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A Colombatti

Publications and source records attributed to A Colombatti.

At least 19 recordsLinked to original sources
PubMed1992

The human type VI collagen gene. mRNA and protein variants of the alpha 3 chain generated by alternative splicing of an additional 5-end exon.

The amino- and carboxyl-terminal globular domains of type VI collagen are composed of several homologous modules similar to the type A collagen-binding modules present in von Willebrand factor. The human alpha 3(VI) chain that contributes most of the amino-terminal globule appears heterogeneous in size as a result of alternative splicing of two exons (Stokes D. G., Saitta, B., Timpl, R., and Chu, M.-L. (1991) J. Biol. Chem. 266, 8626-8633). In the present study, we report a further characterization of the 5'-end of the gene of the human alpha 3(VI) chain and show that transcription initiates at multiple sites. Southern blotting and DNA sequencing indicate that there is an additional type A exon (A9/N10) at about 1.8 kilobase pairs downstream of the exon coding for the signal peptide. The open reading frame of this additional exon reveals 1 cysteine and three potential N-glycosylation sites. Polymerase chain reaction, Northern blotting, and RNase protection assays demonstrate that exon A9/N10 is subject to alternative splicing in normal and tumor cell lines and that this generates more protein variants of the alpha 3(VI) chain than expected before. A comparison with the corresponding amino-terminal globule of the chicken alpha 3(VI) chain shows the presence of 1 additional cysteine in this portion of the molecule and suggests that human type VI collagen has more possibilities for structural and functional variations compared to chicken type VI collagen.

Alternative Splicing↗
PubMed1992

Stable expression of chicken type-VI collagen alpha 1, alpha 2 and alpha 3 cDNAs in murine NIH/3T3 cells.

As a component of an extensive network of microfibrils interwoven with large collagen fibers and in close contact with cell surfaces, type VI collagen plays an important role in cell-matrix interactions. To investigate the behaviour of chicken type VI collagen chains in heterologous host cells as a means to understanding the pattern of assembly of this collagen, we transfected murine NIH/3T3 cells with cDNAs encoding chicken alpha 1(VI), alpha 2(VI) and alpha 3(VI) chains. Cell lines that constitutively expressed the individual chains were analyzed by metabolic labeling and immunoprecipitation with specific antibodies. No self-association was observed for either alpha 1(VI) or alpha 2(VI) chains which were secreted as monomeric polypeptides. Furthermore, neither the chicken alpha 1(VI) nor alpha 2(VI) chains associated with the endogenous murine chains to form chimeric chicken/murine heterotrimers. In contrast, chimeric chicken/murine heterotrimers were detected in cell lines transfected with chicken alpha 3(VI) cDNA. These chimeric forms appeared to be properly aligned since their triple helices were stable to pepsin digestion. In addition, the chimeric heterotrimers coassembled and gave rise to disulfide-linked type VI collagen molecules.

3T3 Cells↗
PubMed1991

Expression of myelomonocytic antigens is associated with unfavourable clinicoprognostic factors in B-cell chronic lymphocytic leukaemia.

The cross-lineage expression of five myelomonocytic antigens (CD11b, CD11c, CD13, CD14, and CD15) was analysed in neoplastic lymphocytes from 100 consecutive B-cell chronic lymphocytic leukaemia (B-CLL) patients. CD14 antigen was detected on lymphocytes from more than 50% of patients whilst smaller percentages of samples were positive for CD11b (21%), CD11c (26%), CD13 (22%), and CD15 (7%). The presence of the CD13 antigen on neoplastic lymphocytes showed a statistically significant association with the two most important unfavourable clinicoprognostic factors in B-CLL: advanced clinical stage (CD13, P less than 0.01 by the Rai staging system; P less than 0.05 by the Binet staging system) and the diffuse pattern of bone marrow infiltration (CD13, P less than 0.001). A multiple logistic regression analysis showed that the increased risk for CD13-positive patients (13.7-fold higher than CD13-negative cases; P = 0.001) of presenting a diffuse pattern of bone marrow infiltration is independent of all other prognostic factors analysed including sex, age, lymphocyte counts, and clinical stage. A statistically significant association of CD11c (P = 0.002) and CD11b (P = 0.032) expression with the pattern of bone marrow infiltration was also found. Our results indicate for the first time a statistically significant association of CD13, CD11c, and CD11b antigens with unfavourable prognostic factors in B-CLL. They suggest that the cross-lineage expression of myeloid-associated surface peptidase (CD13-aminopeptidase N) and/or cell adhesion molecules (CD11c-LeuCAMc, CD11b-LeuCAMb) may influence the biological and clinical behaviour of chronic lymphoproliferative disorders of B cells.

