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R A Colbert

Publications and source records attributed to R A Colbert.

33 records · Page 2Linked to original sources
PubMed1996

Polymorphism in the LMP2 gene influences disease susceptibility and severity in HLA-B27 associated juvenile rheumatoid arthritis.

OBJECTIVE: To determine the potential contribution of the MHC class II region proteasome subunit gene, LMP2, to disease susceptibility, severity, and phenotype in patients with juvenile rheumatoid arthritis (JRA). METHODS: A CfoI restriction site polymorphism in the coding region of the LMP2 gene was evaluated in 279 patients with JRA and 107 healthy controls of similar ethnicity. Patients were divided into 5 groups on the basis of clinical presentation; 46% had early onset pauciarticular disease, 10% early onset polyarticular, 10% late onset pauciarticular, 20% late onset polyarticular, and 11% systemic onset arthritis. The influence of this LMP2 polymorphism on susceptibility to disease, clinical subtype of disease at onset (age and number of joints involved), progression and severity of joint disease (pauci to polyarticular course and radiographic changes), and occurrence of inflammatory eye disease was evaluated. RESULTS: Comparison of genotypes revealed a significantly increased prevalence of homozygosity for the LMP2 B allele (LMP2 BB genotype) in patients who were older (> or = 6 years) at onset of disease (65%, p < 0.05), particularly in those with pauciarticular (71%) involvement at presentation (p < 0.05), compared to controls (51%). The BB genotype was also more prevalent in patients with a polyarticular course, either from onset (63%) or those who progressed from pauciarticular disease (69%), compared with controls, (p = 0.05 and < 0.05, respectively). Stratification for HLA-B27 and DR4, the HLA alleles most frequently associated with late onset pauciarticular and late onset polyarticular JRA, respectively, revealed a persistent effect of LMP2 BB homozygosity on disease susceptibility and phenotype that remained statistically significant in HLA-B27 positive children, and was not due to linkage disequilibrium. CONCLUSION: We show that homozygosity of the B allele of the proteasome subunit LMP2 increases susceptibility to certain subgroups of JRA, and influences the phenotype of disease, predisposing to more progressive and severe articular disease.

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PubMed1994

Differences in peptide presentation between B27 subtypes: the importance of the P1 side chain in maintaining high affinity peptide binding to B*2703.

Susceptibility to spondyloarthropathies is strongly associated with the MHC class I molecule HLA-B27, and is hypothesized to result from the presentation of arthritogenic peptides. Subtypes of B27 that differ structurally but are disease-associated ought to be capable of presenting such peptides, while nondisease-associated subtypes would not. We demonstrate that B*2703, the predominant West African B27 subtype that may not predispose to disease, is not recognized by most B*2705-alloreactive CTL, and does not efficiently present a known B*2705-restricted influenza A nucleoprotein (NP) peptide. We show inefficient presentation is due to a reduced binding affinity of B*2703 for the NP peptide. Furthermore, substituting Arg for the naturally occurring Ser at P1 of the NP peptide, restores high affinity binding and efficient presentation by B*2703. Our results suggest that B*2703 will bind and present efficiently only a subset of the peptides that bind to B*2705, in particular those with Arg or Lys at P1. The apparent lack of disease in individuals with B*2703 may be due to an inability to bind and present putative arthritogenic peptides.

Alleles↗
PubMed1994

Vasoactive intestinal peptide stimulates neuropeptide Y gene expression and causes neurite extension in PC12 cells through independent mechanisms.

