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J R Cook

Publications and source records attributed to J R Cook.

At least 55 records · Page 3Linked to original sources
PubMed1997

Quantitation of the cell surface level of Ld resulting in positive versus negative selection of the 2C transgenic T cell receptor in vivo.

The 2C transgenic TCR is positively selected on Kb and is alloreactive for and negatively selected on Ld. To test an avidity model for positive selection, mice were bred to express different levels of surface Ld by varying the number of gene copies encoding beta 2-microglobulin (beta 2m) or Ld heavy chain. Whereas mice expressing 35% Ld (beta 2m+/- Ld+/-) negatively selected the 2C TCR, mice expressing 2% Ld (beta 2m-/- Ld+/-) positively selected the 2C TCR. Furthermore, 2C cytotoxic T lymphocytes selected on 2% Ld showed peptide-specific cytolytic activity against Ld/p2Ca targets. These findings provide clear in vivo evidence that positive selection can occur on very low levels of the same class I antigen capable of negative selection when expressed at higher levels.

Animals↗
PubMed1997

The interferon gamma (IFN-gamma) receptor: a paradigm for the multichain cytokine receptor.

With the purification and cloning of the interferon gamma (IFN-gamma) receptor chains the mechanism of IFN-gamma action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-gamma) receptor complex consists of two chains: IFN-gammaR1, the ligand-binding chain, and IFN-gammaR2, the accessory chain. Binding of IFN-gamma causes oligomerization of the two IFN-gamma receptor subunits, IFN-gammaR1 and IFN-gammaR2, which initiates the signal transduction events: activation of Jak1 and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-gammaR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1alpha, the latent transcriptional factor. With all these steps established, the IFN-gamma receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Animals↗
PubMed1997

Neighbors' perceptions of group homes.

Neighbors often presume that group homes (GHs) have negative effects on their neighborhoods, but it is rather unclear how often GHs actually have adverse effects. Neighbors of GHs and a matched set of people who did not live near GH were interviewed. Neighbors of GHs were asked about their experiences with the specific GH near them, while "non-neighbors" were asked similar questions about their expectations of what it would be like to live near a GH. For both negative (e.g., noise, traffic) and positive effects (e.g., leaning about disabilities) of GHs, non-neighbors expected GHs would have a much greater impact on them than what was actually reported by neighbors. This research supports prior findings that expectations of negative effects are much greater than what is actually experienced by neighbors. It also suggests that GH operators might wish to capitalize on the positive expectations that may be over-shadowed by the more commonly voiced negative expectations.

Adult↗
PubMed1996

Mouse macrophages carrying both subunits of the human interferon-gamma (IFN-gamma) receptor respond to human IFN-gamma but do not acquire full protection against viral cytopathic effect.

Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-gamma receptor (hu-IFN-gammaR) requires the formation of a complex consisting of ligand (IFN-gamma), a ligand binding receptor chain (IFN-gammaR1), and a signal transducing receptor chain (IFN-gammaR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-gamma can exert on fibroblasts. The current report assesses the ability of hu-IFN-gammaR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-gammaR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-gammaR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-gamma with an affinity comparable to that of human cells. Binding of either human or mouse IFN-gamma to its respective receptor elicits classic IFN-gamma responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2- radicals, interleukin-6, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-gamma could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-gamma could. These results demonstrate that while co-expression of hu-IFN-gammaR1 and hu-IFN-gammaR2 is necessary and sufficient for most IFN-gamma-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-gamma receptor complex. The nature and potential role of such factors in IFN-gammaR signaling is discussed.

Animals↗
PubMed1996

The mechanisms of peptide exchange and beta 2-microglobulin exchange on cell surface Ld and Kb molecules are noncooperative.

