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R Smith

Publications and source records attributed to R Smith.

At least 595 records · Page 33Linked to original sources
PubMed1995

Three-dimensional structure of the second cysteine-rich repeat from the human low-density lipoprotein receptor.

The ligand-binding domain of the low-density lipoprotein receptor comprises seven cysteine-rich repeats, which have been highly conserved through evolution. This domain mediates interactions of the receptor with two lipoprotein apoproteins, apo E and apo B-100, putatively through a calcium-dependent association of the ligands with a cluster of acidic residues on the receptor. The second repeat (rLB2) of the receptor binding domain has been expressed as a thrombin-cleavable GST fusion protein, cleaved, and purified. On oxidation the protein refolded to give a single peak on reverse-phase HPLC. The aqueous solution structure of rLB2 has been determined using two-dimensional 1H NMR spectroscopy. In contrast to the amino-terminal repeat, rLB1, rLB2 has a very flexible structure in water. However, the conformation of rLB2 is markedly more ordered in the presence of a 4-fold molar excess of calcium chloride; the proton resonance dispersion and the number of NOESY cross-peaks are greatly enhanced. The three-dimensional structure of rLB2, obtained from the NMR data by molecular geometry and restrained molecular dynamics methods, parallels that of rLB1, with an amino-terminal hairpin structure followed by a succession of turns. However, there are clear differences in the backbone topology and structural flexibility. As for rLB1, the acidic residues are clustered on one face of the module. The side chain of Asp 37, which is part of a completely conserved SDE sequence thought to be involved in ligand binding, is buried, as is its counterpart (Asp 36) in rLB1. These results provide the first experimental support for the hypothesis that each of the repeats in the ligand-binding domain has a similar global fold but also highlight significant differences in structure and internal dynamics.

Cysteine↗
PubMed1995

Analysis of CD2 and TCR-beta gene expression in Jurkat cell mutants suggests a cis regulation of gene transcription.

Thirty CD2- J32 stable clones, derived by mutagenesis and subsequent immunoselection with anti-CD2 Ab, were used to study the regulation of CD2 and TCR gene expression. Analysis of RNA expression revealed that the loss of surface expression of CD2 was due to a lack of expression of CD2 mRNA and was associated with a lack of expression of VDJ TCR-beta transcripts in 12 of these mutants, sparing the expression of DJ TCR-beta, TCR-alpha, CD3 gamma, delta, epsilon, and zeta RNA. The expression of other differentiation molecules was unaffected, except for CD1, CD4, and CD5, which were either decreased or absent in most of these mutants. A gain in the expression of TCR-gamma transcripts was observed in each of these mutants, while, as expected, no TCR-gamma transcripts were detected in wild-type J32 cells. Several mutants were able to use the human CD2 enhancer and the murine TCR-beta enhancer and promoter to activate transcription from reporter genes in the context of heterologous promoters, indicating that the mutation(s) does not affect transcription pathways. Consistent with this finding is the adequate expression in these mutants of several lineage-specific transcription factors. The expression of CD2 in several of these mutants was rescued by gene transfer using a genomic 28.5-kb CD2 fragment, suggesting that the enhancer function of this gene may be dependent on the enhancer site. These observations suggest that the coordinate expressions of CD2 and TCR-beta genes share common regulatory mechanisms involving factors regulating chromatin structure and accessibility.

Antigenic Variation↗
PubMed1995

Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain.

The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate the endocytosis of multiple ligands in mammalian cells. Its ligand-binding domain consists of seven cysteine-rich ligand-binding repeats, each approximately 40 amino acid residues long. Ligand-binding repeats occur in other members of the LDL receptor (LDLR) gene family and in a number of functionally unrelated proteins. As a first step toward an understanding of the structure and function of LB repeats, we have expressed the amino-terminal ligand-binding repeat (LB1) of the human LDLR as a recombinant peptide (rLB1) and have determined its disulfide-pairing scheme. Oxidative folding of rLB1 yielded a single isomer which contained three disulfide bonds. This isomer reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDLR, suggesting that rLB1 was correctly folded. rLB1 was resistant to digestion with trypsin, chymotrypsin, and V8 protease, consistent with a tightly folded structure. Disulfide bond connections were established using two separate approaches. Digestion with the nonspecific proteolytic enzyme proteinase K yielded an 8 amino acid peptide with a single disulfide bond which connected Cys(IV) and Cys(VI). In the second approach, disulfide bonds were sequentially reduced with tris(2-carboxyethyl)phosphine and the resulting cysteine residues alkylated with iodoacetamide. An analysis of peptides which contained two cysteinylacetamide residues, derived from a single reduced disulfide bond, showed that Cys(I) and Cys(III) were disulfide-bonded and confirmed the presence of a disulfide bond between Cys(IV) and Cys(VI). We infer that the remaining disulfide bond bridges Cys(II) and Cys(V).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Expression and disulfide-bond connectivity of the second ligand-binding repeat of the human LDL receptor.

