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M Hollis

Publications and source records attributed to M Hollis.

At least 19 recordsLinked to original sources
PubMed1996

Monocyte chemoattractant protein-1: receptor interactions and calcium signaling mechanisms.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the Cys-Cys chemokine family. Two related MCP-1 receptors have been identified (CC-CKR2A and CC-CKR2B), although the precise kinetics of ligand binding and calcium signaling of these receptors has yet to be investigated. To examine this more closely, the human MCP-1 receptors were cloned and expressed in Chinese hamster ovary (CHO) cells. Membranes prepared from cells expressing CC-CKR2B bind MCP-1 selectively and with high affinity (Kd = 120 pM). MCP-1 stimulation of recombinant CHO cells expressing CC-CKR2B induces a rapid increase in intracellular Ca2+ through both receptor-operated Ca2+ channels and mobilization of Ca2+ from intracellular stores, and leads to a rapid temperature-dependent internalization of the ligand/receptor complexes. In contrast, recombinant CHO cells expressing CC-CKR2A, and membranes prepared from these cells, fail to bind detectable levels of MCP-1. However, MCP-1 stimulation of cells expressing CC-CKR2A induces a small but significant increase in intracellular Ca2+. Repeated stimulation of these cells with MCP-1 leads to a potentiation of the response to a level comparable to that seen in cells expressing CC-CKR2B. These observations suggest that the levels of cell surface CC-CKR2A are controlled by novel mechanisms.

Animals↗
PubMed1996

High level expression of human MCP-1 using the LCR/MEL expression system.

We have expressed human monocyte chemoattractant protein-1 (hMCP-1) in preerythroid mouse erythroleukemia (MEL) C88 cells using the locus control region/MEL expression system and studied the biological activity of the purified protein in a range of in vitro experimental systems. The recombinant hMCP-1 is expressed at high levels (approximately 10 mg/liter) in this system and is modified in a manner which is very similar to native hMCP-1. We have developed a simple high-yielding two-step purification route employing dye ligand and ion exchange chromatographies which enables us to separate glycosylated and unglycosylated hMCP-1. The purified glycosylated and unglycosylated forms of hMCP-1 have equivalent biological activities in all of the assay systems tested.

Animals↗
PubMed1996

Myelin gene expression in immortalized Schwann cells: relationship to cell density and proliferation.

Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions, [3H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P0 glycoprotein (P0) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and PO than sparse ones. PO mRNa and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.

Animals↗
PubMed1995

Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization.

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.

Adenylyl Cyclases↗
PubMed1994

Computer-generated presentations: current status and future directions.

The personal computer allows the user to create professional presentations as good as any created by commercially available services. With the new generation of inexpensive software, myriad fonts, layouts, graphics, and imported images can all be used by the novice. The technology is currently available that can project images directly from the computer, obviating the need for slides. This presentation will discuss the relative merits and costs of such systems and the problems that remain concerning implementation at a national level.

Audiovisual Aids↗
PubMed1994

A microchip for quantitative detection of molecules utilizing luminescent and radioisotope reporter groups.

Through the marriage of microelectronics and molecular biology, a miniaturized device is presented for ultrasensitive detection of labeled molecules. The novelty of the approach is the direct integration of a charge-coupled device (CCD) and a probe-based assay. Specifically, a CCD detector serves as an active solid support that quantitatively detects and images the distribution of labeled target molecules near the spatially addressable pixels. The device exploits the inherent characteristics of microelectronics that accommodate highly parallel assays, ultrasensitive detection, high throughput, integrated data acquisition and computation. Hence, the technology presented offers substantial practical utility to both research and clinical diagnostic applications that require quantitative analysis of bound molecules. Specifically for probe-based assays such as reverse dot blots, hybridization of radiolabeled or fluorescently labeled, target DNA can be quantitatively assessed within seconds due to the high sensitivity and direct coupling employed.

DNA Probes↗
PubMed1994

Noninvasive determination of bone stiffness for distraction osteogenesis by quantitative computed tomography scans.

Apparent density of canine tibial specimens was correlated to measured quantitative computed tomography (QCT) numbers, and a conversion equation was derived. Finite element analysis models of canine tibiae were constructed using spatial coordinates of converted QCT data. Two QCT-derived finite elemental analysis models were constructed for a pair of matched tibiae: one lengthened 15% by distraction osteogenesis and one contralateral control. Two equations relating the modulus of elasticity to apparent density were used. These models were then verified mechanically by measuring strains of the two specimens under applied load. Equation I correlated best with measured strains in the less-dense regenerate bone of the distracted tibia (R2 = 0.914, p = 0.0028, n = 6); Equation II performed best in the denser control tibia (R2 = 0.820, p = 0.0129, n = 6). These results demonstrate that the stiffness of tubular bones, including cortical and heterogenous cancellous structure, can be accurately predicted by a noninvasive QCT scan.

Animals↗
PubMed1993

Regulated expression of K+ channel genes in electrically silent mammalian cells by linkage to beta-globin gene-activation elements.

Fundamental studies of the mechanism of human beta-like globin gene-expression have identified DNA sequences (locus control regions or LCRs) which directly influence the specific activation of beta-globin genes in erythroid cells. Here we report the first applications of LCR-mediated gene-activation to stable electrophysiological expression of several homomultimeric ion channel proteins. We describe expression driven from a native K+ channel gene promoter region, contiguous to an intronless coding sequence, within a single excised human genomic DNA fragment. In addition, cDNAs encoding both inactivating and non-inactivating mammalian K+ currents have been expressed by insertion between the promoter and second intron of the human beta-globin gene. Expression levels are characteristically independent of integration position and are proportional to LCR-gene copy number, a parameter specific for each clonal cell line. K+ channel expression is inducible by erythroid differentiation and has been demonstrated by electrophysiological recordings of cells taken directly from culture.

