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M L Tanzer

Publications and source records attributed to M L Tanzer.

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

Detection of a type IX collagen-related mRNA in an invertebrate, the marine annelid Nereis virens.

Fibrous and non-fibrous collagens have been described in both vertebrate and invertebrate animals. However, there has been limited characterization of non-fibrous collagens and their corresponding genes in invertebrate animals. In the present study we have used as a probe an avian cDNA clone which encompasses the COL3, NC3 and part of the COL2 domain of the collagen alpha 3(IX) subunit. This probe hybridized to mRNA obtained from the cuticle and body of the marine annelid, Nereis virens. Northern blot hybridization exhibited an mRNA of ca. 7.5-8 kilobases which in situ hybridization shows to be most abundant over cuticle-associated cells. Dot-blot hybridization, comparing cuticle mRNA and body mRNA, indicates that this collagen mRNA is five times more abundant in the cuticle. The composite data suggest evolutionary conservation, in both vertebrate and invertebrate animals, of a non-fibrillar collagen.

Animals↗
PubMed1993

Laminin oligosaccharides play a pivotal role in cell spreading.

The basement membrane glycoprotein laminin promotes cell adhesion, spreading and neurite outgrowth. We can uncouple cell adhesion and spreading (or neurite outgrowth) when unglycosylated laminin is used as a substratum. Mouse melanoma cells, B16F1 line, readily attach to unglycosylated laminin but fail to spread once adherent. Spreading can be restored by titration with glycosylated laminin or with laminin glycopeptides. When the laminin substratum is absent in the test chambers, the cells do not adhere when either intact laminin or its glycopeptides are then added. Analyses show that these added substances are recoverable from the culture medium and do not bind to the chamber surfaces. Use of selective inhibitors which interfere with carbohydrate processing yields several glycoforms of laminin which we have isolated and examined for their ability to support cell adhesion and spreading. Laminin which is enriched in high mannose oligosaccharides is much more effective in promoting cell spreading than laminin which is enriched in hybrid oligosaccharides. These results are consistent with earlier studies which showed that ConA, which primarily recognizes mannose residues, could also uncouple cell adhesion and spreading. Although mono- and disaccharides failed to restore cell spreading, we have found that addition of various mannose oligosaccharides to adherent cells effectively reestablishes their spreading behavior. The extent of cell spreading which is achieved by the added saccharides is related to their amount, their duration of addition, and their molecular structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Molecular cloning of chicken aggrecan. Structural analyses.

The large, aggregating chondroitin sulphate proteoglycan of cartilage, aggrecan, has served as a generic model of proteoglycan structure. Molecular cloning of aggrecans has further defined their amino acid sequences and domain structures. In this study, we have obtained the complete coding sequence of chicken sternal cartilage aggrecan by a combination of cDNA and genomic DNA sequencing. The composite sequence is 6117 bp in length, encoding 1951 amino acids. Comparison of chicken aggrecan protein primary structure with rat, human and bovine aggrecans has disclosed both similarities and differences. The domains which are most highly conserved at 70-80% identity are the N-terminal domains G1 and G2 and the C-terminal domain G3. The chondroitin sulphate domain of chicken aggrecan is smaller than that of rat and human aggrecans and has very distinctive repeat sequences. It has two separate sections, one comprising 12 consecutive Ser-Gly-Glu repeats of 20 amino acids each, adjacent to the other which has 23 discontinuous Ser-Gly-Glu repeats of 10 amino acids each; this latter region, N-terminal to the former one, appears to be unique to chicken aggrecan. The two regions contain a total of 94 potential chondroitin sulphate attachment sites. Genomic comparison shows that, although chicken exons 11-14 are identical in size to the rat and human exons, chicken exon 10 is the smallest of the three species. This is also reflected in the size of its chondroitin sulphate coding region and in the total number of Ser-Gly pairs. The putative keratan sulphate domain shows 31-45% identity with the other species and lacks the repetitive sequences seen in the others. In summary, while the linear arrangement of specific domains of chicken aggrecan is identical to that in the aggrecans of other species, and while there is considerable identity of three separate domains, chicken aggrecan demonstrates unique features, notably in its chondroitin sulphate domain and its keratan sulphate domain. Thus different variants of chondroitin sulphate and keratan sulphate domains may have evolved separately to fulfil specific biochemical and physiological functions.