The human LDL receptor (LDLR) has a binding domain which consists of seven contiguous ligand-binding (LB) repeats, each approximately 40 amino acids long with three disulfide bonds. The second LB repeat, which is required for full binding of LDL, has been expressed, purified and folded to yield a single, fully oxidized isomer. By selective reduction and alkylation, we have shown that the cysteine residues have a I-III, II-V, IV-VI connectivity, matching that recently determined for the amino-terminal repeat. We suggest that the first two LB repeats of the LDLR, with their unique disulfide-bonding pattern, serve as a structural paradigm for other LB repeats.

Amino Acid Sequence↗
PubMed1995

Subareolar breast cancer: long-term results with conservative surgery and radiation therapy.

PURPOSE: It has been suggested that patients presenting with breast cancers within 2 cm of the nipple areolar complex represent a relative contraindication to conservative management due to either a compromised cosmetic result associated with sacrifice of the nipple areolar complex, reluctance to include the entire nipple areolar complex in the conedown field, or increased risk of multicentricity. We have reviewed our experience of conservatively treated patients with specific reference to the subset of patients presenting with tumors within 2 cm of the nipple areolar complex. METHODS AND MATERIALS: Between January 1970 and December 1989, 1014 patients with early stage breast cancer were treated at Yale-New Haven Hospital by excisional biopsy with or without axillary lymph node dissection. Of the 1014 charts reviewed, a total of 98 patients fulfilled the criteria of having a central/ subareolar breast cancer. Reexcision was performed on only 16 patients. Following conservative surgery, patients were treated with radiation therapy to the intact breast to a total median dose of 48 Gy with conedown to a total of 64 Gy. adjuvant systemic therapy and regional nodal irradiation were administered as clinically indicated. RESULTS: As of December 1993, the median follow-up for the 98 patients in this study was 9.03 years. The majority of patients had presented with either a palpable mass or a mammographically detected lesion. Three patients presented with Paget's disease, five with nipple discharge, and seven with nipple inversion. Ten of the 98 patients had the nipple areolar complex sacrificed at the time of surgery, while the remaining 88 patients had the entire nipple areolar complex included in the conedown field. Four of these 88 patients had the nipple partially blocked during the electron conedown. There were no significant complications associated with including the entire nipple areolar complex within the conedown field to a median dose of 64 Gy. Six of the 98 patients experienced a local recurrence, three experienced a regional recurrence, and nine experienced distant metastasis. The actuarial 10-year survival (0.79 +/- 0.06), distant disease-free survival (0.88 +/- 0.04) and breast recurrence-free survival (0.84 +/- 0.07) were not significantly different from those patients who presented with cancers in other parts of the breast. CONCLUSIONS: Patients presenting with subareolar breast cancers within 2 cm of the nipple areolar complex are suitable candidates for conservative surgery and radiation therapy. In the majority of patients in this study, the nipple areolar complex did not need to be sacrificed and could be safely included in the electron conedown field with acceptable complications and cosmesis. A subareolar breast cancer does not represent a relative contraindication to conservative management in patients with early stage breast cancer.

Breast Neoplasms↗
PubMed1995

Inhibition of Drosophila EGF receptor activation by the secreted protein Argos.