Vasoactive intestinal peptide (VIP) is widely recognized as a regulator of tyrosine hydroxylase via a mechanism of trans-synaptic activation. Subsets of adrenal medullary cells and postganglionic sympathetic nerves coexpress the peptide neurotransmitter neuropeptide Y (NPY) with catecholamines. Using PC12 cells transiently expressing a fusion gene in which the bacterial enzyme chloramphenicol acetyltransferase (CAT) is under the control of 700 base pairs of the 5' flanking region of the NPY gene, we have studied the role of VIP and the related peptide pituitary adenylate cyclase activating peptide (PACAP) in regulating NPY gene transcription. Both VIP and PACAP stimulated expression of the NPY gene through activation of cAMP-dependent protein kinase. PACAP was 1000-fold more potent in eliciting this response compared to VIP and activity resided in its N-terminal 27 amino acids. Both VIP and PACAP caused a subpopulation (approximately 50%) of PC12 cells to undergo profound morphological changes in that the cells extended long, slender neurites with prominent growth cones. This change in morphology was unaffected by preincubating cells with inhibitors of either cAMP-dependent protein kinase or calcium/phospholipid-dependent protein kinase. A trophic role for either VIP or PACAP in regulating sympathetic nerve function is proposed.

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PubMed1993

Allele-specific B pocket transplant in class I major histocompatibility complex protein changes requirement for anchor residue at P2 of peptide.

To investigate the role of an anchoring pocket in allele-specific peptide presentation by a major histocompatibility complex class I molecule, we "transplanted" a B pocket from HLA-A*0201 into HLA-B*2705 by site-directed mutagenesis. The resulting protein, designated B27.A2B, binds a different set of endogenous peptides than B*2705 as evidenced by complete loss of allorecognition as well as restored expression in the antigen processing-defective mutant cell line T2. B27.A2B also fails to present an HLA-B27-restricted influenza virus peptide [nucleoprotein (383-391)] to cytotoxic T lymphocytes (CTLs). However, substitution of leucine, the predominant P2 anchor residue in A*0201-restricted peptides, for arginine, the P2 anchor in nucleoprotein-(383-391) and other B*2705-restricted peptides, restores recognition of B27.A2B by the same B*2705-restricted peptide-specific CTLs. These results demonstrate that a dominant polymorphic pocket in a class I molecule, through interaction with the anchor residue of an antigenic peptide, can distinguish among peptides differing by only a single amino acid and thus determine the allelic specificity of peptide presentation.

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PubMed1987

Alpha subunit of eukaryotic translational initiation factor-2 is a heat-shock protein.

The use of ultra high resolution giant two-dimensional gel electrophoresis has expanded the number of recognizable heat-shock proteins to 68 inductions in rat thymic lymphocytes, many of which are among the less abundant cellular proteins (Maytin, E. V., Colbert, R. A., and Young, D. A. (1985) J. Biol. Chem. 260, 2384-2392). Previous studies also show that cells receiving a prior heat shock recover more rapidly from the inhibition of protein synthesis induced by a second heat shock. In this report we use a monoclonal antibody to identify the alpha subunit of eukaryotic initiation factor-2 (eIF-2 alpha) as a heat-shock protein. Its relative rate of synthesis increases approximately 40% in the 2nd h and 5-fold in the 4th h of a continuous heat shock and is stimulated more dramatically, 15-fold, in the 3rd h of recovery from a 1-h heat shock. These results suggest that the induction of eIF-2 alpha in the heat-shock response may be important for restoring the cell's ability to initiate protein synthesis. In addition to identifying a function for one of the heat-shock proteins, our findings draw attention to the likelihood that other low-abundance heat-shock proteins may play critical roles in the heat-shock response.

Animals↗
PubMed1987

Detection of mRNAs coding for translationally regulated heat-shock proteins in non-heat-shocked thymic lymphocytes.

