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Biomedical subjects

J D Turner

Publications and source records attributed to J D Turner.

At least 37 records · Page 2Linked to original sources
PubMed1997

Transgenic animals as models of neurodegenerative diseases in humans.

Neurodegenerative diseases are of major socioeconomic importance and represent an enormous challenge for the scientific and medical communities. Advances in molecular genetics during the past decade have begun to provide approaches for the establishment of animal models for these disorders using transgenic technology. Their analysis will lead to better understanding of disease pathogenesis and will be invaluable for the identification of novel diagnostic and therapeutic agents. With the current pace of genomic research, the generation of transgenic animal models, reproducing in full the pathology and symptoms of even complex disorders such as Alzheimer's disease, must now be considered achievable.

Alzheimer Disease↗
PubMed1997

Role of CCR5 in infection of primary macrophages and lymphocytes by macrophage-tropic strains of human immunodeficiency virus: resistance to patient-derived and prototype isolates resulting from the delta ccr5 mutation.

The alpha-chemokine receptor fusin (CXCR-4) and beta-chemokine receptor CCR5 serve as entry cofactors for T-cell (T)-tropic and macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains, respectively, when expressed with CD4 in otherwise nonpermissive cells. Some M-tropic and dual-tropic strains can also utilize other beta-chemokine receptors, such as CCR2b and CCR3. A mutation of CCR5 (delta ccr5) was recently found to be common in certain populations and appears to confer protection against HIV-1 in vivo. Here, we show that this mutation results in a protein that is expressed intracellularly but not on the cell surface. Primary CD4 T cells from delta ccr5 homozygous individuals were highly resistant to infection with prototype M-tropic HIV-1 strains, including an isolate (YU-2) that uses CCR5 and CCR3, but were permissive for both a T-tropic strain (3B) and a dual-tropic variant (89.6) that uses CXCR-4, CCR5, CCR3, or CCR2b. These cells were also resistant to M-tropic patient isolates but were readily infected by T-tropic patient isolates. Primary macrophages from delta ccr5 homozygous individuals were also resistant to infection with M-tropic strains, including YU-2, but the dual-tropic strain 89.6 was able to replicate in them even though macrophages are highly resistant to CXCR-4-dependent T-tropic isolates. These data show that CCR5 is the essential cofactor for infection of both primary macrophages and T lymphocytes by most M-tropic strains of HIV-1. They also suggest that CCR3 does not function for HIV-1 entry in primary lymphocytes or macrophages, but that a molecule(s) other than CCR5 can support entry into macrophages by certain virus isolates. These studies further define the cellular basis for the resistance to HIV-1 infection of individuals lacking functional CCR5.

CD4 Antigens↗
PubMed1997

A monoclonal antibody (12G5) directed against CXCR-4 inhibits infection with the dual-tropic human immunodeficiency virus type 1 isolate HIV-1(89.6) but not the T-tropic isolate HIV-1(HxB).

We used a monoclonal antibody (12G5) directed against an extracellular domain of CXCR-4 to investigate the role of this receptor in infection of immortalized lymphoid cell lines, peripheral blood mononuclear cells (PBMCs), and primary brain microglia with a dual-tropic strain of human immunodeficiency virus (HIV-1(89.6)) and a T-tropic strain (HIV-1(IIIB)). Addition of antibody 12G5 to cells prior to and during infection with HIV-1(89.6) inhibited p24 production 100- to 10,000-fold in CEMx174 and 174-CD4 cells and about 10-fold in PBMC cultures but had no activity against infection of either monocyte-derived macrophages or brain microglia. In contrast, 12G5 had little or no effect on infection of CEMx174 cells with HIV-1(IIIB) or HIV-1(HxB). To identify the region of the HIV-1(89.6) envelope that confers sensitivity to 12G5, we used chimeric molecular clones. Chimeras containing the V3 loop region of HIV-1(89.6) were inhibited by 12G5 to the same degree as wild-type HIV-1(89.6) whereas replication of those viruses containing the V3 loop of HIV-1(HxB) was not inhibited by the antibody. A similar pattern was seen in infections of a U87 glioblastoma line that coexpresses CD4 and CXCR-4. Antibody 12G5 was also able to block fusion between HeLa-CD4 cells and CEMx174 cells chronically infected with HIV-1(89.6) but had no effect on fusion mediated by cells chronically infected with HIV-1(IIIB). Taken together, these results suggest that different strains of HIV-1 may interact with different sites on CXCR-4 or may have different binding affinities for the coreceptor.