The Drosophila homologue of the mammalian epidermal growth factor (EGF) receptor (DER) is a receptor tyrosine kinase involved in many stages of fly development, including photoreceptor determination, and wing-vein formation. Its primary activating ligand is the Spitz protein, which is similar to mammalian TGF-alpha. Argos is a secreted protein that, like Spitz, contains a single EGF motif. It is a repressor of cell determination in the eye, and acts in other tissues, including the wing. Because Argos has the opposite effects to DER in the eye (the former blocks photoreceptor determination, the latter promotes it) we have tested whether it acts by blocking the DER pathway. We show that Argos does indeed repress this pathway in vivo and find that, in vitro, Argos protein can inhibit the activation of DER by Spitz. Thus the determination of cells by the DER pathway is regulated by a balance between extracellular activating and inhibiting signals. This is the first in vivo example of an extracellular inhibitor of a receptor tyrosine kinase.

Animals↗
PubMed1995

Isolation and photodynamic effects of hematoporphyrin derivative components: a chromatographic analysis of the starting materials.

Twenty different fractions of hematoporphyrin derivatives (HpD) and eight fractions of an HpD dimer mixture were isolated utilizing isocratic reversed-phase ion-pair high-performance liquid chromatography. These fractions were characterized by UV-visible and fluorescence spectrophotometry. Fluorescence quantum yields and photokill efficiency for each fraction in PTK2 epithelial cells were obtained. Results indicate that some part of the photoactivity exhibited by HpD may be due to impurities present in the HpD starting material, hematoporphyrin-IX dihydrochloride, depending on its source. It was also found that hematoporphyrin D, a commercial acetylated product formed during synthesis of HpD, contained a higher percentage of monomers than would be expected.

Animals↗
PubMed1995

X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II complexed with beryllium and aluminum fluoride and magnesium ADP are reported at 2.0 and 2.6 A resolution, respectively. Crystals of the beryllium fluoride-MgADP complex belong to space group P2(1)2(1)2 with unit cell parameters of a = 105.3 A, b = 182.6 A, and c = 54.7 A, whereas the crystals of the aluminum fluoride complex belong to the orthorhombic space group C222(1) with unit cell dimensions of a = 87.9 A, b = 149.0 A, and c = 153.8 A. Chemical modification was not necessary to obtain these crystals. These structures reveal the location of the nucleotide complexes and define the amino acid residues that form the active site. The tertiary structure of the protein complexed with MgADP.BeFx is essentially identical to that observed previously in the three-dimensional model of chicken skeletal muscle myosin subfragment-1 in which no nucleotide was present. By contrast, the complex with MgADP.AlF4- exhibits significant domain movements. The structures suggest that the MgADP.BeFx complex mimics the ATP bound state and the MgADP.AlF4- complex is an analog of the transition state for hydrolysis. The domain movements observed in the MgADP.AlF4- complex indicate that myosin undergoes a conformational change during hydrolysis that is not associated with the nucleotide binding pocket but rather occurs in the COOH-terminal segment of the myosin motor domain.

Adenine↗
PubMed1995

Three-dimensional structure of a cysteine-rich repeat from the low-density lipoprotein receptor.

The low-density lipoprotein (LDL) receptor plays a central role in mammalian cholesterol metabolism, clearing lipoproteins which bear apolipoproteins E and B-100 from plasma. Mutations in this molecule are associated with familial hypercholesterolemia, a condition which leads to an elevated plasma cholesterol concentration and accelerated atherosclerosis. The N-terminal segment of the LDL receptor contains a heptad of cysteine-rich repeats that bind the lipoproteins. Similar repeats are present in related receptors, including the very low-density lipoprotein receptor and the LDL receptor-related protein/alpha 2-macroglobulin receptor, and in proteins which are functionally unrelated, such as the C9 component of complement. The first repeat of the human LDL receptor has been expressed in Escherichia coli as a glutathione S-transferase fusion protein, and the cleaved and purified receptor module has been shown to fold to a single, fully oxidized form that is recognized by the monoclonal antibody IgG-C7 in the presence of calcium ions. The three-dimensional structure of this module has been determined by two-dimensional NMR spectroscopy and shown to consist of a beta-hairpin structure, followed by a series of beta turns. Many of the side chains of the acidic residues, including the highly conserved Ser-Asp-Glu triad, are clustered on one face of the module. To our knowledge, this structure has not previously been described in any other protein and may represent a structural paradigm both for the other modules in the LDL receptor and for the homologous domains of several other proteins. Calcium ions had only minor effects on the CD spectrum and no effect on the 1H NMR spectrum of the repeat, suggesting that they induce no significant conformational change.

Amino Acid Sequence↗