Amino Acid Sequence↗
PubMed1992

GAL4 fusion vectors for expression in yeast or mammalian cells.

We describe two sets of vectors, one for yeast (pY1, pY2 and pY3) and one for mammalian cells (pM1, pM2, and pM3), that simplify the production of fusion proteins containing the DNA-binding domain of GAL4. This protein fragment, consisting of GAL4 amino acid (aa) residues 1-147, binds to a specific 17-bp nucleotide sequence, but is incapable of activating transcription unless fused to a protein that can contribute an activating function. Genetic strategies exploiting this property of GAL4 (aa 1-147) have been developed to characterize transcription factor functional domains, protein-protein interactions, and site-specific proteolysis. The vectors we describe allow fusion to the C terminus of GAL4 (aa 1-147) in any reading frame, and thus facilitate these experimental strategies.

Amino Acid Sequence↗
PubMed1992

LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells.

We have used the human globin locus control region (LCR) to assemble an expression system capable of high-level, integration position-independent expression of heterologous genes and cDNAs in murine erythroleukaemia (MEL) cells. The cDNAs are inserted between the human beta-globin promoter and the second intron of the human beta-globin gene, and this expression cassette is then placed downstream of the LCR and transfected into MEL cells. The cDNAs are expressed at levels similar to those of the murine beta-globin in the induced MEL cells. Heterologous genomic sequences can also be expressed at similar levels when linked to to the LCR and beta-globin promoter. In addition we demonstrate that, after induction of differentiation, MEL cells are capable of secreting heterologous proteins over a prolonged time period, making this system suitable for use in continuous production systems such as hollow fibre bioreactors. The utility of the LCR/MEL cell system is demonstrated by the expression of growth hormone at high levels (greater than 100 mg/l) 7 days after induction. Since the expression levels seen do not depend upon gene amplification and are independent of the integration position of the expression cassette, it is possible to obtain clones with stable high-level expression within 3-4 weeks after transfection.

Animals↗
PubMed1989

Generation of a recombinant mouse-human chimaeric monoclonal antibody directed against human carcinoembryonic antigen.

A procedure was devised for the identification and specific cloning of functionally rearranged variable region immunoglobulin (Ig) gene segments from genomic DNA of a murine hybridoma cell line which produces a high-affinity monoclonal antibody (MAb) directed against human carcinoembryonic antigen (CEA). The cloned, functionally-rearranged murine Ig H-chain and L-chain variable region gene segments were incorporated into plasmid vectors capable of directing the expression of a chimaeric mouse-human antibody molecule with human (gamma 4, kappa) constant region sequences. Expression plasmids were transfected into a mouse myeloma cell line by electroporation and transfectomas secreting functional chimaeric antibody selected. Chimaeric antibody generated by transfectomas was analysed and shown to compete effectively with its murine counterpart for binding to the CEA epitope, and to have an equivalent antigen-binding affinity. This anti-CEA recombinant antibody should find application in in vivo diagnosis by immunoscintigraphy of human colonic carcinoma, and possibly also in therapy of the disease, overcoming some of the difficulties associated with the repeated use of non-human immunoglobulins in human patients.

Animals↗
PubMed1988

The yeast UASG is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator.

The yeast trans-activator protein GAL4, when expressed in HeLa cells, stimulates transcription from several class B (II) eukaryotic promoters containing GAL4 binding sites either as the full UASG or as synthetic 17-mers. The characteristics of this activation are indistinguishable from those of the SV40 enhancer. Transcription was similarly stimulated from either complex promoter regions containing multiple upstream elements or from a simple promoter region composed of only a TATA box. Addition of a 17-mer GAL4 binding site to the SV40 enhancer resulted in a synergistic enhancement of transcription in the presence of GAL4. Furthermore, chimeras of the human estrogen receptor DNA binding domain and either GAL4 or GCN4 activating "acidic" regions can activate a promoter region controlled by an estrogen-responsive enhancer. Together, these data indicate that the molecular mechanisms responsible for transcriptional enhancement have been conserved from yeast to man.

Animals↗
PubMed1988

A repressor heterodimer binds to a chimeric operator.

Replacement of the solvent-exposed residues of the DNA recognition helix of the 434 repressor with the corresponding residues of the P22 repressor generates a hybrid protein, 434R[alpha 3(P22R)], which binds specifically to P22 operators. We show here that a new DNA-binding specificity is generated by combining 434 and 434R[alpha 3(P22R)] repressor monomers to form a heterodimer. The heterodimer specifically recognizes a chimeric P22/434 operator that lacks two-fold rotational symmetry.

Base Sequence↗
PubMed1986

Human Sau3A repeated DNA is enriched in small polydisperse circular DNA from normal lymphocytes.

Representatives of the Sau3A family of short human repeated sequences [Meneveri et al., J. Mol. Biol. 186 (1985) 483-489] have been isolated from the small polydisperse circular DNA (spcDNA) of peripheral human lymphocytes. The prototype repeat is a 72-bp element which is at least partially tandemly repeated in spcDNA and human genomic DNA. In comparison with three major families of human repeated DNA, the Sau3A repeats are enriched in spcDNA. The function of spcDNA in normal and transformed eukaryotic cells is not understood and most studies have attempted to resolve this problem by molecular analysis of circular DNA isolated from cells in culture [see Rush and Misra, Plasmid 14 (1985) 177-191 for references]. We have studied the spcDNA present in normal uncultured human lymphocytes and present data pointing to the selective accumulation of the Sau3A family of repeated DNA within this population. The sequences of twelve of these repeats, the consensus sequence for this family and the sequence of a genomic repeat, are presented.

Base Sequence↗
PubMed1982

Post natal support.

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Continuity of Patient Care↗