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Biomedical subjects

S Benson

Publications and source records attributed to S Benson.

At least 55 records · Page 3Linked to original sources
PubMed1990

The synthesis and secretion of collagen by cultured sea urchin micromeres.

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.

Animals↗
PubMed1989

Assessment of the genotoxicity of calcium cyclamate and cyclohexylamine.

Calcium cyclamate and its major metabolite cyclohexylamine have been subjected to numerous evaluations for genetic activity. With the exception of studies for chromosome damage, the results have been negative. Results from a wide range of in vitro and in vivo cytogenetic assays ranged from clearly negative to various degrees of clastogenicity. Interpretation of the cytogenetic studies has been complicated by the conflicting responses, although some of the positive effects seem to be the consequence of secondary effects produced by high ion levels and excessive toxicity. In the studies presented here calcium cyclamate and cyclohexylamine were tested for mutagenic activity using an in vitro mammalian cells assay for gene mutation and an in vitro unscheduled DNA synthesis assay in rat hepatocytes with the Drosophila sex-linked recessive lethal assay. Calcium cyclamate was not genetically active in any of the three assays when tested to the maximum possible concentrations. The compound was largely nontoxic but did show some evidence of cytotoxicity in rat hepatocytes at concentrations of 1 mg/ml and higher. Cyclohexylamine was also negative in the three assays, but was considerably more cytotoxic at the concentrations used. The results from the three studies conducted in this evaluation are in general agreement with the majority of published genetic toxicology data for these two chemicals and indicated that the calcium cyclamate and cyclohexylamine have no direct, intrinsic genotoxicity of the type measured by these assays.

Animals↗
PubMed1989

Expression of an embryonic spicule matrix gene in calcified tissues of adult sea urchins.

The sea urchin spicule is composed of CaCO3 associated with an organic matrix containing at least 10 proteins. We have previously shown that one of these proteins, a 50-kDa glycoprotein (SM 50), is encoded by a gene which is only transcribed in cells of the micromere-mesenchyme lineage. In this report we examined RNA from five adult tissues: ovary, coelomocytes, intestine, tube feet, and spine for the presence of SM 50 transcripts and protein. Only RNA from cells of spine and tube feet hybridized to a SM 50-specific probe on Northern blots. These same two tissues undergo biomineralization and contain a protein which is immunologically related to the SM 50 protein. The restricted accumulation of SM 50 transcripts and protein suggests that the differentiation of biomineralizing mesenchyme cells in embryonic and adult tissue may utilize the same genetic program.

Animals↗
PubMed1987

A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. I. Authentication of the cloned gene and its developmental expression.

The developing sea urchin embryo forms endoskeletal CaCO3 containing spicules which are elaborated by the primary mesenchyme cells, descendants of the micromeres, beginning at gastrulation. In this and the accompanying paper [H. M. Sucov, S. Benson, J. J. Robinson, R. J. Britten, F. Wilt, and E. H. Davidson (1987) Dev. Biol. 120, 507-519] the isolation and characterization of a gene that encodes a 50-kDa spicule matrix glycoprotein that we call SM50 are described. A cloned cDNA isolated from a lambda gt11 library was used in hybrid-selected translation and hybrid arrest of translation experiments to verify that the cDNA encodes a spicule matrix protein. The cognate RNA transcript encodes a 50-kDa protein which is precipitated by polyclonal antisera against spicule matrix proteins and is present only in polyadenylated RNA at stages known to be making a spicule. The cloned cDNA sequence described in the accompanying paper was used to follow the time of expression of the cognate gene by RNA blotting analysis. The 2.2-kb mRNA is first detected at late cleavage stages and rapidly accumulates as the primary mesenchyme forms, reaching an apparent maximum concentration in the late gastrula and pluteus stages. The cDNA was also used to identify the cells that contain the transcripts by hybridization in situ. Hybridization to cellular transcripts is first detected in primary mesenchyme cells as they enter the blastocoel, and transcripts are confined to these cells during spicule formation and subsequent development.

Animals↗
PubMed1987

A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. Structure of the gene and derived sequence of the protein.