Age Factors↗
PubMed1990

Differential expression of CD54/intercellular adhesion molecule-1 in myeloid leukemias and in lymphoproliferative disorders.

Indirect immunofluorescence staining with monoclonal antibody (MoAb) CL203.4 of malignant cells from 269 patients with hematologic malignancies showed a heterogeneous expression of CD54/intercellular adhesion molecule-1 (ICAM-1). This marker was expressed by malignant cells of 57 out of 118 patients with myeloid malignancies and 69 out of 135 with B-lymphoid malignancies. On the other hand, CD54 was not detected on malignant cells of 16 patients with T-lymphoid malignancies. In myeloid malignancies, CD54 is preferentially expressed by "stem cell-derived" malignancies, being detectable on blast cells from almost all patients affected by chronic myelogenous leukemia in blast phase or myelodysplastic syndromes and by only 34% of patients with de novo acute myeloid leukemia (AML). The expression of CD54 did not correlate with any specific myeloid FAB subtype, although three cases of highly undifferentiated AML (FAB MO) displayed maximal levels of the antigen. The expression of CD54 in AML was significantly associated with that of CD34 and HLA-DR antigens. In B-lymphoid malignancies, CD54 expression appears to correlate with the differentiation stage of malignant cells, since B-origin acute lymphoblastic leukemias and conventional B-chronic lymphocytic leukemias (B-CLL; ie, "dim SIg" CLL) expressed lower levels of CD54 than more mature lymphoproliferative disorders ("bright SIg" CLL, prolymphocytic leukemias, and lymphoplasmacytic tumors). "High-grade" B-cell non-Hodgkin's lymphomas (B-NHL) express in general a higher level of CD54 than "low-grade" ones. This finding in conjunction with the expression of CD54 in all 17 patients with "bright SIg" CLL investigated (characterized by marked organomegaly and poor prognosis) suggest that the differential expression of CD54 in lymphoproliferative disorders may also relate to their degree of malignancy.

Antigens, Surface↗
PubMed1990

Structural and functional features of the alpha 3 chain indicate a bridging role for chicken collagen VI in connective tissues.

Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.

Amino Acid Sequence↗
PubMed1990

Efficient expression of chicken alpha 1(VI) collagen chain in transiently transfected mammalian cells.

Type VI collagen is a component of the extracellular matrix made of three subunits, alpha 1(VI) and alpha 2(VI) (Mr = 140,000), and alpha 3(VI) (Mr = greater than 300,000). Triple helical monomers assemble intracellularly into disulfide-linked dimers and tetramers, with the tetramers being the "building blocks" that give rise to higher order extracellular structures by head-to-head association, the microfilaments. To study the pattern of assembly and the structure-function relationships of type VI collagen, we transfected mammalian cells with a full-length cDNA coding for chicken alpha 1(VI) under the control of SV40 early and late promoters and assayed the expression, secretion, and assembly of the protein by immunoperoxidase and immunoprecipitation of metabolically labeled cells. First, conditions were determined that allowed efficient transfection both in African monkey kidney COS-1 and CV-1 cells and in mouse fibroblasts. In our hands the late promoter was most efficient in CV-1 cells; whereas the early promoter was efficient in L cells at three days post-transfection. Chicken alpha 1(VI) could be isolated from cell extracts as well as from cell medium. Both the intracellular and the secreted forms of alpha 1(VI) are present as a monomer polypeptide and as disulfide-linked dimers and trimers that migrate in SDS gels with apparent Mr of about 130,000, 240,000 and 360,000, respectively. In L cells, endogenous mouse type VI collagen also was isolated by immunoprecipitation with specific antibodies. However, heterologous molecules made of the chicken alpha 1(VI) chain and the mouse alpha 2(VI) and alpha 3(VI) chains were not detected in the present experiments. Digestion with pepsin of the non-reduced chicken alpha 1(VI) polypeptides immunoprecipitated from the cell medium resulted in the disappearance of the bands, suggesting improper or non-stable assembly of alpha 1(VI) homotrimers. These data support predictions from sequence analysis that type VI collagen heterotrimeric molecules are more stable than other assembly alternatives.

Animals↗
PubMed1990

Multiple forms of chicken alpha 3(VI) collagen chain generated by alternative splicing in type A repeated domains.