It has previously been shown that the presence of exogenous beta 2-microglobulin (beta 2m) can dramatically enhance the binding of exogenous peptide to cell surface class I MHC. However, the mechanism by which this enhancement takes place is unknown. Two models have been proposed to explain this phenomenon. In the first model, the exchange of peptide and the exchange of beta 2m are cooperative processes. In the alternative model, beta 2m stabilizes free class I heavy chains to increase the total number of peptide binding sites available. In this report, we have examined the relationship between peptide exchange and beta 2m exchange. Comparisons of Ld and Kb complexes formed with peptides possessing widely disparate affinities revealed a reciprocal correlation between the peptide off-rate and the rate of beta 2m exchange. This result indicates that peptide exchange and beta 2m exchange are noncooperative processes that may, in fact, antagonize one another. These findings provide the first demonstration of peptide-specific influences on the rate of beta 2m exchange and suggest that exogenous beta 2m promotes peptide binding by maintaining class I heavy chains in a peptide-receptive state.

Animals↗
PubMed1996

Differential responsiveness of a splice variant of the human type I interferon receptor to interferons.

Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (alphaYAC) respond to all type I human interferons including IFN-alphaA, IFN-beta, and IFN-omega. The alphaYAC contains at least two genes encoding interferon-alpha receptor (IFN-alphaR) chains that are required for response to type I human interferons: Hu-IFN-alphaR1 and Hu-IFN-alphaR2. We previously isolated a splice variant of the Hu-IFN-alphaR1 chain designated Hu-IFN-alphaR1s. Chinese hamster ovary cells containing a disrupted alphaYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-alphaR1 and Hu-IFN-alphaR1s chains. With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-alphaR1s) and with the Hu-IFN-alphaR1 chains: Hu-IFN-alphaA and IFN-omega. Two other type I interferons, Hu-IFN-alphaB2 and Hu-IFN-alphaF, are capable of signaling through the Hu-IFN-alphaR1 chain only and cannot utilize the splice variant Hu-IFN-alphaR1s. Hu-IFN-alphaR1 and Hu-IFN-alphaR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene. These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-alphaR1 receptor chain, and that the splice variant chain (Hu-IFN-alphaR1s) is functional.

Amino Acid Sequence↗
PubMed1996

Disruption of microfilaments alters laminin synthesis but not laminin trafficking in NHEK in vitro.

Laminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK) in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis rates of most cytosolic proteins as revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug, however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent of the type of collagen matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were secreted basally than apically, an observation consistent with laminin's role in basal lamina formation, cytochalasin D had no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole, showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network does not alter laminin synthesis or secretion.

Actin Cytoskeleton↗
PubMed1996

Interferon-gamma differentially regulates antigen-processing functions in distinct endocytic compartments of macrophages with constitutive expression of class II major histocompatibility complex molecules.

RAW264.7 cells were transfected to express constitutively the murine class II major histocompatibility complex (MHC-II) molecule, I-Ak. The resulting RAW.Ak cells presented HEL(46-61) peptide to 3A9 T hybridoma cells, but they were unable to process and present HEL protein in their resting state. However, IFN-gamma stimulation induced the ability of RAW.Ak to process and present HEL protein, with little effect on their ability to present HEL(46-61) peptide. Antigen catabolism showed little change with IFN-gamma stimulation, suggesting that the production of peptides was not the regulated step in the processing pathway. Furthermore, HEL(46-61) peptide delivered directly into lysosomes by acid-resistant liposomes was also presented only upon IFN-gamma stimulation, while the presentation of peptides delivered into endosomes by acid-sensitive liposomes showed a lesser dependence on IFN-gamma stimulation. Thus, IFN-gamma regulated the ability of peptides delivered into certain lysosomal compartments to meet with MHC-II molecules and form peptide-MHC complexes, or to transport subsequently to the plasma membrane. Two other antigens, ribonuclease A and haemoglobin, were processed by RAW.Ak cells without IFN-gamma stimulation, suggesting that these antigens could be processed by different mechanisms, perhaps in earlier endocytic compartments. Thus, different antigens may be processed in distinct endocytic compartments, and an IFN-gamma-regulated mechanism controls the rescue of peptides from lysosomal compartments for presentation at the plasma membrane.

Animals↗
PubMed1996

Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury.

Delayed re-epithelialization of the cornea after injury usually precedes stromal ulceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adhesive structures at the basement membrane zone. In this study, we provide additional evidence for this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibitions of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermally injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both could participate in dissolution of this structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types of corneal injury.

Animals↗
PubMed1995

Interaction between the components of the interferon gamma receptor complex.

