, the effect of PMA on ICAM-1 delta 3 mRNA was recovered by ligating multiple AUUUA sequences derived from a heterologous gene fragment. The stability of this chimeric mRNA and the full-length ICAM-1 mRNA was markedly increased by PMA treatment, indicating that the AUUUA multimers in the 3'UTR are important in the PMA-induced upregulation of ICAM-1 mRNA.

Animals↗
PubMed1994

A new ATP-independent DNA endonuclease from Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers and 6-4 photoproducts.

We have discovered a new DNA endonuclease in the fission yeast Schizosaccharomyces pombe which recognizes cyclobutane pyrimidine dimers and (6-4) pyrimidine-pyrimidone photoproducts. S. pombe DNA endonuclease (SPDE) catalyzes a single ATP-independent incision immediately 5' to the UV photoproduct and generates termini containing 3' hydroxyl and 5' phosphoryl groups. Based on these properties, we propose that SPDE may function in a DNA repair capacity, representing the initial recognition/cleavage step of a DNA excision repair pathway.

Adenosine Triphosphate↗
PubMed1994

Membrane topology of liver microsomal cytochrome P450 2B4 determined via monoclonal antibodies directed to the halt-transfer signal.

The membrane topology of cytochrome P450 2B4 from the endoplasmic reticulum has been studied with highly-purified liver microsomes in a site-directed immunochemical approach. Microsomes were prepared from phenobarbital-induced rabbits, and the resulting microsomal fraction was washed 6 additional times with 0.1 M pyrophosphate buffer to effect removal of significant quantities of adventitiously-bound protein. Monoclonal antibodies were prepared against residues 18-29 of P450 2B4 (Leu18-Leu-Phe-Arg-Gly-His-Pro-Lys-Ala-His-Gly-Arg29), essentially corresponding to the halt-transfer signal. This region was chosen due to its mutually-exclusive location in the two alternative membrane topology models currently tenable [Black, S.D. (1992) FASEB J.6, 680-685]. Model "A" contains a single transmembrane anchor peptide with the amino terminus projecting into the lumen of the endoplasmic reticulum, while model "B" exhibits a hairpin loop of the first approximately 46 residues inserted into the membrane with the amino terminus located on the cytosolic side of the lipid bilayer; the halt-transfer signal peptide would be located at the cytosolic surface of the membrane in model "A" or as a loop on the lumenal side of the membrane in model "B". Nine antibodies, denoted as MmAbA, MmAbC, MmAbD, MmAbF, MmAbH, MmAbI, MmAbK, MmAbL, and MmAbP, were produced, and all were identified as IgM/kappa subtypes. Western blotting demonstrated that the antibodies could readily recognize P450 2B4 in microsomes. ELISA assays showed that all of the antibodies exhibited strong binding to intact microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Structure of copper- and oxalate-substituted human lactoferrin at 2.0 A resolution.

The three-dimensional structure of human dicupric monooxalate lactoferrin, Cu(2)oxLf, has been determined to 2.0 A resolution, using X-ray diffraction data collected by diffractometry to 2.5 A resolution, and oscillation photography on a synchrotron source to 2.0 A resolution. Difference electron-density maps calculated between Cu(2)oxLf and both dicupric lactoferrin, Cu(2)Lf, and diferric lactoferrin, Fe(2)Lf, showed that the oxalate had replaced a carbonate in the C-terminal binding site, and that, relative to Cu(2)Lf, there were no significant differences in the N-terminal site. The structure was then refined crystallographically by restrained least-squares methods. The final model, in which the r.m.s. deviation in bond distances is 0.017 A, contains 5314 protein atoms (691 residues), two Cu(2+) ions, one bicarbonate ion, one oxalate ion, 325 solvent molecules and one sugar residue. The crystallographic R factor of 0.193 is for 46 134 reflections in the range 8.0 to 2.0 A resolution. The oxalate ion is coordinated to copper in a 1,2-bidentate fashion, and the added bulk of the anion results in the rearrangement of the side chains of nearby arginine and tyrosine residues. No other major alterations in the molecule can be observed, the overall protein structure being the same as that for Cu(2)Lf and Fe(2)Lf.

