Biomedical subjects

M Zain

Publications and source records attributed to M Zain.

9 recordsLinked to original sources

PubMed1995

Granulocyte colony-stimulating factor induction of normal human bone marrow progenitors results in neutrophil-specific gene expression.

We have used a combination of hematopoietic growth factors to induce in vitro granulocytic maturation. A fraction of marrow cells enriched for hematopoietic progenitor cells (CD34+, HLA-DR+) was isolated from normal human bone marrow by monoclonal antibody staining and fluorescence-activated cell sorting. Cells were cultured in a suspension system for 3 days in the presence of stem cell factor and interleukin-3 (IL-3), after which granulocyte colony-stimulating factor (G-CSF) was added. Cells were harvested daily and analyzed for phenotypic maturation by morphologic criteria, and total RNA was obtained for analysis of myeloid gene expression. Maturation was observed to progress to the late metamyelocyte and band stage over a period of 10 to 12 days. Neutrophil-specific gene expression was assayed by reverse transcription-polymerase chain reaction (RT-PCR). Induction with G-CSF resulted in sequential expression of primary and secondary granule proteins, with asynchronous expression of primary granule proteins starting from days 1 to 5, and synchronous expression of lactoferrin and transcobalamin I (secondary granule proteins) from days 7 to 8. Interestingly, myeloperoxidase (MPO) mRNA expression was easily detected in both the freshly isolated CD34+, HLA-DR+ cells and cells at all subsequent stages of induction. This suggests that MPO mRNA is expressed very early during neutrophil development, perhaps before the development of significant numbers of phenotypically recognizable granules. This recapitulation of a program of sequential expression of primary and secondary granule protein genes suggests that in vitro marrow culture suspensions to which appropriate growth factors are added can mimic normal granulocytic maturation. This system should provide an important model for the study of neutrophil-specific gene expression.

Antigens, CD↗

PubMed1994

Are primed CD4+ T lymphocytes different from unprimed cells?

Primed and unprimed lymphocytes are usually classified as separate subsets of cells, based on phenotypic and functional distinctions. In the case of CD4+ T lymphocytes, primed cells are thought to proliferate more vigorously, quickly and easily, and to release a different profile of cytokines, than their naive equivalent. However, most of these data were obtained from studies in which populations of lymphocytes were compared before and after antigenic stimulation, and therefore did not distinguish between the effects resulting from the clonal expansion of specific precursor cells within such populations and those due to cell differentiation per se. We have investigated the contribution of precursor cell frequency to some of the functional changes observed in populations of CD4+ T cells following antigenic stimulation, using approaches in which antigen-specific precursor frequencies are high in both primary and secondary stimulations: mixed leukocyte reaction responses and cells from alpha beta T cell receptor transgenic mice. Our data suggest that when equivalent numbers of antigen-specific naive and previously primed CD4+ responder T cells are compared, there is no difference in their potency to proliferate but only the previously activated subset can generate cytokines such as interferon-gamma.

Animals↗

PubMed1993

Alternatively spliced CD44 transcripts in diffuse large-cell lymphomas: characterization and comparison with normal activated B cells and epithelial malignancies.