Heat shock induces 31 proteins in thymic lymphocytes in 1 h, 11 of which are not blocked by cordycepin, suggesting that their induction may be regulated at the level of translation (Maytin, E.V., Colbert, R.A., and Young, D.A. (1985) J. Biol. Chem. 260, 2384-2392). The possibility that mRNAs coding for these 11 cordycepin-insensitive heat-shock proteins would be found in non-heat-shocked thymus cells was investigated. Analysis of 1500 in vitro translation products separated by giant two-dimensional gel electrophoresis revealed that poly(A)+ RNA isolated from non-heat-shocked thymus cells coded for proteins corresponding to 10 of the 11 non-cordycepin-inhibitable heat-shock proteins. Comparison of the relative rates of synthesis of these 10 proteins in whole cells incubated at 37 and 42 degrees C, with their synthesis in vitro directed by poly(A)+ RNA isolated from cells incubated at 37 degrees C, suggests that mRNAs for 7 of them are present in sufficient amounts in non-heat-shocked cells to account for their increased synthesis during heat shock. These results indicate that part of the response of thymic lymphocytes to heat shock involves a rapid increase in the translation of a group of pre-existing mRNAs that are normally translated at very low rates or not at all.

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PubMed1986

Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs.

The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 [35S]methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.

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PubMed1986

Glucocorticoid-mediated induction of glucocortin: a rapid primary response common to major target tissues.

Glucocorticoids are known to rapidly induce four proteins in rat thymus cells in time to account for the earliest known metabolic hormone effects. We report here an additional protein, which we refer to as "glucocortin" (Mr = 17,000, pI = 4.7). It is of special interest since it is the only protein rapidly induced in all of the glucocorticoid target cells we have examined. We have characterized the kinetics of glucocortin mRNA induction in isolated thymus cells. Giant two-dimensional gel electrophoretic analysis of in vitro translation products reveals a 2-fold increase in the level of translatable mRNA within 15 min of dexamethasone addition, with maximal stimulation (approximately equal to 7-fold) by 45 min. Cycloheximide does not reduce the hormone-mediated increase in glucocortin mRNA, suggesting that the induction represents a primary response to glucocorticoids. This protein is induced by dexamethasone in a dose-dependent manner, with maximal induction at 10(-6) M and partial inductions at concentrations as low as 10(-10) M. It is strongly induced by cortisol at 10(-6) M, but it is not induced by estradiol or testosterone or by thyroid hormone, even at concentrations as high as 10(-6) M. Deoxycorticosterone has no effect at 10(-8) M but does generate a half-maximal effect at 10(-6) M, a finding consistent with its status as a partial glucocorticoid agonist. In summary, glucocortin appears to be a primary glucocorticoid-induced protein that represents the most rapid induction so far detected, and it appears to be the only one that may be common to all glucocorticoid target cells.

Animals↗
PubMed1986

Glucocorticoid-induced messenger ribonucleic acids in rat thymic lymphocytes: rapid primary effects specific for glucocorticoids.

We examined the mechanism underlying the rapid (15-120 min) glucocorticoid-mediated increase in the synthesis of proteins envisioned as mediators of the initial steroid effects in thymic lymphocytes. Analysis of about 1500 in vitro translation products on giant two-dimensional gels revealed rapid hormonal influences on only 7 mRNAs, 6 whose translation products have mol wt and pIs identical to those proteins whose synthesis has been found to be increased after the addition of glucocorticoids to thymus cells. The additional mRNA codes for glucocortin, whose induction in thymus as well as other target cells has been reported. mRNA inductions for proteins 1, 2, and 1N are detectable 15-30 min after the addition of dexamethasone to isolated thymus cells and continue to increase until 2 h, whereas the mRNAs for proteins 4 and 5 are not increased until after 1 h. The mRNA for protein 3 is elevated by 1 h, but attempts to quantitate the change at earlier times have failed because this protein focuses poorly. The parallel between these in vitro translation results and the increased synthesis of individual proteins seen in whole cells demonstrates that increased mRNA levels underlie the cellular changes. Other hormones, such as estradiol, testosterone, and T3, at 10(-6) M and deoxycorticosterone at 10(-8) M do not induce these messages. Partial inductions of 1 and 2, but not 1N, are seen with deoxycorticosterone at 10(-6) M, consistent with its classification as a partial glucocorticoid agonist. Cycloheximide does not block the rise in mRNA for the most rapid inductions, 1, 2, and 1N. The results indicate that dexamethasone rapidly and specifically induces 6 thymus cell mRNAs in addition to the one coding for glucocortin. The cycloheximide results suggest that at least 3 of these represent primary steroid hormone responses.