Interferon gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different chains: the IFN-gamma receptor binding subunit (IFN-gamma R, IFN-gamma R1), and a transmembrane accessory factor (AF-1, IFN-gamma R2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-gamma receptor complex, we examined the function of the receptor components in signal transduction upon IFN-gamma treatment. A specific IFN-gamma R2:IFN-gamma cross-linked complex was observed in cells expressing both IFN-gamma R1 and IFN-gamma R2 indicating that IFN-gamma R2 (AF-1) interacts with IFN-gamma and is closely associated with IFN-gamma R1. We show that the intracellular domain of IFN-gamma R2 is necessary for signaling. Cells coexpressing IFN-gamma R1 and truncated IFN-gamma R2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-gamma R1 alone were unresponsive to IFN-gamma treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1 alpha were activated, and IFN-gamma R1 was phosphorylated only in cells expressing both IFN-gamma R1 and IFN-gamma R2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-gamma R2.

Amino Acid Sequence↗
PubMed1995

New vectors for manipulation and selection of functional yeast artificial chromosomes (YACs) containing human DNA inserts.

A set of fragmentation vectors is described which produce a deletion series of smaller yeast artificial chromosomes (YACs) from a larger parent YAC with the insertion of a eukaryotic selectable marker. In addition, new vectors were designed to permit integration of the genes encoding neomycin (neo) or hygromycin B (hyg) resistance into YACs containing inserts of human DNA. All these vectors are compatible with the yeast host strain AB1380, in which most human genomic YAC libraries are maintained. Linearized vector DNA is used to transform yeast cells in which homologous recombination between human DNA in the YAC and the Alu sequence in the fragmentation or integrating vector produces terminal deletions from the acentromeric (URA3) end of the YAC or insertion of the vector into the YAC, respectively. A set of directional deletions of a YAC is useful for genomic mapping, restriction analysis and functional measurements of large chromosomal regions. The neo and hyg eukaryotic markers permit the study of gene function after introduction of deleted YACs into mammalian cells. Transformation of YACs with the fragmentation vectors resulted in fragmentation in 21-46% of the clones examined; transformation with the integrating vector resulted in integration in 46% of the clones examined.

Base Sequence↗
PubMed1995

Induction of peptide-specific CD8+ CTL clones in beta 2-microglobulin-deficient mice.

We have examined the ability of beta 2-m- mice to produce CD4-8+ T cells by generating CD8+ CTLs to a defined ligand. We report here the first demonstration of peptide-specific, self-class I MHC-restricted CTLs from beta 2-m-deficient mice. We have used the KOD mouse, an H-2d beta 2-m- strain, to generate CTLs that recognize the class I MHC molecule Ld in association with one of two Ld-binding immunogenic peptides. Testing of these CTLs on a panel of Ld-binding peptides reveals a high degree of peptide specificity. Peptide-specific CTL bulk cultures from KOD mice differ from those generated in beta 2-m+ mice in that they possess altered affinities for their peptide ligands. In addition, we show that CTLs generated from beta 2-m- mice in the presence of beta 2-m+ stimulator cells and exogenous peptide are specific either for the exogenous peptide or for endogenous peptides that are present in association with Ld on the surface of beta 2-m+ cells, but are not present at detectable levels on beta 2-m- cells. These results demonstrate that positive selection of CD8+ CTLs can occur in vivo on the very low levels of class I MHC found in the KOD mouse. Furthermore, CTLs from the KOD mouse maintain a high degree of peptide specificity despite reduced levels of class I MHC.

Amino Acid Sequence↗
PubMed1995

Computer-assisted mapping of infarcted and viable regions of gross cardiac sections following experimental myocardial infarction.

We have developed a computer-assisted method that creates digital maps of the viable and infarcted regions of tetrazolium-stained gross cardiac sections. Here we describe and test the method, using a canine occlusion-reperfusion infarction model. Quantitative image analysis showed that the method accurately recorded differences between infarcted and viable regions at a spatial resolution of 5-20 pixels per mm2. Microscopic analysis of tissue samples taken from the sections showed that the maps were accurate. In 15 of 15 cases the histology of the samples matched that predicted by the maps. A comparison of infarct area measurements derived from the maps showed that the method was reproducible. The average intraoperator standard deviation was +/- 8% of a slice's infarcted area and the average interoperator standard deviation was +/- 20%. We conclude that the method creates accurate, detailed, and reproducible maps of the infarcted and viable regions of tetrazolium-stained cardiac sections.

