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Biomedical subjects

G A White

Publications and source records attributed to G A White.

At least 19 recordsLinked to original sources
PubMed2001

Improvements in circulating cholesterol, antioxidants, and homocysteine after dietary intervention in an Australian Aboriginal community.

BACKGROUND: Poor nutrition contributes to high rates of coronary heart disease among Australian Aboriginal populations. Since late 1993, the Aboriginal community described here has operated a healthy lifestyle program aimed at reducing the risk of chronic disease. OBJECTIVE: We evaluated the effectiveness of a community-directed intervention program to reduce coronary heart disease risk through dietary modification. DESIGN: Intervention processes included store management policy changes, health promotion activities, and nutrition education aimed at high-risk individuals. Dietary advice was focused on decreasing saturated fat and sugar intake and increasing fruit and vegetable intake. Evaluation of the program included conducting sequential, cross-sectional risk factor surveys at 2-y intervals; measuring fasting cholesterol, lipid-soluble antioxidants, and homocysteine concentrations; and assessing smoking status. Nutrient intakes were estimated from analysis of food turnover in the single community store. RESULTS: There was a significant reduction in the prevalence of hypercholesterolemia (age-adjusted prevalences were 31%, 21%, and 15% at baseline, 2 y, and 4 y, respectively; P < 0.001). There were significant increases in plasma concentrations of alpha-tocopherol, lutein and zeaxanthin, cryptoxanthin, and beta-carotene across the population. Retinol and lycopene concentrations did not change significantly. Mean plasma homocysteine concentrations decreased by 3 micromol/L. There was no significant change in smoking prevalence between the 2 follow-up surveys. There was an increase in the density of fresh fruit and vegetables and carotenoids in the food supply at the community store. CONCLUSION: This community-directed dietary intervention program reduced the prevalence of coronary heart disease risk factors related to diet.

Adolescent↗
PubMed2000

Effectiveness of a community-directed 'healthy lifestyle' program in a remote Australian aboriginal community.

OBJECTIVE: To assess the sustainability and effectiveness of a community-directed program for primary and secondary prevention of obesity, diabetes and cardiovascular disease in an Aboriginal community in north-west Western Australia. METHOD: Evaluation of health outcomes (body mass index, glucose tolerance, and plasma insulin and triglyceride concentrations) in a cohort of high-risk individuals (n = 49, followed over two years) and cross-sectional community samples (n = 200 at baseline, 185 at two-year and 132 at four-year follow-ups), process (interventions and their implementation) and impact (diet and exercise behaviour). RESULTS: For the high-risk cohort, involvement in diet and/or exercise strategies was associated with protection from increases in plasma glucose and triglycerides seen in a comparison group; however, sustained weight loss was not achieved. At the community level, significant reductions were observed in fasting insulin concentration but no change in prevalence of diabetes, overweight or obesity. Weight gain remained a problem among younger people. Sustainable improvements were observed for dietary intake and level of physical activity. These changes were related to supportive policies implemented by the community council and store management. CONCLUSIONS: Community control and ownership enabled embedding and sustainability of program, in association with social environmental policy changes and long-term improvements in important risk factors for chronic disease. IMPLICATIONS: Developmental initiatives facilitating planning, implementation and ownership of interventions by community members and organisations can be a feasible and effective way to achieve sustainable improvements in health behaviours and selected health outcomes among Aboriginal people.

Adult↗
PubMed1999

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.

Adoptive Transfer↗
PubMed1999

An autologous self-antigen differentially regulates expression of I-A glycoproteins and B7 costimulatory molecules on CD4- CD8- T helper cells.

During inflammation, T helper cells transiently express class II major histocompatibility complex (MHC) glycoproteins and present antigens to other T cells. To assess involvement of self-antigens in the generation of T cell antigen-presenting cell (T-APC) activity, rat (R) myelin basic protein (MBP) was used to stimulate a rat CD4-CD8- T cell clone. RMBP induced T cell surface expression of class II MHC glycoproteins and T-APC activity, although RMBP did not elicit interleukin (IL-2) production or proliferation. When added to culture with the strong agonist guinea pig (GP) MBP, RMBP acted as a partial antagonist and inhibited responses of IL-2 production, proliferation, and T cell expression of B7.1. RMBP did not, however, efficiently antagonize GPMBP-induced I-A expression on T cells. These findings indicate that the self-antigen RMBP specifically induces accumulation of I-A/peptide complexes at signaling thresholds that inhibit pathogenic autoimmune responses. Overall, this study suggests a role for self-antigens in the generation of B7-deficient T-APC activity as a mechanism of tolerance in experimental autoimmune encephalomyelitis.