Antibodies, Monoclonal↗
PubMed1997

Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses.

Feline immunodeficiency virus (FIV) induces a disease state in the domestic cat that is similar to AIDS in human immunodeficiency virus (HIV)-infected individuals. As with HIV, FIV can be divided into primary and cell culture-adapted isolates. Adaptation of FIV to replicate and form syncytia in the Crandell feline kidney (CrFK) cell line is accompanied by an increase in the net charge of the V3 loop of the envelope glycoprotein, mirroring the changes observed in the V3 loop of HIV gp120 with the switch from a non-syncytium-inducing phenotype to a syncytium-inducing phenotype. These data suggest a common mechanism of infection with FIV and HIV. In this study, we demonstrate that cell culture-adapted strains of FIV are able to use the alpha-chemokine receptor CXCR4 for cell fusion. Following ectopic expression of human CXCR4 on nonpermissive human cells, the cells are able to fuse with FIV-infected feline cells. Moreover, fusion between FIV-infected feline cells and CXCR4-transfected human cells is inhibited by both anti-CXCR4 and anti-FIV antibodies. cDNAs encoding the feline CXCR4 homolog were cloned from both T-lymphoblastoid and kidney cell lines. Feline CXCR4 displayed 94.9% amino acid sequence identity with human CXCR4 and was found to be expressed widely on cell lines susceptible to infection with cell culture-adapted strains FIV. Ectopic expression of feline CXCR4 on human cells rendered the cells susceptible to FIV-dependent fusion. Moreover, feline CXCR4 was found to be as efficient as human CXCR4 in supporting cell fusion between CD4-expressing murine fibroblast cells and either HIV type 1 (HIV-1) or HIV-2 Env-expressing human cells. Previous studies have demonstrated that feline cells expressing human CD4 are not susceptible to infection with HIV-1; therefore, further restrictions to HIV-1 Env-dependent fusion may exist in feline cells. As feline and human CXCR4 support both FIV- and HIV-dependent cell fusion, these results suggest a close evolutionary link between FIV and HIV and a common mechanism of infection involving an interaction between the virus and a member of the seven-transmembrane domain chemokine receptor family of molecules.

3T3 Cells↗
PubMed1997

Toward altering milk composition by genetic manipulation: current status and challenges.

The implementation of large-scale genome mapping and sequencing has improved the understanding of animal genetics. A large number of gene sequences are now available to serve as regulatory elements or genes of interest. Although the central thrust of this work is focused on understanding disease states, the manipulation of normal metabolic processes is feasible. To date, the genetic manipulation of livestock has been limited to the permanent addition of genes of clinical interest. This study explores the utility of genetically engineered cattle as a means of altering milk composition to improve the functional properties of milk, increasing marketability. Improvements would include increasing the concentration of valuable components in milk (e.g., casein), removing undesirable components (e.g., lactose), or altering composition to resemble that of human milk as a means of improving human neonatal nutrition. The protracted time lines of genetically modifying dairy cattle has prompted the development of animal models. A model for dwarf goats is discussed in terms of circumventing the lengthy time lines involved in generating transgenic cattle and allowing for an accelerated expansion of research in molecular genetics of dairy animals. Thus, the genetic manipulation of dairy cattle is feasible and could have significant impacts on milk quality, attributes of novel dairy products, and human health.

Animals↗
PubMed1996

CD4-independent infection by HIV-2 is mediated by fusin/CXCR4.

Several members of the chemokine receptor family have been shown to function in association with CD4 to permit HIV-1 entry and infection. However, the mechanism by which these molecules serve as CD4-associated cofactors is unclear. In the present report, we show that one member of this family, termed Fusin/ CXCR4, is able to function as an alternative receptor for some isolates of HIV-2 in the absence of CD4. This conclusion is supported by the finding that (1) CD4-independent infection by these viruses is inhibited by an anti-Fusin monoclonal antibody, (2) Fusin expression renders human and nonhuman CD4-negative cell lines sensitive to HIV-2-induced syncytium induction and/or infection, and (3) Fusin is selectively down-regulated from the cell surface following HIV-2 infection. The finding that one chemokine receptor can function as a primary viral receptor strongly suggests that the HIV envelope glycoprotein contains a binding site for these proteins and that differences in the affinity and/or the availability of this site can extend the host range of these viruses to include a number of CD4-negative cell types.

Animals↗
PubMed1996

Inhibition of cellular proliferation and modulation of insulin-like growth factor binding proteins by retinoids in a bovine mammary epithelial cell line.

