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Biomedical subjects

J B Lorens

Publications and source records attributed to J B Lorens.

At least 19 recordsLinked to original sources
PubMed2004

Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

Amino Acid Motifs↗
PubMed2001

The use of retroviruses as pharmaceutical tools for target discovery and validation in the field of functional genomics.

Retrovirally mediated functional genomics enables identification of physiologically relevant cellular therapeutic targets. Unique properties of retroviruses make them ideal tools for the introduction of large and diverse libraries of potential genetic effectors to a variety of cell types. The identification and recovery of intracellular library elements responsible for altered disease responses establishes a direct basis for pharmaceutical development. Recent innovations in retroviral infection efficiency and expression control have broadened application of the methodology to include libraries of mutagenized cDNAs, peptides and ribozyme genetic effectors.

Animals↗
PubMed2001

Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Amino Acid Motifs↗
PubMed2000

Optimization of regulated LTR-mediated expression.

Retroviral vectors are ideally suited to the study of gene function, allowing efficient, stable expression. Many biological systems (e.g., cell cycle, apoptosis) require the use of regulated expression systems. We therefore developed a regulated retroviral vector system, TRA99, based on a tetracycline transactivator-dependent LTR, where the MMLV enhancer was replaced with a tetracycline-response element. Using fluorescence-activated flow cytometric analysis of a destabilized green fluorescent protein to monitor expression levels, we optimized the minimal promoter configuration with respect to both activated and repressed transcription. The TRA99 vectors demonstrate regulated expression with activated levels comparable to those of standard retroviral vectors and repressed levels indistinguishable from background. This was achieved without using an internal promoter cassette, thus retaining the cis-packaging elements requisite for helper-mediated transfer.

Animals↗
PubMed2000

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Amino Acid Motifs↗
PubMed1999

A novel paired domain DNA recognition motif can mediate Pax2 repression of gene transcription.

The paired domain (PD) is an evolutionarily conserved DNA-binding domain encoded by the Pax gene family of developmental regulators. The Pax proteins are transcription factors and are involved in a variety of processes such as brain development, patterning of the central nervous system (CNS), and B-cell development. In this report we demonstrate that the zebrafish Pax2 PD can interact with a novel type of DNA sequences in vitro, the triple-A motif, consisting of a heptameric nucleotide sequence G/CAAACA/TC with an invariant core of three adjacent adenosines. This recognition sequence was found to be conserved in known natural Pax5 repressor elements involved in controlling the expression of the p53 and J-chain genes. By identifying similar high affinity binding sites in potential target genes of the Pax2 protein, including the pax2 gene itself, we obtained further evidence that the triple-A sites are biologically significant. The putative natural target sites also provide a basis for defining an extended consensus recognition sequence. In addition, we observed in transformation assays a direct correlation between Pax2 repressor activity and the presence of triple-A sites. The results suggest that a transcriptional regulatory function of Pax proteins can be modulated by PD binding to different categories of target sequences.

5' Untranslated Regions↗
PubMed1999

Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.

BACKGROUND: Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes. METHODS: Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay. RESULTS: Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein. CONCLUSIONS: The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.

Androstadienes↗
PubMed1999

Neurokinin-3 receptor distribution in rat and human brain: an immunohistochemical study.

Autoradiographic and immunohistochemical studies have shown that the neurokinin-3 receptor is widely distributed in the rodent CNS. Expression of the neurokinin-3 receptor in human brain, however, has been debated. These conflicting findings, as well as the poor resolution of autoradiographic images, prompted us to develop a polyclonal antibody against an oligopeptide derived from the carboxy-terminus consensus sequence of both the rat and human neurokinin-3 receptor ([C]ASTTSSFISSPYTSVDEYS, amino acids 434-452 of the rat neurokinin-3 receptor). Western blot analysis of both human and rat brain tissue revealed a major band in the molecular weight range 65,000-67,000, the proposed molecular weight of the neurokinin-3 receptor based on its amino acid sequence and presumed glycosylation state. The distribution of selective high affinity neurokinin-3 receptor agonist [3H]senktide binding and neurokinin-3 receptor immunoreactivity were virtually identical in the brains of male Fischer 344 rats. The highest concentrations of neurokinin-3 receptors were observed in cortical layers IV-V; the basolateral amygdaloid nucleus; the hypothalamic paraventricular, perifornical and supraoptic nuclei; the zona incerta; and the entopeduncular and interpeduncular nuclei. [3H]senktide binding and neurokinin-3 receptor immunoreactivity were compared in homologous cortical areas of the human and rat brain. In contrast to the rat, autoradiographic analysis of normal control human brains (35-75 years) revealed a distinct and predominant superficial cortical labeling in the glia limitans and the cortical layer I. However, neurokinin-3 receptor immunoreactivity could be found not only in the superficial cortical layers, but also on pyramidal neurons and astrocytes in the neuropil and white matter. These findings suggest species differences in both the cellular and anatomical distribution of the neurokinin-3 receptor.