Animals↗
PubMed1985

The hepatic glucocorticoid domain: evidence for early and late hormone-mediated changes in the synthesis of individual protein gene products.

Studies were conducted to determine the extent of rapidly evolving effects of glucocorticoids on the transcriptional activity of individual hepatocyte genes through comparisons of the relative rates of synthesis of the more than 3000 protein gene products that are resolved in giant two-dimensional separatory gels. During the first 20 h in primary culture normal hepatocytes displayed substantial spontaneous changes in over 80 proteins. One effect of an added glucocorticoid, dexamethasone, was to retard or reverse the progression of roughly half of these. However, such long-term hormone treatment also caused 27 inductions and 26 repressions, many of which occurred in proteins that do not change spontaneously. Some of these coincide with the previously reported glucocorticoid domain of hepatoma cells. In contrast to such long-term changes, short-term (4 h) incubation with dexamethasone induced 10 proteins and repressed 6 others. Five of these early hormone inductions and all of the early repressions were maintained or enhanced by 20 h. However, the remaining five early glucocorticoid inductions appeared to be transient, since by 16-20 h the effects were either markedly reduced or absent. These results show the existence of an early glucocorticoid domain, qualitatively different from that seen at later times, which may be more representative of the primary steroid hormone responses.

Animals↗
PubMed1985

Early heat shock proteins in primary thymocytes. Evidence for transcriptional and translational regulation.

Primary isolates of thymic lymphocytes maintained in vitro provide a physiologically well-characterized system in which to study induction of proteins by heat shock; this response is agent-specific and separable from inductions by glucocorticoids or heavy metals (Maytin, E. V., and Young, D. A. J. Biol. Chem. 258, 12718-12722). Here we identify 68 heat shock protein inductions among more than 2,500 individual proteins separated on giant two-dimensional gels and further describe their time course of appearance, sensitivity to cordycepin (3'-deoxyadenosine), and reversibility during recovery. Thirty-one changes are detectable within 1 h. Among these early increases, 20 are inhibitable by cordycepin. However, 11 early changes are not affected by cordycepin; all represent proteins found in relatively low abundance. Five of these inductions are rapidly reversible during recovery from heat shock, in contrast to most other heat shock proteins whose synthesis is maintained or enhanced. One protein identified here appears to be increased by recovery per se. Overall, these results provide evidence for two separate classes of heat shock inductions in normal mammalian cells, i.e. a transcriptionally regulated group, not readily reversible during recovery, and a translationally regulated group in which several inductions rapidly revert to normal during recovery.

Animals↗
PubMed1984

Changes in the expression of hepatocyte protein gene-products associated with adaptation of cells to primary culture.

Expression of hepatocyte protein gene-products changes as cells adapt to the environment of tissue culture. We examined such changes, using "giant" two-dimensional (2-D) gel electrophoresis. Over 80 cellular and secreted proteins, about 3% of the 2500 we examined, were found to change spontaneously during the 20-h culture period. The magnitude of these changes was estimated to range from 1.5- to nearly 100-fold. If dexamethasone is included in the culture medium, the overall production of secreted proteins is maintained, but in addition it exerts major influences on the expression of individual protein gene-products. Ten of the spontaneous changes are enhanced and eight are unaffected, but most are substantially retarded (32) or even reversed (27). There are also large inductions or repressions in 12 proteins that do not change spontaneously in culture. We have tentatively identified tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, gamma-fibrinogen, and alpha 1-acid glycoprotein to be among those induced by dexamethasone. Evidently, many more changes occur as these cells adapt to tissue culture than was previously appreciated. These results emphasize the power of 2-D gel analysis in monitoring the protein phenotype of cells. We have also shown that glucocorticoids are important in maintaining the overall synthesis of secreted proteins and the protein phenotype of isolated hepatocytes in primary culture.

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