Type VI collagen is a structurally unique component widely distributed in connective tissues. Its molecular structure consists of monomers that have the potential to assemble intracellularly into dimers and tetramers which, once secreted, can form microfilaments by end-to-end association. Individual monomers are composed of chains of Mr = approximately 140,000 (alpha 1 and alpha 2) and greater than 300,000 (alpha 3). Type VI collagen molecules contain a short triple helix with large globular domains at both ends. These domains are made for their greatest part of repetitive units similar to type A repeats of von Willebrand Factor. The alpha 3(VI) chain, contributing most of the mass of the NH2-terminal globule, appeared heterogenous both at the mRNA and protein level. Several alpha 3(VI)-specific clones that lack the sequences corresponding to repeats A8 and A6 were isolated from a chicken aorta cDNA library. Northern blot hybridization of poly (A+)-enriched RNA from chicken gizzard with cDNA fragments corresponding to several individual type A repeats showed that A8- and A6-specific probes did not hybridize to the lower Mr transcripts. Clones spanning approximately 20 kb of the 5'-end of the alpha 3(VI) gene were isolated from a chicken genomic library and subjected to analysis by restriction mapping, Southern blotting, and selective sequencing of the intron-exon boundaries. At the most 5'-end of the gene an additional type A repeat (A9), previously undetected in cDNA clones, was identified. Furthermore, it was determined that the presumed signal peptide and repeats A9 through A6 are encoded within individual exons. Reverse transcription and polymerase chain reaction of aorta RNA suggested that a mechanism of alternative mRNA splicing by a phenomenon of exon skipping generates alpha 3(VI) isoform variants that contain different numbers of type A repeats. Immunohistochemistry of frozen sections of chicken embryo tissues with repeat-specific mAbs showed that an antibody directed against a conditional exon has a more restricted tissue distribution compared to an antibody against a constitutive exon.

Amino Acid Sequence↗
PubMed1989

The carboxyl terminus of the chicken alpha 3 chain of collagen VI is a unique mosaic structure with glycoprotein Ib-like, fibronectin type III, and Kunitz modules.

The primary amino acid sequence of the carboxyl-terminal portion of the alpha 3 chain of chick type VI collagen as deduced from the nucleotide sequence is reported. This carboxyl-terminal segment is not present in the alpha 1 and alpha 2 chains of chick type VI collagen and is specific for a mosaic region with extensive similarities to several other proteins. This unique segment, beginning with a stretch (73 residues) very rich in serine and threonine, is preceded by sequences analogous to the platelet glycoprotein Ib. This region is followed by one segment that closely resembles the type III domains of fibronectin. At the end of the sequence, there is a 58-residue motif very similar to sequences characteristic of the Kunitz-type proteinase inhibitors. The present findings and our recent observation that the alpha 3(VI) chain contains 11 repeats similar to type A repeats of von Willebrand factor raise interesting questions about the peculiar mosaic structure and the multiple functions that this unique collagen might play in growth and remodeling of connective tissues.

Amino Acid Sequence↗
PubMed1989

Differential role of distinct determinants of intercellular adhesion molecule-1 in immunologic phenomena.

This study analyzed 1) the relationship between the molecules recognized by anti-intercellular adhesion molecule-1 (ICAM-1) mAb RR 1/1 and by anti-96K melanoma-associated Ag mAb CL203.4 in lymphoid cells, 2) the induction of ICAM-1 on activated PBMC, and 3) the functional activity of distinct and spatially distant determinants recognized by mAb CL203.4 and RR1/1. Sequential immunoprecipitation experiments showed that the determinant recognized by mAb CL203.4 is expressed on a slightly broader population of ICAM-1 molecules than that defined by mAb RR1/1. Serologic and immunochemical assays have shown that ICAM-1 is induced on lymphocytes activated with Con A, PHA-M, IL-2, allogeneic HLA mismatched lymphocytes and autologous PHA-M-activated T cells. However, ICAM-1 was not detected on lymphocytes incubated with IFN-gamma. Incubation of monocytes with LPS induced ICAM-1 in the subpopulation which lacks it and increased its density on the cells which express it. Induction of ICAM-1 is an early event in the activation process and precedes the appearance of IL-2 and transferrin receptors. Comparison of the functional activity of the anti-ICAM-1 mAb CL203.4 and RR1/1 showed that both of them inhibit to a similar extent proliferation of lymphocytes stimulated with PHA-M and with allogeneic lymphocytes, but that only mAb RR1/1 inhibits PMA-induced aggregation of cultured B lymphoid cells JY, of promonocytic cells U-937 and of PHA-blasts as well as LAK cell-mediated cytotoxicity of target cells. mAb CL203.4 represents the first example of anti-ICAM-1 mAb without inhibitory effect on the aggregation of lymphoid cells. The differential functional activity of mAb CL203.4 and RR1/1 does not reflect differences in their affinity, because they display a similar affinity constant to lymphoid cells. These results suggest that distinct determinants of ICAM-1 play a different role in immunologic phenomena.

