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J Gorski

Publications and source records attributed to J Gorski.

At least 127 records · Page 7Linked to original sources
PubMed1991

Transfer of polymorphic monoclonal antibody epitopes to the first and second domains of HLA-DR beta-chains by site-directed mutagenesis.

We used site-directed mutagenesis of HLA-DR beta-chains to localize the binding sites for two polymorphic DR-binding mAb to residues in the first and second external domains, respectively. Transfer of three first domain alpha-helical residues, G73, R74 and N77, normally present in DR3a and DRw52a, to a DR4 beta-chain was sufficient for recognition of this mutant DR molecule by a DR3-specific mAb, NDS 9. A polymorphism controlling the binding of a DR4-specific mAb, GS 359-13F10, was mapped to a tyrosine at position 96 of the DR4 beta-chain second domain by the construction fo a chimeric DR molecule containing a DR2-first domain and DR4-second domain. The mapping of these two polymorphic epitopes to specific positions on the DR beta-chain will allow further structural and functional analysis of the DR molecule.

Amino Acid Sequence↗
PubMed1991

Detection of 14 HLA-DQB1 alleles by oligotyping.

Fourteen alleles have been identified by analysis of the nucleotide sequences encoding the HLA-DQB1 chain. This study describes the detection of these 14 alleles by selective gene amplification and sequence-specific hybridization with nonradioactive oligonucleotide probes. These techniques were employed to create an oligotyping system with two levels of resolution that provide versatility and the capacity for comprehensive detection of all polymorphic sequences. An initial low-resolution assay was employed to detect four major groups of alleles that are associated with the DQw1-w4 serological specificities. A further high-resolution assay was then employed to differentiate 14 individual DQB1 alleles. Appropriate control measures were also included to detect carry-over and to confirm hybridization specificity. This system was used to analyze the allele frequencies of DQB1-0301, -0302, and -0303 in 115 DQB1-03** Caucasian blood donors. Allele frequencies of oligotypes DQB1-05** and DQB1-06** were analyzed in 112 DQB1-01** Caucasian blood donors. This system was also utilized to identify oligotypes designated DQB1-0501 through DQB1-0503 and DQB1-0601 through DQB1-0605 in 35 Tenth International Histocompatibility Workshop DQw1 cell lines to examine the correlation with serological and cellular specificities. In one of these cell lines, an unexpected linkage was discovered between the DRB1 and DQB1 loci, suggesting a recombination event. Oligotyping is a precise and accurate method for directly defining polymorphic sequences and it promises to have a major impact on the future direction of HLA typing.

Alleles↗
PubMed1991

Inhibitory effects of serum and stimulatory effects of estrogen on prolactin mRNA levels in GH3 rat pituitary tumor cells.

We investigated the effects of serum and estrogen on prolactin (PRL) mRNA accumulation in GH3 cells under different cell culture conditions. Hybridization analysis of GH3 cellular RNA indicated that PRL mRNA levels decreased more than 20-fold in cells cultured for 1 week in medium containing dextran-charcoal-treated serum (stripped serum). No effects on actin mRNA levels were observed under these conditions. Furthermore, this inhibition of PRL mRNA accumulation depended on the concentration of stripped serum in the medium. Although incubation in stripped-serum medium inhibited cell growth, these culture conditions did not appear to irreversibly affect the GH3 cell population. These data indicate that a potent inhibitor of PRL mRNA accumulation is present in stripped serum. GH3 cells grown in stripped-serum medium were shown to be responsive to estrogen. Treatment of these cells with 10(-9) M estradiol resulted in a 6.6-fold stimulation of PRL gene expression. However, estrogen had no effect on cell growth under these conditions, suggesting that estrogen stimulates PRL gene expression and cell proliferation by independent mechanisms.

Animals↗
PubMed1991

Growth and cell cycle regulation of mRNA levels in GH3 cells.