Aggrecans↗
PubMed1991

The major proteoglycan of adult rabbit skeletal muscle. Relationship to small proteoglycans of other tissues.

We have been interested in examining the putative biological role(s) of the major proteoglycan of adult skeletal muscle. The small proteoglycans of adult rabbit skeletal muscle and tendon were extracted and purified by sequential density-gradient ultracentrifugation, ion-exchange chromatography and gel filtration. They appeared to be homogeneous by the criterion of gel electrophoresis in SDS and to yield one major product, the core protein, after digestion with chondroitin ABC lyase, also observed after gel electrophoresis. Two major products were obtained when the intact proteoglycans were cleaved by CNBr, and those peptides were separated by SDS/PAGE and by ion-exchange chromatography. Sequencing of the N-terminal amino acids of either the intact proteoglycans or the CNBr-cleaved products allowed for comparison of the muscle and tendon proteoglycan with derived amino acid sequences previously reported for bovine bone proteoglycan. The bone and tendon proteoglycan sequences were remarkably similar, whereas those of the muscle proteoglycan differed from the other two molecules. The major site of glycosaminoglycan substitution was on a peptide fragment distant from the N-terminus, and a presumptive serine residue at position 4 from the N-terminus also appeared to be substituted, perhaps with a small glycosaminoglycan chain. These results provide some insight into the diversity of small proteoglycans of the PG-II class and provide a basis for exploring their mode of genetic expression.

Amino Acid Sequence↗
PubMed1991

Laminin carbohydrates are implicated in cell signaling.

We have examined how laminin carbohydrates participate in cellular responses and have focused upon cell spreading and neurite outgrowth. Our earlier studies showed that unglycosylated laminin fully supported cell adhesion but did not promote subsequent spreading of mouse melanoma cells or neurite outgrowth of rat pheochromocytoma cells (Dean et al. (1990): J Biol Chem 265:12553-12562). In the present experiments, we determined whether those cellular responses could be restored to adherent cells. When a mixture of unglycosylated and glycosylated laminins was used as a substratum for mouse melanoma cells, some cells began to spread when 30% glycosylated laminin was present. At least 65% glycosylated laminin was required to elicit a maximal spreading response by the majority of the cells. In separate experiments, we found that cell spreading was fully restored by a pronase digest of glycosylated laminin; a similar digest of unglycosylated laminin had no effect. These results indicate that laminin carbohydrates, rather than polypeptide sequences, were responsible for cell spreading. We also conclude that substrate attachment of the carbohydrate moieties was not essential. In other experiments, laminins containing immature oligosaccharides were produced using two glycosylation pathway inhibitors, swainsonine or castanospermine. When such laminins were used to study cell spreading or neurite outgrowth, laminin containing immature oligosaccharides was as effective as laminin which contains fully processed oligosaccharides. In contrast, laminin with partially processed oligosaccharides had incomplete activity. These composite reconstitution experiments show that laminin carbohydrates provide essential information to responsive cells, enabling them to progress from an adherent state to a spread form or to extend neurite processes.

Animals↗
PubMed1990

A biological role of the carbohydrate moieties of laminin.