3T3 Cells↗
PubMed1998

Partial agonism elicits an enduring phase of T-cell-medicated antigen presentation.

Previous studies have shown that the anti-CD4 mAb W3/25 strongly enhances T cell APC (T-APC) activity. In this study, single positive CD4+ and double negative (DN) (CD4-CD8-) T-helper cells specific for the 55-69 or 72-86 sequence of guinea pig (GP) myelin basic protein (GPMBP) were used to study CD4 regulation of T-APC activity. Clones were cultured with irradiated SPL and GPMBP or rat (R) MBP for 2-3 days, were propagated in IL-2 for another 1-3 days, were irradiated, and were used as T-APC. DN T cells specific for GP55-69 effectively presented GPMBP and were superior APC compared to other CD4+ T cells for presentation of this antigen. In contrast, DN T cells specific for the dominant encephalitogenic 72-86 determinant did not effectively present the agonist GPMBP but potently presented the partial agonist RMBP. The heightened APC activity of DN T cells reflected the lack of CD4 because the anti-CD4 mAb W3/25 promoted T-APC activity of CD4+ T cells to those levels expressed by DN T cells. Overall, T cells with potent reactivity to GPMBP or RMBP were subsequently unable to present that antigen, whereas T cells exhibiting partial or low antigen reactivities were highly effective APC for presentation of that antigen. The unrelated antigen conalbumin was presented by MBP-specific clones only when added to culture with a specific partial agonist. Together, these data indicate that partially agonistic MHC ligands promote prolonged expression of T-APC activity and that DN T cells may be specialized to mediate postactivational antigen presentation.

Amino Acid Sequence↗
PubMed1998

Class II MHC/peptide complexes on T cell antigen-presenting cells: agonistic antigen recognition inhibits subsequent antigen presentation.

Previous studies have shown that tolerogenic anti-CD4 (W3/25) and anti-LFA-1 mAb (LRTC1) which block T cell activation paradoxically enhance T cell-mediated antigen presentation. Lasting T cell APC (T-APC) activity requires and initial exposure of T cells to these mAb in the presence of professional APC and antigen. This study revealed a central mechanism regulating the duration of T-APC activity. T cell recognition of class II MHC complexes of T-APC catalyzed a rapid decay in the presentation of agonistic antigens, whereas partial agonistic signals decayed at a shower rate. Likewise, blockade of agonistic T-T cell autorecognition by these mAb led to the persistence of agonistic MHC/antigen on T-APC. The best predictor of T-APC activity was related to the ability of clonal T cells to respond to antigen presented by neighboring T cells. Strong responders were inefficient T-APC, whereas inefficient responders were strong T-APC. Addition of irradiated myelin basic protein (MBP0-specific responders to T-APC cultures specifically inhibited the subsequent presentation of MBP but not conalbumin, and vice versa. T-APC presentation of antigen to responder T cells also resulted in reduced surface expression of class II MHC I-A glycoproteins on T-APC. These findings indicate that agonistic recognition of antigen of T-APC specifically inhibits subsequent presentation of that antigen, whereas antagonistic MHC/antigen complexes are preserved for an enduring T-APC activity.

Animals↗
PubMed1996

T-helper lymphocytes specific for myelin basic protein: low-density activation prolongs a postactivation refractory phase marked by decreased pathogenicity and enhanced sensitivity to anergy.

Regulation of experimental autoimmune encephalomyelitis (EAE) in Lewis rats may involve activation-dependent negative feedback pathways of T-helper cells. Previous studies have shown that T-helper cells specific for myelin basic protein exhibit a postactivation refractory phase during which antigenic restimulation elicits proliferation without production of IL-2. Herein, we show that postactivation refractoriness inhibits regeneration of EAE transfer activity and is manifest by a lack of IL-2 mRNA accumulation despite induction of normal levels of IL-4 mRNA. Preactivated refractory T cells were substantially more susceptible than resting T cells to the induction of anergy. Low-density T cell activation or subcloning prolonged the duration of the refractory phase and engendered long-term desensitization of T cells marked by a blockade of IL-2 production and by enhanced susceptibility to anergy. Overall, these results support the concept that postactivation refractoriness controls the pathogenicity and differentiation of T-helper cells.