In the rat anterior pituitary gland, estrogen increases both prolactin (PRL) mRNA levels and stimulates the proliferation of PRL-producing cells. The temporal sequence of these events suggests that PRL gene expression may be coordinated with cell proliferation. We investigated the relationship between cell cycle progression and the accumulation of the PRL mRNA, as well as several other mRNAs, in the rat pituitary tumor GH3 cell line. Serum-deprived cells progressed from G0 to S phase in 20-24 h following serum stimulation. During this time, beta-actin mRNA levels increased 7-fold in 5 h, then returned to basal levels prior to the beginning of S phase. Histone H1 mRNA levels increased approximately 3-fold as cells entered S phase. These data are consistent with the cell cycle-dependent regulation of beta-actin and histone H1 gene expression reported for other cell types. Glucocorticoid receptor mRNA levels were barely detectable in serum-deprived cells but rapidly increased 3- to 5-fold following serum stimulation. This increase resulted in glucocorticoid receptor mRNA levels that were equivalent to those seen in cells maintained in serum-containing medium, suggesting that serum factors regulate glucocorticoid receptor gene expression. In contrast to these changes in gene expression, the levels of PRL and growth hormone (GH) mRNAs gradually increased 2-fold while the cells progressed through G1 phase. Similarly, in cells synchronized to progress through S and G2 phases following aphidicolin treatment, histone H1 gene expression showed a specific increase in S phase cells, whereas PRL and GH mRNA levels changed little with cell cycle progression. These results indicate that the levels of PRL and GH mRNAs are not regulated in a cell cycle-dependent manner. When changes in estrogen responsiveness were determined during the cell cycle, we found that estradiol treatment was capable of increasing PRL mRNA accumulation independent of cell cycle progression and cell cycle distribution in synchronized GH3 cells. These results support the hypothesis that the hormonal regulation of PRL gene expression is not significantly affected by cell growth.

Actins↗
PubMed1991

The role of ligand in estrogen receptor regulation of gene expression.

We believe that steroid binding is not required for receptor binding to DNA, but instead induces a conformational change in the receptor domains involved in the protein-protein interactions proposed above. Data from Hansen and Gorski (1986), and more recent studies (M. Fritsch and J. Gorski, unpublished results) strongly suggest that the steroid binding domain when bound to estrogens undergoes a dramatic change in conformation characterized by a loss of hydrophobic surface. This marked change in the steroid binding domain probably affects the so-called dimerization region located in this domain and thus the interaction of receptor with nuclear proteins in vivo. In our working model, ER is bound to specific DNA sequences or response elements of a variety of genes with or without estrogen. Ligand binding induces conformational changes in the steroid binding and perhaps other domains of the receptor that in turn change receptor interaction with the transcriptional machinery. The nature of this change is not at all clear at present, and the possibility of enzymatic modification of receptor or associated transcription factors should not be excluded. Whatever the mechanism of receptor action on transcription, we expect it kinetically will be closely related to the occupancy of the receptor with estrogen. Finally, any model of ER interactions with target genes also needs to account for the drastic ligand effect on the extractability of all ER from the nucleus.

Cell Nucleus↗
PubMed1991

Differential binding of GH3 and pituitary protein extracts to the estrogen-dependent enhancer of the rat prolactin gene.

Extracts of two prolactin (PRL) producing cells (GH3 cells and rat pituitary tumors) were compared for their ability to bind to a PRL DNA fragment that contained sequences important for estrogen enhancement of PRL transcription. The ability of estradiol (E2) to alter concentrations of existing PRL enhancer binding proteins or to promote the appearance of additional binding proteins was also examined. When diethylstilbestrol (DES)-induced pituitary tumor extracts were examined for protein binding to the same DNA fragment, a different banding pattern was observed than the banding pattern seen using protein extracts from GH3 cells, suggesting that PRL DNA binding proteins differ between GH3 cells and rat pituitary tumors. The differences in PRL DNA binding may result from the fact that approximately 20% of GH3 cells are lactotrophs, while 80% of DES-induced pituitary tumor cells are lactotrophs. In addition, no change in the relative concentrations of proteins binding to the upstream regulatory element was observed following treatment of GH3 cells with E2.

Animals↗
PubMed1991

Nucleotide sequence of the human autoantibody 2E7 specific for the platelet integrin IIb heavy chain.