Animals↗
PubMed1995

The matrix form of collagen and basal microporosity influence basal lamina deposition and laminin synthesis/secretion by stratified human keratinocytes in vitro.

The ability of the collagen matrix form to support the formation of a basal lamina by cultured normal human epidermal keratinocytes (NHEK) was determined using transmission electron microscopy. The collagen matrix forms tested in this study were a) a dry type I collagen film and b) a type I collagen gel. NHEK were grown for 14 days on the following five different substrates: plain plastic culture dishes without the addition of collagen (PP); plain plastic culture dishes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-P); plain plastic culture dishes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-P); Millipore Millicell CM microporous membranes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-CM); and Millipore Millicell CM microporous membranes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-CM). NHEK maintained for 2 wk on PP and DCF-P were unable to secrete a basal lamina. NHEK grown for 2 wk on the GEL-P and GEL-CM substrates, however, secreted a contiguous basal lamina at the GEL-NHEK interface. To determine if the appearance of this basal lamina correlated with laminin synthesis, laminin was immunoprecipitated from cellular extracts, as well as media from the apical and basal chambers. NHEK grown on the GEL-P substrate synthesized more laminin than did NHEK grown on the other four alternative substrates. In addition, NHEK grown on GEL-CM were able to direct more laminin to the basal compartment than NHEK grown on DCF-CM substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Conformational changes induced in the MHC class I molecule by peptide and beta 2-microglobulin.

Assembly of the class I MHC molecule is inextricably linked to the antigen presentation function of the class I molecule. Association of the class I MHC molecule with beta 2-microglobulin (beta 2m) is a prerequisite for association with the heterodimeric protein TAP, and once peptide is acquired, the class I molecule folds and begins its sojourn to the cell surface. To maintain its folded conformation, class I MHC requires peptide but not beta 2m, and the sequence of the peptide bound exercises a subtle influence on the structure of the class I molecule that is likely to be a factor in T cell receptor discrimination of MHC/peptide complexes.

Animals↗
PubMed1994

Molecular cloning and structural analysis of the human gene encoding cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) appears to play a role in the vertebrate visual process as a substrate-routing protein, influencing the enzymatic partitioning of 11-cis-retinol at a key branch point in the visual cycle. Genomic clones spanning 29 kilobases and encompassing the human CRALBP gene have been isolated by screening two human genomic libraries and from polymerase chain reaction amplification of human leukocyte DNA. The sequence of 13,647 contiguous nucleotides has been determined, including 3130 and 516 bases from the 5'- and 3'-flanking regions, respectively. The human CRALBP gene exists as a single copy in the genome based on Southern analyses and localization to a single site on human chromosome 15 (Sparkes, R. S., Heinzmann, C., Goldflam, S., Kojis, T., Saari, J. C., Mohandes, T., Klisak, I., Bateman, J. B., and Crabb, J. W. (1992) Genomics 12, 58-62). The gene is composed of eight exons and seven introns with average lengths of 198 base pairs and 1.2 kilobases, respectively, and which exhibit conventional vertebrate splicing. Alu repetitive sequences exist in introns 4 and 5 as well as in the 5'- and 3'-flanking regions of the gene. RNase protection and primer extension analyses indicate that the human CRALBP gene transcription start site is 922 bases upstream of the initiation codon. The first exon is entirely untranslated and both exon 2 and exon 8 contain untranslated regions. The proximal 5'-flanking region lacks GC boxes and consensus TATA and CCAAT boxes at the usual positions. The 3'-untranslated region of CRALBP exon 8 is essentially identical to a partial cDNA clone reportedly isolated from a human hippocampus cDNA library, suggesting that the protein may be expressed in a wider spectrum of tissues than previously recognized. The human CRALBP genomic clones and structure provide valuable tools for studying the physiological role of the protein in vision and visual disorders.

Animals↗