Animals↗
PubMed1996

Anergy-associated T cell antigen presentation. A mechanism of infectious tolerance in experimental autoimmune encephalomyelitis.

CD4+ T cells promote immune responses against foreign Ags while actively suppressing responses against self Ags. To address how CD4+ T cells ensure self-tolerance, we focused on two CD4+ T helper cells specific for myelin basic protein (MBP). GP2.E5/R1 T cells recognized rat MBP (RMBP) as a partial agonist and mediated mild experimental autoimmune encephalomyelitis (EAE), whereas R2 T cells recognized RMBP with full efficacy and mediated severe EAE. GP2.E5/R1 T cells were more susceptible to anergy induction than R2 T cells. Anergic GP2.E5/R1 T cells lacked proliferative reactivity, but expressed both I-A glycoproteins and high levels of radioresistant APC activity. During induction of anergy, these T cells acquired the ability to present MBP. In a separate subsequent culture without further addition of Ag, anergic GP2.E5/R1 T cells elicited full proliferative and IL-2 production responses by R2 T cells. Unlike activations induced via irradiated splenocytes, irradiated anergic T cells elicited anergy in R2 T cells in the form of a postactivational phase of nonresponsiveness. Anergic GP2.E5/R1 T cells not only transferred anergy to pathogenic R2 T cells in vitro, but these anergic T cells also transferred resistance to EAE in Lewis rats subsequently challenged with guinea pig MBP in CFA. Antagonistic signaling by autologous RMBP was more tolerogenic than that of guinea pig MBP in both in vitro and in vivo models of infectious anergy. We conclude that in the presence of tolerogenic mAb, antagonistic signaling by a self protein elicited the coordinate expression of anergy and T cell-mediated APC activity as a mechanism for the genesis and spread of infectious tolerance.

Animals↗
PubMed1996

Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence.

Adenovirus (Ad) vectors for gene therapy are made replication defective by deletion of E1 region genes. For isolation, propagation, and large-scale production of such vectors, E1 functions are supplied in trans from a stable cell line. Virtually all Ad vectors used for clinical studies are produced in the 293 cell, a human embryonic kidney cell line expressing E1 functions from an integrated segment of the left end of the Ad type 5 (Ad5) genome. Replication-competent vector variants that have regained E1 sequences have been observed within populations of Ad vectors grown on 293 cells. These replication-competent variants presumably result from recombination between vector and 293 cell Ad5 sequences. We have developed Ad2-based vectors and have characterized at the molecular level examples of replication-competent variants. All such variants analyzed are Ad2-Ad5 chimeras in which the 293 cell Ad5 E1 sequences have become incorporated into the viral genome by legitimate recombination events. A map of Ad5 sequences within the 293 cell genome developed in parallel is consistent with the proposed recombination events. To provide a convenient vector production system that circumvents the generation of replication-competent variants, we have modified the Ad2 vector backbone by deleting or rearranging the protein IX coding region normally present downstream from the E1 region such that the frequency of recombination between vector and 293 cell Ad5 sequences is greatly reduced. Twelve serial passages of an Ad2 vector lacking the protein IX gene were carried out without generating replication-competent variants. In the course of producing and testing more than 30 large-scale preparations of vectors lacking the protein IX gene or having a rearranged protein IX gene, only three examples of replication-competent variants were observed. Use of these genome modifications allows use of conventional 293 cells for production of large-scale preparations of Ad-based vectors lacking replication-competent variants.

Adenovirus E1 Proteins↗
PubMed1995

Autologous rat myelin basic protein is a partial agonist that is converted into a full antagonist upon blockade of CD4. Evidence for the integration of efficacious and nonefficacious signals during T cell antigen recognition.