The platelet glycoprotein (GP) IIb-IIIa complex figures prominently as an immunogen in autoimmune (idiopathic) thrombocytopenic purpura (ITP). 2E7 is a human monoclonal IgM autoantibody, derived from splenocytes of a patient with ITP, that recognizes a specific octapeptide amino acid sequence, Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser, on the heavy chain of GPIIb. This represents the first precise identification of an epitope on GPIIb-IIIa recognized by a human antibody. In this study, we have isolated total mRNA from 2E7, synthesized the corresponding cDNA using reverse transcriptase, and amplified the immunoglobulin mu and kappa chain cDNA by the Taq 1 polymerase chain reaction (PCR) using specific primers. The 2E7 mu chain variable region is encoded by a VH3 gene segment that is 98% homologous to the germline gene VH1.9III, a D-gene that is not homologous to any of the germline D-genes reported to date, and a JH6 gene segment that is essentially germline. The heavy-chain sequence, save for the unique D-gene, is similar to that of a number of human autoantibodies. The 2E7 kappa variable region is encoded by a Vk1 gene segment linked to a Jk1 gene segment. The Vk1 sequence of 2E7, with the exception of one nucleotide, is identical to that of autoantibody HF2-1/17, a prototype of SLE-associated anti-DNA autoantibodies bearing the 16/6 idiotype. The single base substitution results in a relatively conservative exchange of Asp for Glu at position 70 of the protein sequence. Despite this near identity in sequence, 2E7 does not bind to either single-stranded or double-stranded DNA. From these results, we conclude that specificity of 2E7 is likely to reside in either or both the D-JH region (CDR3) of the mu chain and the Jk region (CDR3) of the kappa chain. In addition to the identification of a novel D-gene, we also provide evidence that the 2E7 VHIII gene is probably a prototype of a VHIII subfamily, that the germline Vk1 gene shared by 2E7 and autoantibodies of the 16/6 idiotype probably represents a separate Vk family, and that this Vk gene cannot itself attribute specificity for DNA.

Amino Acid Sequence↗
PubMed1991

Immunodetection of estrogen receptors in fetal and neonatal female mouse reproductive tracts.

An immunocytochemical assay for estrogen receptor (ER) was used to study the distribution of receptor in fetal and immature female mouse reproductive tracts. Immunoblots confirmed that a single band, the size of the ER, immunostained in extracts from day 15 and 17 fetal reproductive tracts. Staining was observed over nuclei of epithelial cells of the Mullerian duct and over nuclei of cells of the developing connective tissue (mesenchymal cells) of the reproductive tract on fetal day 15. By day 17 when a primitive uterus could be distinguished, ERs were detected in nuclei of mesenchymal cells, but in only a small portion of epithelial cells. A different pattern of immunocytochemical staining was observed in uteri from animals killed on the day of birth; cells of the connective tissue contained ER, but the epithelial cells did not. By 4 or 6 days after birth, more nuclei in the connective tissue stained for ER with a greater intensity compared to nuclear staining in epithelial cells. ERs were detectable in nuclei of both uterine epithelial cells and connective tissue cells on days 10 and 19 after birth.

Animals↗
PubMed1991

Body fat accretion: a rat model.

A rat model for studying causes of body fat accretion was developed. Employing this model, it was possible to produce a 108 g difference in carcass fat content between two groups of rats simply by selecting the seven fattest and the seven leanest animals from a group of 100. The results of this study provide evidence that the gastrointestinal tract was not a site of caloric wastage, that total energy excreted in feces and urine was directly proportional to caloric intake, that differences in brown adipose tissue thermogenesis were not responsible for the twofold difference in adiposity between the lean and fat rats, and that a family of hepatic lipogenic enzymes was not up-regulated even though the habitual consumption of a diet rich in carbohydrate was greater in the fat vs lean rats. The fat rats also had a greater lean body mass. Therefore, the additional calories consumed by the fat rats could have contributed to their greater adiposity and/or could have been channeled to sustain more active tissue. Finally, evidence was presented to show that the experimental selection process is an important consideration when determining the relationship between caloric intake and body fat content.