Retinoids are potent inhibitors of growth and tumor progression in many mammary carcinoma cell lines, though regulation of growth in nontumorigenic mammary epithelial cells by retinoids is less clear. Here, we have characterized the inhibition of MAC-T (a nontransformed bovine mammary epithelial cell line) cellular proliferation by retinoids and their role in regulating insulin-like growth factor binding proteins (IGFBPs). Retinoic acid (RA) (100 nM) was a potent inhibitor of MAC-T cell proliferation. Retinol was 10-100 times less effective. Neither retinoid could completely arrest growth at noncytotoxic concentrations. Retinoic acid inhibited cellular proliferation by 1 h (P < .05), but inhibition was fivefold greater by 24 h (P < .01). This second stage of growth inhibition (after 12 h) was dependent upon protein synthesis. However, RA-induced inhibition of cellular proliferation did not persist, with thymidine incorporation increasing toward control levels by 4 days in culture. Retinoic acid was less effective in inhibiting thymidine incorporation when cells were stimulated with insulin, des(1-3) IGF-I, or Long(R3) IGF-I when compared to cells stimulated with native IGF-I or serum. Inhibition of proliferation by RA was associated with increased levels of IGFBP-2 in conditioned media and in plasma membrane preparations. Treatment with insulin or des(1-3) IGF-I resulted in the appearance of IGFBP-3 in conditioned media and on the cell surface. However, RA significantly reduced IGFBP-3 levels in conditioned media and eliminated IGFBP-3 associated with the plasma membrane. Thus, RA is a potent but transient inhibitor of bovine mammary epithelial cell proliferation, and this growth inhibition is correlated with increased IGFBP-2 accumulation and inhibition of IGF-I stimulated IGFBP-3 protein secretion.

Animals↗
PubMed1996

Steroidogenic properties of a spontaneously established porcine granulosa cell line (PGC-2).

A spontaneously established porcine granulosa cell line (PGC-2) was cloned through the continuous culturing of primary granulosa cells collected from equine chorionic gonadotropin (eCG)-treated prepubertal gilts. This established cell line has undergone approximately 100 passages and shows contact-inhibition of growth. PGC-2 stained with a monoclonal antibody (mAb) directed against cytokeratin, indicating its epithelial nature, but not with a mAb directed against vimentin, suggesting that it is not fibroblast-derived. Immunoblotting revealed that PGC-2 expresses cadherin, an epithelial Ca+2-dependent cell adhesion molecule. The cells were dependent on serum for growth and had a doubling time of approximately 20 hr when cultured with 10% fetal bovine serum. The cell line was examined for the presence of FSH receptors, cAMP responses, and steroidogenic capabilities. The cell line lacks FSH receptors as assessed by radiolabelled-ligand binding, and no transcripts for FSH receptor were detected by Northern blotting of total cellular RNA. Neither FSH nor cholera toxin (0.5 ng/mL) stimulated increases in cAMP levels in these cells, whereas forskolin (10 microM) induced a fivefold increase in cAMP production. When a higher concentration of cholera toxin (300 ng/mL) was used, however, cAMP levels doubled by 2 hr. Despite a lack of responsiveness to purified of SH or oLH, the cells were capable of progesterone and estradiol production when provided with the appropriate substrates. We conclude that PGC-2 display properties that are similar to immature granulosa cells and may provide a suitable in vitro model for the study of granulosa cell function.

Animals↗
PubMed1996

Correlations of neutrophil phagocytosis for proven bulls with traits of economic importance of their daughters.

Neutrophil phagocytic parameters, measured on 25 AI Canadian Holstein bulls, were investigated for evidence of association with production and type traits, SCC, and survival of dairy cows. An animal model and REML methodologies were used to evaluate the bulls for their neutrophil potential phagocytic activity and capacity. A total of 42,103 first lactation records, collected from 1985 through 1993 for 2919 Quebec dairy herds, were used to obtain EBV for SCC and log SCC for 697 sires. From the same population of cows, 32,900 lactation records were used to calculate EBV for survival after first lactation for 338 sires. Correlations of EBV for SCC and log SCC with all other traits were not significant. Official Canadian ETA for type traits related to the mammary system tended to be positively correlated with phagocytosis parameters. Official Canadian ETA for production traits tended to be negatively correlated. The ETA for milk protein had a significant negative correlation with potential phagocytic activity. Survival after first lactation was significantly associated with ETA for dairy character, milk fat, and fat percentage.