A central question of TCR function is based on the observation that some MHC ligands may bind TCR without stimulating biologic activity. To address the role of CD4 in this mechanism, we studied the interactions of the anti-CD4 mAb W3/25 with an encephalitogenic line of T helper cells. Proliferative responses to guinea pig (GP) myelin basic protein (GPMBP) were mediated by distinct W3/25-sensitive and W3/25-insensitive mechanisms whereas responses to autologous rat (R) MBP (RMBP) were exclusively mediated by W3/25-sensitive pathways. In assays of IL-2 production, RMBP was a partial agonist that stimulated an intermediate level of IL-2 production but antagonized high levels of GPMBP-stimulated IL-2 production to that intermediate level. In the presence of W3/25, RMBP lacked stimulatory activity but instead exhibited inhibitory activity that completely blocked GPMBP-stimulated proliferative responses. The inhibitory mechanism did not involve antigenic competition for MHC glycoproteins, a blockade of mitogenic signaling, or induction of high zone tolerance. Rather, the mechanism involved specific occupancy of TCR with antagonistic MHC ligands derived from the 72-86 region of RMBP. In proliferative assays, GPMBP was approximately 10-fold more active than RMBP. In the presence of W3/25 however, GPMBP-induced agonism and RMBP-induced antagonism exhibited overlapping dose-response curves. RMBP and W3/25 not only fully inhibited GPMBP-stimulated proliferation, this synergistic combination also elicited an extended phase of T cell energy. In conclusion, RMBP-derived MHC ligands occupy TCR to exhibit full efficacy for CD4-dependent signaling pathways while simultaneously lacking efficacy for W3/25-resistant signaling pathways. These data support an "integrative" model of T cell Ag recognition whereby MHC glycoproteins actively guide specific clustering of MHC-ligated TCR to enable quantitative comparisons of efficacious (nonself) and nonefficacious (self) signals by T cells.

Amino Acid Sequence↗
PubMed1992

Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard.

M phase-promoting factor (MPF) consists of a p34cdc2 (cdc2) kinase and cyclin B complex which in its active form promotes G2 to M transition. The role of MPF in G2 arrest following DNA damage, however, has remained largely uncharacterized. We have investigated whether nitrogen mustard (HN2) interfered with either the formation of MPF or its activation. For this purpose, we measured cdc2 kinase activity relative to cdc2 and cyclin B protein turnover and the phosphorylation status of cdc2. Studies were performed in two exceptional human lymphoma cell lines, which differed in HN2 sensitivity by 5-fold (CA46, 50% growth-inhibitory dose = 1.0 microM; JLP119, 50% growth-inhibitory dose = 0.2 microM) but exhibited virtually identical DNA interstrand and DNA-protein cross-link exposure. Following HN2 treatment, CA46 cells ceased to enter mitosis and exhibited a marked delay in G2 phase. Failure to enter mitosis paralleled inhibition of cdc2 kinase. Inhibition was not due to decreased levels of cdc2 or cyclin B protein; rather, G2 arrest correlated with the accumulation of both tyrosine-phosphorylated cdc2 and cyclin B. These findings implied that G2 arrest resulted from a down-regulation of the processes that activate MPF. We also found that JLP119 cells, within a few hours of mitosis at the time of drug treatment, evaded checkpoint control and continued cell division unabated by DNA damage. Furthermore, despite similar DNA cross-link exposure, JLP119 cells within the window of checkpoint control were more susceptible to S phase delay than CA46 cells. Altered cell cycle responses correlated with the greater susceptibility of JLP119 cells to the cytotoxic effects of HN2.

CDC2 Protein Kinase↗
PubMed1991

Isolation, characterization and sequence of a gene conferring resistance to the systemic fungicide carboxin from the maize smut pathogen, Ustilago maydis.

A gene which confers resistance to the systemic fungicide carboxin (Cbx) has been isolated from the maize pathogen, Ustilago maydis, by transferring a plasmid gene library from a Cbx-resistant mutant strain into a sensitive strain and selecting for expression of the resistance gene. Five plasmids, rescued from transformants which exhibited enhanced resistance to Cbx, were shown to have DNA inserts with common restriction enzyme fragments. All the plasmids transformed a sensitive U. maydis strain to Cbx resistance. The gene (Cbxr), sub-cloned on a 3.2 kb EcoR1-HindIII fragment, transformed U. maydis to Cbx resistance at frequencies similar to those obtained with the bacterial Hygromycin B resistance (HygBr) gene. The sequence of the Cbxr gene showed a high degree of homology to succinate dehydrogenase (EC 1.3.99.1) iron-sulphur subunit genes from other organisms.

Amino Acid Sequence↗
PubMed1991

tpr-met oncogene product induces maturation-producing factor activation in Xenopus oocytes.

tpr-met, a tyrosine kinase oncogene, is the activated form of the met proto-oncogene that encodes the receptor for hepatocyte growth factor/scatter factor. The tpr-met product (p65tpr-met) was tested for its ability to induce meiotic maturation in Xenopus oocytes. While src and abl tyrosine kinase oncogene products have previously been shown to be inactive in this assay, p65tpr-met efficiently induced maturation-promoting factor (MPF) activation and germinal vesicle breakdown (GVBD) together with the associated increase in ribosomal S6 subunit phosphorylation. tpr-met-mediated MPF activation and GVBD was dependent on the endogenous c-mosxe, while the increase in S6 protein phosphorylation was not significantly affected by the loss of mos function. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine inhibits tpr-met-mediated GVBD at concentrations that prevent insulin- but not progesterone-induced oocyte maturation. Moreover, maturation triggered by tpr-met is also inhibited by cyclic AMP-dependent protein kinase. This is the first demonstration that a tyrosine kinase oncogene product, p65tpr-met, can induce meiotic maturation in Xenopus oocytes and activate MPF through a mos-dependent pathway, possibly the insulin or insulinlike growth factor 1 pathway.