CD44 exists in a variety of alternatively spliced isoforms that include variable numbers of additional exons from the membrane proximal domain (exons 6-14). Lymphocytes express high levels of a "hematopoietic" isoform (CD44H) with no additional variable exons. CD44H binds lymphocytes to postcapillary venules and promotes lymphocyte extravasation into nodal areas. Epithelial carcinomas also express CD44 variants, including exon 10-containing isoforms associated with metastasis in a rat model. An exon 10-containing CD44 isoform is also transiently expressed by normal activated lymphocytes, suggesting that the protein may function in the trafficking of both normal lymphocytes and metastatic tumors. To identify the specific CD44 transcripts in tumors from patients with primary nodal, extranodal, and disseminated large-cell lymphomas (LCLs) and compare them with CD44 mRNAs in normal Ig-activated splenic B cells and epithelial cells, we used a semiquantitative RNA-based polymerase chain reaction. Specific CD44 variants were identified by size, hybridization with exon-specific probes, and sequence analysis. In comparison to primary nodal LCLs, extranodal and disseminated LCLs had increased levels of CD44H and additional isoforms, including a directly spliced (exon 5-->exon 10-->exon 15) exon 10-containing CD44 variant. The CD44 transcripts in extranodal and advanced-stage LCLs were similar to those in normal Ig-activated splenic B cells, whereas epithelial malignancies contained decreased CD44H and increased amounts of larger CD44 variants with additional exons from the membrane proximal domain. The regulated expression of specific CD44 variants is likely to influence the trafficking of lymphoid and epithelial malignancies.

Alternative Splicing↗

PubMed1993

Functional effects of a G to U base change at position 530 in a highly conserved loop of Escherichia coli 16S RNA.

Any base change at position 530 introduced into Escherichia coli on a multicopy plasmid leads to cell death [Powers & Noller (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1042-1046]. It was suggested that these mutants cannot carry out chain elongation. To define more precisely the function of base 530, we have studied ribosomes in which G530 was mutated to U530. In vivo, U530 16S rRNA was incorporated into 30S subunits and could combine with 50S to make 70S ribosomes. 16S rRNA in vitro transcripts containing U530 were assembled into 30S ribosomes, and their activity was tested in defined steps of protein synthesis. Mutant 30S ribosomes were as active as wild-type in poly(U)-dependent poly(Phe) synthesis, P- and A-site tRNA binding, and 30S initiation complex formation. 30S initiation complexes, in the presence of 50S, could react with puromycin like the wild-type. The rate, extent, and position of cross-linking of AcVal-tRNA in the P site to 16S RNA were identical in mutant and wild-type ribosomes. Although there appeared to be no defect in 70S initiation complex formation or in direct A-site binding of Phe-tRNA dependent on poly(U), U530 30S ribosomes were nevertheless defective in carrying out synthesis of fMet-Val dipeptide using natural mRNA. Mutant 30S ribosomes were also refractory to streptomycin-induced misreading although no misreading was observed in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1992

Malignant transformation of NIH-3T3 cells after subcutaneous co-injection with a reconstituted basement membrane (matrigel).

NIH-3T3 cells are non-tumorigenic when injected into athymic mice. If these cells are mixed with an extract of basement-membrane proteins (matrigel) and injected s.c., they form locally invasive and highly vascularized tumors. Cells cultured from the NIH-3T3-matrigel-induced tumors showed a transformed phenotype and lacked contact inhibition. When cultured in a gel of matrigel, they proliferated and formed branched and invasive colonies. In contrast, the parental NIH-3T3 cells cultured on matrigel remained as cell aggregates and were not invasive. I.V. injections of the tumor-derived NIH-3T3 cells produced many colonies on the surface of the lungs, whereas the parental NIH-3T3 cells were not metastatic. Zymographic analysis of the conditioned media obtained from both the tumor-derived and parental NIH-3T3 cells demonstrated higher amounts of the 72-kDa gelatinase (type-IV collagenase) enzyme in the tumor-derived cells. Also, tumor-derived NIH-3T3 cells, but not parental NIH-3T3 cells, secreted the 92-kDa type-IV collagenase. These studies suggest that the interaction of pre-malignant NIH-3T3 cells with extracellular matrix components may contribute to the process of tumor progression.

Animals↗

PubMed1992

Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo.