Amino Acid Sequence↗
PubMed1997

Local delivery of interleukin 4 by retrovirus-transduced T lymphocytes ameliorates experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system which serves as a model for the human disease multiple sclerosis. We demonstrate here that encephalitogenic T cells, transduced with a retroviral gene, construct to express interleukin 4, and can delay the onset and reduce the severity of EAE when adoptively transferred to myelin basic protein-immunized mice. Thus, T lymphocytes transduced with retroviral vectors can deliver "regulatory cytokines" in a site-specific manner and may represent a viable therapeutic strategy for the treatment of autoimmune disease.

Animals↗
PubMed1996

Two variants of the pituitary specific transcription factor Pit-1 in Atlantic salmon.

We have cloned two 1.6 kb cDNAs encoding variants of the POU-type pituitary-specific transcription factor Pit-1 from Atlantic salmon. Sequence comparison with mammalian Pit-1 revealed that the POU domain was highly similar while flanking regions were less conserved. The N-terminal region contained three insertions relative to mammalian Pit-1, one of these corresponded to the insertion found in the alternatively spliced Pit-1a isoform. While two different salmon Pit-1 transcripts were expressed, alternative splicing in the 5'-region did not appear to contribute to further transcript diversity. Both salmon Pit-1 cDNAs encoded 39.5 kDa proteins that specifically bind a consensus Pit-1 recognition sequence in vitro. The salmon Pit-1 proteins also recognized the classical octamer motif; however, a point mutation in the POU homeodomain abolished this interaction.

Alternative Splicing↗
PubMed1995

Embryonic expression and DNA-binding properties of zebrafish pax-6.

Zebrafish pax-6 (pax[zf-a]) and its murine homologue are structurally and functionally related to the Drosophila paired box gene eyeless, a master control gene for eye development. This report details the zebrafish pax-6 embryonic expression pattern both at the mRNA and protein level. Transcripts are first detected in the presumptive forebrain and hindbrain regions of the neural plate. After formation of the neural keel, Pax-6 protein accumulates within the same two domains. Expression is also observed in the optic vesicles and lens placodes, confirming that the Pax-6 protein is expressed in those areas of the eye where it is assumed to control differentiation. The relative DNA-binding affinity of the zebrafish Pax-6 protein to different categories of Pax recognition sites is shared with the murine homologue.

Animals↗
PubMed1995

Molecular cloning and characterization of anionic and cationic variants of trypsin from Atlantic salmon.

Pancreatic cDNA libraries from Atlantic salmon (Salmo salar) were constructed and screened with salmon trypsin-specific probes. Five clones containing near full-length transcripts were selected for further characterization. Comparison of deduced amino acid sequences revealed that all variants possessed the canonical serine protease catalytic triad, consisting of histidine, aspartic acid and serine residues, a substrate-binding pocket with aspartic acid at the bottom, and 12 cysteine residues comprising six disulphide bridges. Translation in vitro of one of the trypsin clones produced a protein with the expected molecular mass of 24.5 kDa. Three of the Atlantic salmon trypsins (SalTRP-I, SalTRP-IA and SalTRP-IB) possessed very similar sequences and may represent allelic variants encoded by the same gene focus; however, existence as tetraploid loci or isoloci where disomic inheritance is incomplete may also exist in Atlantic salmon and cannot be excluded. Two other trypsin clones (SalTRP-II and SalTRP-III) are probably encoded by separate gene loci. Analysis of genomic DNA by Southern blotting and hybridization to a trypsin probe showed a complex pattern, indicative of a large number of gene loci for trypsin in Atlantic salmon. The charged amino acid distribution showed that four of the Atlantic salmon trypsin clones encoded anionic forms of the enzyme, while the fifth clone represented a cationic variant. Multiple alignments of the Atlantic salmon trypsin sequences with trypsin, chymotrypsin and elastase from different species placed all Atlantic salmon sequences approximately equidistant from trypsins of other species. Interestingly, the distance between the anionic and cationic variants from Atlantic salmon was similar to the distance between salmon and mammalian trypsins, revealing an early separation of these two types of trypsin, possibly prior to the derivation of fish during evolution. A structural model based on X-ray diffraction studies of the salmon trypsin protein was very similar to that of the mammalian enzyme. All residues which differ in charge between anionic and cationic trypsins were located at exposed regions of the proteins.