1-Methyl-3-isobutylxanthine↗
PubMed1991

p34cdc2 is physically associated with and phosphorylated by a cdc2-specific tyrosine kinase.

The mammalian homologue of the yeast cdc2 gene encodes a 34-kilodalton serine/threonine kinase that is a subunit of M phase-promoting factor. Recent studies have shown that p34cdc2 is also a major tyrosine-phosphorylated protein in HeLa cells and that its phosphotyrosine content is cell cycle regulated and related to its kinase activity. Here, we show that cdc2 is physically associated with and phosphorylated in vitro by a highly specific tyrosine kinase. Tyrosine phosphorylation of cdc2 in vitro occurs at tyrosine 15, the same site that is phosphorylated in vivo. The association between the two kinases takes place in the cytosolic compartment and involves cyclin B-associated cdc2. Evidence is presented that a substantial fraction of cytosolic cdc2 is hypophosphorylated, whereas nuclear cdc2 is hyperphosphorylated. Finally, we show that the tyrosine kinase associated with cdc2 may be a 67-kilodalton protein and is distinct from src, abl, fms, and other previously reported tyrosine kinases.

Amino Acid Sequence↗
PubMed1990

Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.

The gene associated with cystic fibrosis (CF) encodes a membrane-associated, N-linked glycoprotein called CFTR. Mutations were introduced into CFTR at residues known to be altered in CF chromosomes and in residues believed to play a role in its function. Examination of the various mutant proteins in COS-7 cells indicated that mature, fully glycosylated CFTR was absent from cells containing delta F508, delta 1507, K464M, F508R, and S5491 cDNA plasmids. Instead, an incompletely glycosylated version of the protein was detected. We propose that the mutant versions of CFTR are recognized as abnormal and remain incompletely processed in the endoplasmic reticulum where they are subsequently degraded. Since mutations with this phenotype represent at least 70% of known CF chromosomes, we argue that the molecular basis of most cystic fibrosis is the absence of mature CFTR at the correct cellular location.

Animals↗
PubMed1990

The amino-terminal region of pp60c-src has a modulatory role and contains multiple sites of tyrosine phosphorylation.

The c-src gene product from either platelet-derived growth factor treated cells or from polyomavirus-infected cells migrates anomalously on gels, displays enhanced kinase activity, and contains additional sites of tyrosine phosphorylation within its amino-terminus. To probe the importance of these post-translational modifications, each of the five amino-terminal tyrosine residues (residues 90, 92, 131, 136, and 149) was altered to phenylalanine using site-directed mutagenesis. Except for the 136F variant, the mutants displayed enhanced kinase activity, albeit at a low level shown insufficient to induce focus formation in NIH3T3 cells. Mutagenesis of residues adjacent to tyrosines 90 and 92 also resulted in variants with enhanced kinase activity indicating that the region and not the tyrosines per se, may be involved in regulating this activity. Fingerprinting analysis demonstrated that the enhanced kinase activity brought about by these mutations occurred through a mechanism which appears to be independent of the phosphorylation state of Tyr 416 and possibly Tyr 527. Upon treatment of cells with vanadate both amino-terminal variants and transforming mutants of pp60c-src displayed a slower migrating form, designated p60+. Hence, the appearance of these p60+ proteins may be elicited either by mutations that enhance the transforming activity of pp60c-src or by perturbations within the amino-terminal region of the enzyme. The retarded mobility of p60+ was shown to be due in part to additional tyrosine phosphorylations residing at its amino-terminus. The demonstration that p60+ could be resolved into multiple bands and that amino-terminal fragments containing phosphorylated tyrosine residues were obtained regardless of which of the five tyrosines in the region was altered to phenylalanine indicates that there are multiple sites of tyrosine phosphorylation within the amino-terminal region of pp60c-src.

Amino Acid Sequence↗