A lambda gt11 cDNA clone isolated by use of a polyclonal antispicule matrix protein antiserum is shown in the accompanying paper [S. C. Benson, H. M. Sucov, L. Stephens, E. H. Davidson, and F. Wilt (1987) Dev. Biol. 120, 499-506] to encode a prominent 50-kDa spicule matrix protein (SM50). This clone was used to select homologous genomic recombinants, and the structure of the gene was determined. The SM50 gene occurs once per haploid genome. It contains a single intron located within the 35th codon. A unique transcription initiation site 110 nucleotide pairs prior to the translation start signal was mapped by primer extension. The mRNA is 1895 nucleotides in length, excluding the 3' poly(A) sequence, and contains a single open reading frame 450 codons in length. Though rare in whole embryo RNA the prevalence of the SM50 mRNA is calculated to be about 1% of the total mRNA in skeletogenic mesenchyme cells. The derived peptide sequence indicates a typical N-terminal signal peptide, and an N-linked glycosylation site near the C terminus. About 45% of the length of the protein is included in a domain composed of consecutive approximate repetitions of a 13-amino-acid element, the consensus sequence of which is Trp-Val-Gly-Asp-Asn-Gln-Ala-LeuTrp-Val-IleAsp-Asn-GlnPro+ ++-ValGlu. The protein also contains an internal domain unusually rich in proline residues and a very basic C-terminal region.

Amino Acid Sequence↗
PubMed1987

The role of lysyl oxidase and collagen crosslinking during sea urchin development.

Lysyl oxidase, the only enzyme involved in collagen crosslinking, is shown to be present in embryos of the sea urchin Strongylocentrotus purpuratus. The enzyme specific activity increases over six-fold during development, showing the greatest rise during gastrulation and prism larva formation. The enzyme is inhibited by the specific inhibitor, beta-aminoproprionitrile (BAPN). Continuous BAPN treatment of S. purpuratus and Lytechinus pictus embryos from late cleavage stages onward increases the amount of noncrosslinked collagen present in prism larvae. When BAPN is added at the 128- or 256-cell stage it causes developmental arrest at the mesenchyme blastula stage. Embryos can be maintained in the arrested state for at least 96 h and will resume normal development and morphogenesis following BAPN removal. If BAPN is added after the mesenchyme blastula stage, it has little adverse effect on development; consequently nonspecific toxic effects of the drug are unlikely. The results suggest that lysyl oxidase and collagen crosslinking play a vital role in primary mesenchyme migration, gastrulation, and morphogenesis during sea urchin development and indicate that BAPN may be very useful in studying the extracellular matrix-cell interactions at the cellular and molecular level.

Amino Acid Oxidoreductases↗
PubMed1987

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.

Bacterial Proteins↗
PubMed1984

Collagen metabolism and spicule formation in sea urchin micromeres.

The role of collagen or collagen-like protein(s) in the in vitro formation of the sea urchin embryonic skeleton was investigated using isolated micromeres of Strongylocentrotus purpuratus. Micromeres were cultured in sea water containing 4% horse serum on tissue culture plastic or an extracellular matrix of type I collagen. The effect of proline analogs and an inhibitor of collagen hydroxylation on in vitro spicule formation in both culture systems was monitored. When micromeres are cultured in the presence of proline analogs L-azetidine-2-carboxylic acid and L-3, 4-dehydroproline which disrupt collagen metabolism, spicule formation is significantly less inhibited on a collagen substratum than on plastic. Culturing micromeres on plastic in the presence of alpha, alpha'-dipyridyl, an inhibitor of collagen hydroxylation, resulted in almost complete inhibition of spicule formation. The inhibition by alpha, alpha'-dipyridyl can be overcome by culturing micromeres on collagen substratum. These results do not support the idea of collagen being the calcified organic matrix of the spicule. Rather, they suggest that micromeres synthesize a collagen-like extracellular matrix which is necessary for spicule formation. Inhibition of this activity by proline analogs or a collagen processing inhibitor can be overcome by providing the cells with a previously deposited extracellular matrix.

2,2'-Dipyridyl↗
PubMed1983

Genetic mapping of antigenic determinants on a membrane protein.

The antigenic determinants recognized by two monoclonal antibodies were mapped on LamB, an outer membrane protein of Escherichia coli. The procedure consisted of performing immunoprecipitation experiments with extracts of strains which produced truncated fragments of LamB, either in a free form (deletion and nonsense mutants) or fused to another polypeptide (malK-lamB and lamB-lacZ fusion strains). The conclusion is that the two antigenic determinants are located within 70 residues from the COOH-terminal end of LamB, which contains a total of 421 amino acids. Since these two antigenic sites were previously demonstrated to be exposed at the cell surface, it follows that a COOH-terminal portion of LamB must be located on the outer surface of the outer membrane.

ATP-Binding Cassette Transporters↗
PubMed1983

Morphology of the organic matrix of the spicule of the sea urchin larva.

The morphology of the organic matrix of the skeletal spicule of the sea urchin pluteus larva has been analyzed by light and electron microscopy. Purified isolated spicules can be demineralized, and they reveal lamellae of an irregular fibrillar nature with overall outlines similar to the shape of the intact spicule. Sections through the spicule show the fibrous lamella is probably composed of interconnected concentric sleeves.

Animals↗