Journal Article↗
PubMed1994

A lymphotoxin-beta-specific receptor.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses. The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta (LT-beta), a type II transmembrane protein that is another member of the TNF ligand family. Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons; however, these receptors do not recognize the major cell surface LT-alpha-LT-beta complex. A receptor specific for human LT-beta was identified, which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha.

Amino Acid Sequence↗
PubMed1994

Pleiotropic effects of the CD30 ligand on CD30-expressing cells and lymphoma cell lines.

CD30 is a member of the tumor necrosis factor receptor superfamily. CD30 was originally described as a cell surface antigen on primary and cultured Hodgkin's and Reed-Sternberg cells. In this study, recombinant human CD30 ligand was expressed on the surface of CV-1/EBNA cells and tested for biologic activities on a variety of different CD30+ human lymphoma cell lines. CD30 ligand enhanced Ig secretion of Epstein-Barr virus (EBV)-immortalized, CD30+ lymphoblastoid B-cell lines, but not Burkitt lymphoma lines. Recombinant CD30 ligand enhanced proliferation of "T-cell-like" Hodgkin's disease-derived cell lines and an adult T-cell leukemia cell line, but not "B-cell-like" Hodgkin's disease-derived cell lines, CD30+, EBV-immortalized lymphoblastoid B-cell lines, or CD30+ and EBV+ tumor B-cell non-Hodgkin's lymphoma cell lines. In addition, CD30 ligand mediated reduction of proliferation and viability, by induction of cytolytic cell death, of CD30+, large-cell anaplastic lymphoma cell lines. Two new antibodies, M44 and M67, against the CD30 antigen demonstrated similar biologic activities to the CD30 ligand. Taken together, these data demonstrate pleiotropic biologic activities of the CD30 ligand on different CD30+ lymphoma cell lines and indicate that the CD30-CD30 ligand interaction might have a pathophysiologic role in Hodgkin's and some non-Hodgkin's lymphomas.

Antibodies, Monoclonal↗
PubMed1994

Characterization of (6-4) photoproduct DNA photolyase.

Pyrimidine (6-4) pyrimidone photoproduct is the second most abundant UV photoproduct in DNA. Recently, it was reported that Drosophila melanogaster cell-free extracts restored the biological activity of (6-4) photo-product-containing DNA in a light-dependent reaction (Todo, T., Takemori, H., Ryo, H., Ihara, M., Matsunaga, T., Nikaido, O., Sato, K., and Nomura, T. (1993) Nature 361, 372-374) concomitant with the loss of (6-4) photoproduct antigenic sites and (6-4) photoproduct-caused alkali-labile sites. In the present study we show that the (6-4) photoproduct but not its Dewar isomer is the substrate for the enzyme, that the enzyme has an action spectrum peak at 400 nm, and that the efficiency of repair per incident photon is very low compared with cyclobutane pyrimidine dimer photolyases. Furthermore, we provide evidence that the (6-4) photoproduct photolyase converts the photoproduct to unmodified bases probably through an oxetane intermediate.

Animals↗
PubMed1994

Isolation and characterization of two mouse Pi-class glutathione S-transferase genes.

Pi-class glutathione S-transferases (GSTs) play an important role in the detoxification of chemical toxins and mutagens and are implicated in neoplastic development and drug resistance. In all species characterized to date, only one functional Pi-class GST gene has been described. In this report we have identified two actively transcribed murine Pi-class GST genes, Gst p-1 and Gst p-2. The coding regions of Gst p-1 and the mouse Pi-class GST cDNA (GST-II) reported by Hatayama, Satoh and Satoh (1990) (Nucleic Acids Res. 18, 4606) are identical, whereas Gst p-2 encodes a protein that has not been described previously. The two genes are approximately 3 kb long and contain seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (GGGCGG) respectively. The cDNAs of the two genes were isolated from total liver RNA using reverse PCR. The peptide sequence deduced from the cDNAs share 97% identity and differ in six amino acids. Both genes are transcribed at significantly higher levels in male mouse liver than in female, and Gst p-1 mRNA is more abundant in both sexes than Gst p-2.

Amino Acid Sequence↗