The ways in which the carbohydrate moieties of laminin affect its cellular interactions have been examined by two different experimental approaches. In one approach, we used lectins in order to block specific carbohydrates on laminin which previously had been dried onto a plastic surface. We found that wheat germ agglutinin and Griffonia simplicifolia agglutinin I blocked the binding of the neuron-like rat pheochromocytoma cell line PC12. However, when concanavalin A was used cell binding was unaffected but neurite outgrowth was prevented, compared to controls, over a 24-h period. In the second approach we used unglycosylated laminin as a substratum on the plastic surface. We have developed a method for the purification of unglycosylated laminin from tunicamycin treated cultures of a mouse embryonal carcinoma derived cell line, M1536 B3, and have partially characterized the purified material. A mixture of unglycosylated and glycosylated laminin was selectively purified from the M1536 B3 cell lysate by an anti-EHS laminin monoclonal antibody immunoaffinity column. The unglycosylated laminin was separated from glycosylated laminin using G. simplicifolia lectin affinity chromatography. The lectins, wheat germ agglutinin, G. simplicifolia agglutinin I, and concanavalin A, did not bind to any of the subunits of unglycosylated laminin in Western blots. The unglycosylated laminin migrated as a single band in agarose-gel electrophoresis under nonreducing conditions indicating that it is a fully assembled and disulfide bonded molecule. Circular dichroism studies showed no differences between glycosylated and unglycosylated laminin, indicating similar molecular conformations. Western blots using antibodies specific for the A, B1, and B2 chains of laminin showed that unglycosylated laminin contained each of these subunits. We then performed cell binding and spreading or neurite outgrowth assays using unglycosylated laminin. A mouse melanoma cell line, B16 F1, bound to this laminin in the same numbers as to the control glycosylated laminin, but cell spreading was minimal. When this unglycosylated laminin was used as a substrate for PC12 cells neurite outgrowth was impaired; no effect was noted on the number of cells bound, compared to glycosylated laminin. We conclude from these results that once cells become bound to laminin the carbohydrate residues of that glycoprotein must be available to enable the cells to spread or to extend neurite processes.

Adrenal Gland Neoplasms↗
PubMed1990

Characterization of Chinese hamster ovary cells with impaired spreading properties on fibronectin.

The development of receptor-defective or -deficient mutants can be applied to the investigation of cell-matrix interactions including cell adherence and spreading. In the present study we developed a series of ethyl methyl sulfonate (EMS)-induced Chinese hamster ovary (CHO) cell mutants, which adhere to fibronectin but have impaired spreading characteristics. Using morphometric analysis, a significant suppression in the degree of cell spreading between the wild-type and the mutant cells (P less than 0.001) was seen. This inability of the mutant cells to spread adequately on fibronectin also resulted in a decreased number and diameter of stress fibers as compared to wild-type cells. The decreased cell spreading of the mutant cells was not due to inherent differences in cell size or volume, as determined by fluorescence-activated cell sorter (FACS) analysis. Since integrins, specifically the fibronectin receptor (alpha FN/beta 1), are important in cell adhesion and cell spreading, we carried out a comparative immunochemical analysis, using a monoclonal antibody to the beta 1 subunit of integrin (7E2). Western blot analysis of cell extracts and cell membranes indicated that both wild-type and mutant cells expressed the alpha and beta 1 subunits of the fibronectin receptor; the mutant cells displayed reduced levels of the subunit. Immunohistochemical analysis indicated that, despite the presence of the receptor in both cell types, their patterns of localization and aggregation were different. The wild-type cells showed a needle-like distribution of the receptor, in contrast to the clumped appearance in the mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1988

Partial structure of the gene for chicken cartilage proteoglycan core protein.

A genomic DNA fragment (gCORE-1), encoding a portion of the cartilage proteoglycan core protein, has been isolated from a phage library using cDNA as a probe. The genomic insert is about 17 kilobase pairs; two BamHI fragments of the insert (1.3 and 4.8 kilobase pairs) contain most of the hybridizable sequences found in the cDNA. Sequence analysis of these fragments shows that they contain a total of five exons that encompass 216 amino acid residues, all of which are identical to those of the corresponding cDNA sequence. Three of the exons, which are adjacent to one another, are very similar to the corresponding exons in the gene of a rat hepatic lectin as well as to an exon in the gene of human pulmonary surfactant-associated protein. There is a strong degree of conservation of amino acid sequences encoded in the three genes, although there is no similarity between their introns. The sizes of the five exons in gCORE-1, except for one (which is indeterminate because only a partial cDNA sequence is available), are less than 184 base pairs, whereas the sizes of the introns range from 218 to greater than 2629 base pairs. Four of the introns interrupt an exon codon at either their donor or acceptor sites, between the first and second nucleotides. Only one intron does not split a codon. Intron and exon boundary sites are in agreement with known consensus sequences for introns. The dispersed distribution and relatively small size of the exons, if representative of the entire gene, suggest that the complete gene which codes for the core protein may be quite sizable.