Antibodies, Monoclonal↗
PubMed1989

Alpha 1 chain of chick type VI collagen. The complete cDNA sequence reveals a hybrid molecule made of one short collagen and three von Willebrand factor type A-like domains.

A cDNA library constructed from chick aorta poly(A+) RNA in the expression vector pEX1 was screened with rabbit polyclonal antisera. Additional clones were obtained by DNA-DNA hybridization with subclones from the most 5'- and 3'-ends. The overlapping clones span 4.6 kilobases and code for the entire alpha 1 (VI) chain. The nucleotide sequence reveals a 3057-base pair open reading frame that codes for 1019 amino acids. Analysis of the deduced amino acid sequence predicts that alpha 1 (VI) has one collagenous domain (COL) of 336 residues flanked by three repeated domains of about 200 residues each, one at the amino (A'3) and two at the carboxyl ends (A'2 and A'1), respectively, that are similar to the type A repeats of von Willebrand Factor. The COL domain presents two short interruptions near the carboxyl end of the triple helix and three of the six potential N-asparaginyl-linked carbohydrate attachment sites (Asn-Xaa-Ser/Thr). Furthermore, it contains 1 cysteine at position 89 that could participate in the formation of dimers and 3 Arg-Gly-Asp sequences that might be potential sites for cell adhesion. The COL domain shows an extended region, starting from position 40, within the triple helix, made of 14 Gly-Xaa-Yaa triplets that lack proline in the Y position, suggesting that it might be more flexible than the rest of the domain. At the junction of the COL with the N- and C-terminal domains, there are several cysteines that could confer the well known resistance of type VI collagen to pepsin and collagenase digestion under nonreducing conditions. The present sequence data allow a structural model for type VI collagen assembly to be proposed that is consistent with the structure implied from previous electron microscopic observation by Furthmayr et al. (Furthmayr, H., Wiedemann, H., Timpl, R., Odermatt, R., and Engel, J. (1983) Biochem. J. 221, 303-311).

Amino Acid Sequence↗
PubMed1989

Basement membrane components in lymphoid follicles: immunohistochemical demonstration and relationship to the follicular dendritic cell network.

We analyzed the distribution of two basement membrane components, laminin and type IV collagen, in human reactive and neoplastic lymphoid follicles by using immunoenzymatic and immunofluorescence methods on serial frozen sections from 11 reactive lymph nodes, three palatine tonsils, and 11 immunophenotypically defined B cell non-Hodgkin's lymphomas. The patterns of distribution of these two antigens were compared with that of follicular dendritic reticulum cells (DRCs) as visualized by anti-DRC-1 and anti-desmoplakin 1 and 2 antibodies. Immunostaining for laminin and type IV collagen produced overlapping results. Germinal centers contained some vascular and fiber-like linear staining, but the most striking feature was the presence of a large amount of punctate-granular staining. This staining was present throughout the entire area of most of the germinal centers, although often more densely in the central areas. The stronger punctate-granular staining for laminin, type IV collagen, and desmoplakin 1 and 2 corresponded to the more densely stained areas for DRC-1. Double immunofluorescence assay demonstrated that there was a close similarity between the punctate-granular staining pattern of laminin and the dendritic network of DRC-1-positive cells. In the nine B cell lymphomas of follicular center origin in which DRCs were present, although distributed in different patterns, immunostaining for laminin and type IV collagen could be observed only in those areas showing immunoreactivity for DRC-1. The results of this study suggest that the punctate-granular staining pattern for laminin and type IV collagen is specific for the follicle. This finding may be strictly related to the follicular microenvironment because the pattern of distribution displayed by laminin and type IV collagen seems to be spatially related to the staining pattern of DRCs, as visualized by the anti-DRC-1 and desmoplakins antibodies, both in reactive and neoplastic conditions.

Antibodies, Monoclonal↗
PubMed1989

Type VI collagen: high yields of a molecule with multiple forms of alpha 3 chain from avian and human tissues.