Animals↗
PubMed1996

Quantification of bovine neutrophil migration across mammary epithelium in vitro.

A microassay system was developed to quantify bovine neutrophil transepithelial migration in vitro. The bovine mammary epithelial cell line (MAC-T) formed a confluent monolayer that served as a biologically meaningful barrier for neutrophil migration. Neutrophils added into the upper compartment of an inverted monolayer were driven to migrate across the epithelium from a basal-to-apical direction by the addition of zymosan activated serum (ZAS) into the lower compartment. The numbers of migrated neutrophils were determined by assaying the neutrophil azurophilic granule marker, myeloperoxidase. Results showed that ZAS stimulated neutrophil migration across the epithelium in a time-and dose-dependent manner. In the presence of 5% ZAS and 2 x 10(6) neutrophils, approximately 2.4 x 10(5) neutrophils migrated across the epithelium in 120 min. The procedures we have developed in this study provide a simple precisely controlled system to investigate the normal dynamics of bovine neutrophil transepithelial migration and a means to detect and study impaired neutrophil migration.

Animals↗
PubMed1995

Characterization of transforming growth factor-beta growth regulatory effects and receptors on bovine mammary cells.

Transforming growth factor-beta (TGF-beta) has been shown to inhibit mammary morphogenesis, growth, and differentiation in murine studies. We have characterized TGF-beta receptors and their autoregulation, and the growth response to TGF-beta 1 and TGF-beta 2 in cultured bovine mammary epithelium (MAC-T) and fibroblasts. Affinity labelling studies revealed that fibroblast and epithelial cells contained type I, II, and III (betaglycan) receptors, with the type III receptor being the predominant binding component. On both fibroblasts and epithelial cells, TGF-beta 1 and TGF-beta 2 had equal binding affinities for the type I and II receptors, but TGF-beta 2 had a higher affinity for the type III receptor. Also, preincubation of MAC-T cells with 50 pM TGF-beta 1 or TGF-beta 2 markedly downregulated TGF-beta receptors. Proliferative response was measured using both total DNA and 3H-thymidine incorporation. Both TGF-beta isoforms were effective in inhibiting proliferation of MAC-T cells and fibroblasts. Inhibition of proliferation was not altered following immortalization of fibroblasts with SV-40 Large-T-antigen (LT), even when the cells acquired a transformed phenotype. Inhibition of proliferation was not a result of cytotoxicity, as TGF-beta at concentrations 1,000-fold higher than ED50 levels did not increase cell death. Moreover, the inhibition was reversible as shown by return of cellular proliferation to control levels following TGF-beta removal. Although growth inhibition was not transient as culture of MAC-T cells in TGF-beta resulted in sustained inhibition of proliferation for at least 144 h.

Animals↗
PubMed1995

Characterisation of recombinant serotonin 5-HT1A receptors expressed in Chinese hamster ovary cells: the agonist [3H]lisuride labels free receptor and receptor coupled to G protein.

Serotonin 5-HT1A receptors expressed stably in recombinant Chinese hamster ovary cells have been studied using radioligand binding with the radiolabelled agonist [3H]lisuride. Competition studies with a range of antagonists versus [3H]lisuride confirmed that all of the specific [3H]lisuride binding was to 5-HT1A receptors on the cells. Competition studies with the antagonist spiperone and several agonists gave data that fitted best to two-binding-site models. The affinities of these competing ligands at the two classes of sites were generally in agreement with their corresponding affinities determined in previous work with either 8-[3H]hydroxydipropylaminotetralin ([3H]8-OH-DPAT; labels receptor coupled to G protein) or [3H]spiperone (labels free receptor). Saturation analyses with [3H]lisuride showed that this radioligand labels a single class of binding sites, but the level of radioligand binding was approximately twice that seen when either [3H]8-OH-DPAT or [3H]spiperone was used. [3H]Lisuride binding was partially inhibited by addition of guanine nucleotides, and the extent of inhibition decreased as the [3H]lisuride concentration was increased. This inhibition was due to the effect of guanine nucleotide to decrease slightly the affinity of [3H]lisuride for binding to the 5-HT1A receptors on the cells. It is concluded that [3H]lisuride can label both the free receptor and the receptor coupled to G proteins but with slightly different affinities and that these two states of the receptor exist in roughly equal amounts in the cells. Agonists generally have a higher affinity for the receptor coupled to G protein, whereas antagonists, with the exception of spiperone (which has a higher affinity for the free receptor), have roughly equal affinities for the free receptor and the receptor coupled to G proteins.