Aggrecans↗
PubMed1988

Lectins inhibit cell binding and spreading on a laminin substrate.

This study examined the effects on cell binding and spreading of the exposure of laminin substrates to the lectins Wheat Germ Agglutinin or Concanavalin A. Exposure of laminin to Wheat Germ Agglutinin inhibited binding of mouse B16 F1 melanoma cells in a dose dependent manner. Exposure to Concanavalin A had no deleterious effects on binding but did inhibit cell spreading. Both effects were completely prevented by the specific sugars for each lectin. These effects may be due to the proximity of N-linked oligosaccharides to the known cell binding sites of laminin.

Animals↗
PubMed1988

Temporal and spatial analysis of cartilage proteoglycan core protein gene expression during limb development by in situ hybridization.

As limb mesenchymal cells differentiate into chondrocytes they initiate the synthesis of a cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and other cartilage-specific proteins. In the present study, in situ hybridization with a 32P-labeled cloned cDNA probe complementary to mRNA encoding the core protein of cartilage proteoglycan has been used to visualize and localize the accumulation of cartilage proteoglycan core protein mRNA sequences during development of the chick limb bud in vivo. When the probe was hybridized to sections through 7-day (stage 32) limbs, an intense hybridization signal was observed over the well-differentiated cartilage rudiments of the limb, while no signal above background was observed over nonchondrogenic tissues including muscle, loose connective tissue, and epidermis. At early stages of limb development, an accumulation of silver grains representing hybridizable core protein mRNA first became detectable in the proximal central core of the limb where the prechondrogenic condensation of mesenchymal cells that characterizes the onset of cartilage differentiation was occurring. In fact, the pattern of silver grain accumulation closely followed the pattern of mesenchymal cell condensation, and no hybridizable core protein mRNA sequences were detectable in the limb bud prior to condensation. Cartilage-characteristic type II collagen mRNA was colocalized with core protein mRNA in the condensing central core of the limb suggesting that the genes for these two major constituents of cartilage matrix are coordinately regulated at the onset of chondrogenesis. Furthermore, the appearance of hybridizable core protein mRNA was closely followed by the appearance of the protein for which it codes as detected by immunohistochemical staining with monospecific antibody. These observations support the hypothesis that at the initial stages of limb chondrogenesis core protein gene expression is controlled primarily at the transcriptional level.

Aggrecans↗
PubMed1988

Separation and characterization of the subunits of the laminin of EHS sarcoma.

A rapid and sensitive method was developed for the preparative separation of laminin subunits. Laminin was extracted and purified from mouse EHS sarcoma. On SDS-PAGE, the reduced and carboxymethylated molecule separated into two components corresponding to molecular weights of about 400 KDa (subunit A) and 200 KDa (subunit B). These two subunits were preparatively separated using heparin-agarose affinity chromatography. The larger subunit quantitatively adhered to the affinity column while the smaller one did not adhere. Amino acid analyses of the separated subunits showed distinct differences. Subunit B was further resolved into two distinct polypeptides of 200 KDa, B1 and B2, by means of reverse-phase HPLC. Although the amino acid compositions of B1 and B2 were very similar, the peptide maps generated by digestion of the B1 and B2 chains with Staphylococcus aureus V8 protease or by cyanogen bromide showed B1 and B2 to differ from each other. Thus, at least three different polypeptide subunits are present in this laminin and probably arise from separate gene origins. These studies provide a basis for the subsequent localization and analysis of the specialized structural and functional domains of laminin.