A differential extraction procedure followed by molecular sieve column chromatography for the isolation of large quantities of the tissue form of type VI collagen is described. Recovery of the protein was more than 60% from both chick gizzard and human placenta. On reduced NaDodSO4-gels chick type VI collagen migrated as two major bands at Mr = 140,000 and 150,000 that were present in a 1:1 ratio and five less intense bands between Mr = 230,000 and 180,000. By immunoblotting with a polyclonal antibody against the pepsinized form of chick type VI collagen, all these bands were stained. Furthermore, the amino acid composition of the five higher Mr polypeptides indicated that they all contained hydroxyproline and hydroxylysine. In the chick type VI collagen molecule the five bands of higher Mr belong to the alpha 3 chain since they were recognized by monoclonal antibodies specific for the chick Mr = 260,000 alpha 3 chain. On examination of antigenic activity by solid-phase radioimmunobinding, densitometry of stained NaDodSO4 polyacrylamide gels, and protein content type VI was found to be an abundant collagen since it accounted for up to 0.1% of the tissue wet weight. The yields per tissue wet weight and the migration pattern of human type VI collagen polypeptides were similar to those of the chick. Agarose/polyacrylamide composite gels indicated that the molecular size of the tissue form of type VI collagen molecules under non-reduced conditions corresponded to a basic type of tetrameric molecule.

Amino Acids↗
PubMed1989

Extracellular matrix of lymphoid tissues in the chick.

We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp.

Animals↗
PubMed1988

The elastin associated glycoprotein gp115. Synthesis and secretion by chick cells in culture.

Synthesis of gp115 by aorta smooth muscle cells and tendon fibroblasts isolated from chick embryos was investigated. gp115 was specifically immunoprecipitated by both polyclonal and monoclonal antibodies from cell lysates and culture medium of matrix free cells metabolically labeled with [3H]leucine and [35S]methionine. The component of gp115 isolated from the cell lysate had an apparent Mr in reduced sodium dodecyl sulfate polyacrylamide gels lower (105,000) than the protein isolated from the culture medium (Mr = 115,000). In immunoblot experiments, the latter corresponded in apparent Mr to the form isolated from chick tissues. gp115 was glycosylated in vitro; it was labeled with [3H]fucose, and when cells were cultured and labeled in the presence of tunicamycin, a lower Mr form with an apparent Mr = 90,000 was immunoprecipitated in both the cell lysate and the culture medium. In pulse-chase experiments, the intracellular and the extracellular forms were clearly suggestive of a direct precursor-product relationship in the absence of intermediate forms. The kinetics of secretion appeared very slow compared with that of other proteins of the extracellular matrix investigated in the same system; about 50-70% of gp115 in the form of the Mr = 105,000 species was still cell-associated after 4 h, whereas the half-time for secretion of fibronectin, type VI collagen, and tropoelastin was about 60 min, 3 h, and 60 min, respectively. Newly synthesized and processed cell-associated gp115 migrated in both reduced and non-reduced gels as a monomer. On the contrary, the secreted protein was present in the culture medium as large aggregates that did not enter the gel in the absence of reducing agents.

Animals↗
PubMed1988

Differentiation of human leukaemic cell lines and fresh leukaemia cells by low dose Ara-C: monitoring by expression of c-myc and c-fos oncogenes.

The effects of low dose Ara-C on cellular oncogene expression were measured in HL-60 and U-937 cell lines and in primary cultures from leukaemic patients. Expression of c-myc was completely abolished in U-937 and greatly reduced in HL-60 after a 3 day exposure to the drug, whereas specific c-fos transcripts were increased. In fresh myeloid leukaemia samples, growth and DNA synthesis were reduced as in the two cell lines. Conversely, changes compatible with the induction of differentiation along the myelomonocytic pathway were much less pronounced than in cell lines treated with the same dose of Ara-C. Cells from one patient did not show any appreciable morphological change. The same sample displayed a greatly reduced expression of c-myc accompanied by a concurrent 10-fold increased expression of c-fos. The data suggest that the action of low dose Ara-C on oncogene expression is comparable to that of other differentiation-inducing agents that display both cytostatic and maturation promoting effects. Evaluation of cellular oncogene expression may therefore represent a useful tool for monitoring effects of low dose Ara-C on leukaemia cells.

Cell Differentiation↗
PubMed1988

Monoclonal antibodies for the different chains of chick type VI collagen.

Avian type VI collagen is composed of three subunits of Mr 140,000, 150,000 and 260,000. Monoclonal antibodies were raised against type VI collagen isolated from chick embryo gizzard, and these antibodies were used to immunoprecipitate type VI collagen from metabolically labeled embryo cells. Several antibodies appeared to react with epitopes independent of glycosylation and hydroxylation processes. The antibody-binding sites were identified on the different chains by immunoblotting of total cell extracts. In addition, antibodies that recognized different epitopes on the Mr 260,000 subunit could be grouped in at least three different clusters by competitive inhibition radioimmunobinding assays.

Animals↗