Endothelial cells are known to bind to laminin, and two peptides derived from the laminin A (CTFALRGDNP) and B1 (CDPGYIGSR) chains block the capillary-like tube formation on a laminin-rich basement membrane matrix, Matrigel. In the present study, we have used various in vitro and in vivo assays to investigate the angiogenic-biologic effects of a third active site in the laminin A chain, CSRARKQAASIKVAVSADR (designated PA22-2) on endothelial cells. The SIKVAV-containing peptide was as active as the YIGSR-containing peptide for endothelial cell attachment but was less active than either the RGD-containing peptide or intact laminin. Endothelial cells seeded on this peptide appeared fibroblastic with many extended processes, unlike the normal cobblestone morphology observed on tissue culture plastic. In addition, in contrast to normal tube formation on Matrigel, short irregular structures formed, some of which penetrated the matrix and sprouting was more apparent. Analysis of endothelial cell conditioned media of cells cultured in the presence of this peptide indicated degradation of the Matrigel and zymograms demonstrated active collagenase IV (gelatinase) at 68 and 62 Kd. A murine in vivo angiogenesis assay and the chick yolk sac/chorioallantoic membrane assays with the peptide demonstrated increased endothelial cell mobilization, capillary branching, and vessel formation. These data suggest that the -SIKVAV-site may play an important role in initiating branching and formation of new capillaries from the parent vessels, a behavior that is observed in vivo in response to tumor growth or in the normal vascular response to injury.

Amino Acid Sequence↗

PubMed1991

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.

Amino Acid Sequence↗

PubMed1991

Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.

Previously we found that the reconstituted basement membrane matrix Matrigel, when premixed with human small-cell lung carcinoma cells and injected subcutaneously into athymic mice, permitted tumor growth, whereas cells injected in the absence of Matrigel did not form tumors. In the present study, we examined additional cell types and determined some of the underlying mechanisms involved in the promotion of tumor formation by Matrigel. The tumor cell lines that we studied included transformed mouse Englebreth-Holm-Swarm tumor cells (T-EHS), human submandibular carcinoma A253 cells, mouse melanoma B16F10 cells, human epidermoid carcinoma KB cells, and human primary renal cell carcinoma cells. When coinjected subcutaneously with Matrigel, these cell lines formed rapidly proliferating tumors. Primary biopsy specimens of human colon carcinoma, when dispersed and coinjected with Matrigel, also formed tumors. Only A253, KB, and B16F10 cells formed small tumors in the absence of Martrigel, but a fivefold to tenfold increase in tumor size was observed in the presence of Matrigel. These data demonstrate a useful method for improving the growth of human tumors in athymic mice.

Animals↗

PubMed1991

Basement membrane and the SIKVAV laminin-derived peptide promote tumor growth and metastases.

Laminin, the major glycoprotein component of basement membrane, promotes the malignant phenotype. Cells which are adherent to laminin are more malignant than the non-adherent cells and in certain tumor cells, the number of laminin receptors is positively correlated with malignancy. Laminin also increases collagenase IV activity, an enzyme demonstrated to be critical for tumor spread. A site on laminin, containing the amino acid sequence SIKVAV, has been identified which when injected intravenously with B16F10 melanoma cells, causes an increase in the number of colonies on the surface of the lungs. This peptide does not affect tumor cell arrest in the vasculature or the immune system. It does promote angiogenesis in various in vitro and in vivo models, thereby facilitating tumor cell survival. When a complex mixture of laminin-enriched basement membrane components (Matrigel) is coinjected with tumor cells subcutaneously, tumor incidence and growth increases. Various tumor cell lines and primary isolates, which previously could not form tumors in mice, can be induced to grow rapidly in the presence of Matrigel. Slowly growing tumors or arrested tumors can also be induced to grow more quickly with additional injections of Matrigel. When an SIKVAV-containing synthetic peptide is coinjected with B16F10 tumor cells and Matrigel subcutaneously in mice, larger tumors are formed than that observed with either Matrigel or cells alone. Such studies define the role of laminin in tumor growth and spread and generate new models for studying therapeutic agents. Of particular interest is the ability to grow primary isolates which generally do not grow in mice.

Amino Acid Sequence↗