Animals↗
PubMed1987

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle.

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1986

Structures of the asparagine-linked sugar chains of laminin.

This investigation describes the isolation and characterization of oligosaccharides of the basement membrane glycoprotein, laminin. Pronase-released glycopeptides of isolated laminin, from a mouse Engelbreth-Holm-Swarm tumor, were fractionated using a combination of gel permeation chromatography and Con A-Sepharose affinity chromatography. The glycopeptides were analyzed for sugar linkage patterns by methylation analysis. Glycopeptides and hydrazine-released oligosaccharides were further analyzed using endo-beta-galactosidase, endo-beta-N-acetylglucosaminidase H and specific exoglycosidases in conjunction with calibrated gel permeation chromatography. Based on these experiments, murine tumor laminin was shown to contain asparagine-linked oligosaccharides with the following structures: bi-, tri- and tetraantennary complex-type oligosaccharides; polylactosaminyl side chains containing Gal(beta 1----4)GlcNAc(beta 1----3) repeating units attached to the trimannose core portion of the bi-, tri- and tetraantennary complex-type oligosaccharides; unusual complex-type oligosaccharides terminated at the nonreducing end with sialic acid, alpha-galactose, beta-galactose and beta-N-acetylglucosamine; alpha-galactosyl residues linked to N-acetyllactosamine sequences; high-mannose-type oligosaccharides. These results, in conjunction with analytical data, indicate that most of the carbohydrate of this laminin is N-linked to asparagine and that there are about 43 such N-linked oligosaccharides per laminin molecule.

Animals↗
PubMed1986

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation.

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.

Age Factors↗
PubMed1986

Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein.

A chicken embryo sternal cartilage cDNA library, created in the plasmid expression vector pUC9, was screened for sequences coding for immunologically detectable core protein of the large, major proteoglycan of cartilage. A 1229-base-pair cDNA clone was isolated that contained only one extended open reading frame, which had sequences coding for a polypeptide of 379 amino acid residues. These deduced sequences corresponded to those anticipated from current models of proteoglycan structure; a deduced sequence encompassing 21 amino acids was almost identical to a known sequence of bovine nasal cartilage proteoglycan. Significant homology was found between the deduced amino acid sequence of the proteoglycan and two regions of a chicken hepatic lectin. Immunoprecipitation of the products of cell-free translation yielded a component of about 340 kDa, and transfer blot hybridization of sternal cartilage RNA showed a single mRNA of about 8.1 kilobases. Hybridizable mRNA sequences were readily detectable by dot-blot analyses of the cytoplasm of cartilaginous tissues of the chicken embryo, whereas similar analyses of prechondrogenic limb mesenchymal cells did not demonstrate such hybridizable mRNA signals.

Amino Acid Sequence↗
PubMed1986

Fibronectin glycosylation modulates fibroblast adhesion and spreading.

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.

Adsorption↗
PubMed1985

Peptide repeats in a mussel glue protein: theme and variations.

The adhesive protein from Mytilus edulis contains 75-80 closely related, repeated peptide sequences in its primary structure. These peptides can be resolved following digestion with trypsin by reversed-phase high-pressure liquid chromatography. The most frequently repeated sequence is the decapeptide Ala-Lys-Pro-Ser-Tyr-Hyp-Hyp-Thr-Dopa-Lys (peptide E). Variations of this occur in peptides B with Hyp-3 and Dopa-5, C with Dopa-5, and D with Hyp-3, respectively. Lesser amounts of hexapeptides (A and B') that are lacking residues 4-7 also occur. Peptide A has the sequence Ala-Lys-Pro-Thr-Dopa-Lys, whereas B' contains Tyr instead of Dopa. 4-Hydroxyproline occurs at positions 3 and 7 and occasionally at position 6 of the decapeptide; 3-hydroxyproline occurs only at position 6. Adhesiveness of the protein may be related to the repetition of Dopa residues, the catecholic moiety of which has strong hydrogen-bonding and metal-liganding capabilities.

Amino Acid Sequence↗