Animals↗
PubMed1995

Effects of Staphylococcus aureus toxins on the growth of bovine mammary epithelial cells (MAC-T) in culture.

The effects of Staphylococcus aureus M60 culture supernatant on growth of mammary epithelial cells were tested in vitro. Exposure of MAC-T cells to S. aureus culture supernatant reduced cell proliferation and colony-forming ability because of reduced ability to adhere to plastic. Growth-inhibiting effects of S. aureus culture supernatant were abolished by pretreatment with trypsin. Lysis of S. aureus cells with lysostaphin demonstrated that the inhibition was also present in cell lysates. Treatment of MAC-T cells with culture supernatant from isogeneic mutants of S. aureus M60 that produced either alpha or beta toxins implicated alpha toxin as the main factor inhibiting cell proliferation.

Animals↗
PubMed1995

Comparison of growth hormone-releasing factor and somatotropin: body growth and lactation of primiparous cows.

Growth hormone-releasing factor is the main endogenous stimulator of somatotropin secretion. Our objective was to compare the effects of recombinant growth hormone-releasing factor and recombinant bST on somatotropin secretion, mammary function, and body composition of lactating, primiparous dairy cows. Cows (118 d of lactation) served as uninfused controls or were infused for 63 d with 12 mg/d of releasing factor or with 29 mg/d of bST. These doses elevated somatotropin in serum to concentrations of similar magnitude. The releasing factor and bST each similarly increased milk yield, yield of milk components, weight of most organs, mobilization of adipose tissue, accretion of lean tissue in the carcass, and metabolic activity (RNA) of mammary tissue. Relative to controls, neither releasing factor nor bST significantly affected cell numbers (DNA) in mammary glands or concentrations of plasmin in milk. None of the variables measured provided evidence for galactopoietic effects of releasing factor independent of somatotropin.

Adipose Tissue↗
PubMed1995

Morphologic observation of neutrophil diapedesis across bovine mammary gland epithelium in vitro.

Neutrophils are present in milk of cows as a means of suppressing invading pathogens during mastitis. However, the manner by which neutrophils traverse the secretory epithelia is still not clear: do they diapedese between epithelial cells or do they kill epithelial cells to gain entry into milk? We investigated the process of bovine neutrophil diapedesis across bovine mammary gland epithelium in vitro. The bovine mammary epithelial cell line MAC-T, grown on collagen-coated filters, formed a confluent monolayer with characteristic tight junctions, basal-apical polarity, and functional barriers to the dye trypan blue. Neutrophils added on the apical surface of the monolayer were stimulated to diapedese across the epithelium by the addition of Staphylococcus aureus (10(7) colony-forming units/ml) to the basal compartment. Light and transmission electron microscopy revealed the series of events for neutrophil transmigration: accumulation of neutrophils on the surface of epithelial monolayer; projection of pseudopods into intercellular junctions and movement of neutrophils between adjacent epithelial cells; and reapproximation of the lateral epithelial cell membranes and reformation of the apical tight junctions after neutrophils crossed the epithelium. Morphologically, epithelial cell damage caused by neutrophil diapedesis was not evident. This in vitro model provides a two-dimensional epithelial sheet by which neutrophil diapedesis can be qualitatively studied under defined conditions. Results of the study suggest a major mode by which bovine neutrophils diapedese across the alveolar epithelia into milk during mastitis.

Animals↗
PubMed1994

Coronavirus polyprotein processing.

MHV gene 1 contains two ORFs in different reading frames. Translation proceeds through ORF 1a into ORF 1b via a translational frame-shift. ORF 1a potentially encodes three protease activities, two papain-like activities and one poliovirus 3C-like activity. Of the three predicted activities, only the more amino terminal papain-like domain has been demonstrated to have protease activity. ORF 1a polypeptides have been detected in infected cells by the use of antibodies. The order of polypeptides encoded from the 5' end of the ORF is p28, p65, p290. p290 is processed into p240 and p50. Processing of ORF1a polypeptides differs during cell free translation of genome RNA and in infected cells, suggesting that different proteases may be active under different conditions. Two RNA negative mutants of MHV-A59 express greatly reduced amounts of p28 and p65 at the non-permissive temperature. These mutants may have defects in one or more viral protease activities. ORF 1b, highly conserved between MHV and IBV, potentially contains polymerase, helicase and zinc finger domains. None of these activities have yet been demonstrated. ORF 1b polypeptides have yet been detected in infected cells.

Animals↗