Amino Acid Sequence↗
PubMed1994

Pancreatic carboxylester lipase from Atlantic salmon (Salmo salar). cDNA sequence and computer-assisted modelling of tertiary structure.

We report the isolation and characterization of a 1795-bp cDNA fragment encoding Atlantic salmon pancreatic carboxylester lipase from salmon pancreas mRNA. The nearly full-length cDNA contained a 540-amino-acid open-reading frame, encompassing the mature protein (by similarity to mammalian carboxylester lipase enzymes). The salmon carboxylester lipase primary structure shared 58% identity with mammalian carboxylester lipases, lacking the proline-rich C-terminal repeats found in human and rat carboxylester lipases. Congruent with other esterase B type enzymes, the salmon carboxylester lipase contained a canonical serine-esterase catalytic triad motif consisting of serine, histidine and aspartic acid. Computer-assisted modelling of the tertiary structure for salmon carboxylester lipase was conducted using acetylcholine esterase (Torpedo californica) as a template structure. The model, in conjunction with sequence comparisons and available enzymological data, has been used to locate putative bile-salt-binding and lipid-binding sites. The carboxylester lipase enzymes contain a unique, highly conserved insert region that may be associated with bile-salt binding. In the model structure, this region is located close to the active site, and contains a tyrosine residue with an adjacent carboxylester-lipase-conserved arginine. These traits have previously been predicted for the non-specific (regarding bile-salt hydroxylation) bile-salt-binding site in carboxylester lipase enzymes. At this site, a dihydroxy or trihydroxy bile-salt molecule may bind the tyrosine via hydrophobic interactions, the anionic bile-salt head group may bind the arginine, while hydrogen bonding between the bile-salt 12 alpha hydroxy group and an adjacent aspargine residue is possible. The model does not contain an active site 'lid' structure as found in other lipases. The carboxylester lipase structural homolog to the 'flap' of the lipases from Geotrichum candidum and Candida rugosa contains a carboxylester-lipase-conserved deletion that renders this region unable to cover the active site. Instead, the shortening of this loop leads to solvent exposure of the carboxylester lipase insert region, an additional indication of the functional importance of this region.

Amino Acid Sequence↗
PubMed1993

Expression of growth hormone genes in Atlantic salmon.

Atlantic salmon (Salmo salar) possess two genes encoding GH. We have investigated the expression of these two genes in the salmon pituitary. The transcriptional start site was localized 64 nucleotides upstream of the first methionyl codon using primer extension and 5' specific polymerase chain reaction (PCR) assays. Northern analysis revealed a major Atlantic salmon GH (salGH) transcript band of approximately 1400 nucleotides. As coexpression of the salGH genes is not discernible by transcript length, other techniques were used to assess gene activity; RNase protection analysis revealed GH transcript heterogeneity, while reverse transcription-PCR assays detected transcripts from both genes at approximately equivalent amounts. The encoded salGH protein, generated in vitro and by Escherichia coli, shares electrophoretic and immunoreactive identity with native pituitary salGH.

Animals↗
PubMed1992

The complete nucleotide sequence of the Atlantic salmon growth hormone I gene.

Two closely related genes encoding growth hormone were isolated from Atlantic salmon by genomic cloning. From one of these genes a total of 6500 nucleotides were determined including 3900 nucleotides in exons and introns and about 600 and 2000 nucleotides in 5' and 3' flanking regions. The gene is organized in six exons and encodes a polypeptide of 210 amino acids including a 22 amino acids signal sequence. The promoter region contains a typical TATA box 21 nucleotides upstream from the transcription start site. At the 3' end, three putative poly(A) signal sequences are present. The last two are within a 121 nt inverted repeat.

Amino Acid Sequence↗
PubMed1992

The Atlantic salmon prepro-gonadotropin releasing hormone gene and mRNA.

Screening for the gene encoding salmon gonadotropin releasing hormone (sGnRH) in an Atlantic salmon (Salmo salar) genomic library resulted in isolation of a positive clone designated lambda sGnRH-1. An anchor polymerase chain reaction (PCR) technique was used to amplify GnRH cDNA derived from salmon hypothalamic mRNA. The cDNA sequence was aligned to the 7607 base pair genomic sequence which was shown to encode the entire prepro-GnRH gene. The cDNA proved that the cloned gene is expressed in the hypothalamus of mature salmon. The coding domain of sGnRH differs from the mammalian GnRH by six nucleotide changes which allow the two amino acid differences between the two GnRH variants. Salmon GnRH associated peptide (GAP) differs extensively in sequence and size from the mammalian counterpart. Compared to the GnRH cDNA of a cichlid species the similarity is 69.3% in the protein coding sequence.

Amino Acid Sequence↗