Adipose Tissue, Brown↗
PubMed1990

Relationship of polymorphisms near the rat prolactin, N-ras, and retinoblastoma genes with susceptibility to estrogen-induced pituitary tumors.

Chronic treatment of rats with the synthetic estrogen diethylstilbestrol is known to induce the formation of pituitary tumors, and such tumor induction is highly dependent on the strain of rat used. We examined three previously discovered restriction fragment length polymorphisms in rats to determine whether these correlated with susceptibility to tumor formation. The results indicate that the presence of particular alleles of the polymorphic N-ras and retinoblastoma (Rb) genes does not correlate with tumor susceptibility. A polymorphism upstream of the rat prolactin (Prl) gene is due to the presence or absence of an Alu-like sequence. Results of this study indicate that animals bearing the allele lacking this Alu-like insertion are more likely to develop larger pituitary tumors in response to diethylstilbestrol than are animals in which the Prl allele contains the insertion. In addition, we show that the N-ras, Rb, and Prl genes are dispersed in the rat genome and that the polymorphic alleles of the Prl genes are segregating as classical Mendelian alleles. These results suggest that the difference in the Prl gene itself or in some closely linked gene is related to tumor resistance or susceptibility.

Animals↗
PubMed1990

Mapping of distinct serologic and T cell recognition epitopes on an HLA-DR beta-chain.

A new DR beta-chain allele is defined that is identical to the previously described DR6b molecule except for the first hyperpolymorphic region, where the new allele displays the same polymorphisms found on DR8 and DR12 genes. Two distinct epitopes have been mapped on this new allele. The polymorphism in common with DRw8 and DRw12 is recognized by mAb GS313-9D11. However, alloreactive T cell clones specific for DR6b cells (Dw9) recognize this allele, whereas Dw8-specific T cell clones do not. The mAb determinant maps to the first beta-sheet and probably involves a polymorphic residue lying outside the helix. The binding of mAb 9D11 to this region does not interfere with TCR binding. Alloreactive T cell recognition is associated with polymorphisms located predominantly on the alpha-helical portion of the molecule.

Alleles↗
PubMed1990

Estrogen receptor binding to a DNA response element in vitro is not dependent upon estradiol.

Gel shift assays were employed to distinguish between the contribution of 17 beta-estradiol (E2) and a short heating step to the ability of the rat uterine cytosolic estrogen receptor (ER) to bind to the estrogen response element (ERE) from the vitellogenin A2 gene (vitERE). Despite the popularity of models in which the ER is a ligand-activated DNA-binding protein, these studies find that estrogen does not significantly contribute to receptor-DNA complex formation. An avidin-biotin complex with DNA (ABCD) assay was utilized to obtain quantitative measurement of the affinities of the ER for the vitERE and a mutant sequence. Scatchard analysis gave a dissociation constant of 390 +/- 40 pM for the E2-occupied, heated ER to the vitERE. The data fit a one-site model and evidence for cooperatively was not observed. A dissociation constant of 450 +/- 170 pM was obtained for the unoccupied, heated ER, leading to the conclusion that estrogen was not necessary for specific binding to DNA. The percentage of ER capable of binding vitERE varied with each cytosol preparation, ranging from 60 to 100% and estrogen did not appear to affect this variation. Competition against the vitERE with a 2-bp mutant sequence showed a 250-fold lower relative binding affinity of the receptor for the mutant over the vitERE sequence. This ability of the ER to discriminate between target and nonspecific DNA sequences was also not dependent on the presence of estrogen.

Animals↗
PubMed1990

PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily.

An antibody to a platelet integral membrane glycoprotein was found to cross-react with the previously identified CD31 myelomonocytic differentiation antigen and with hec7, an endothelial cell protein that is enriched at intercellular junctions. This antibody identified a complementary DNA clone from an endothelial cell library. The 130-kilodalton translated sequence contained six extracellular immunoglobulin (Ig)-like domains and was most similar to the cell adhesion molecule (CAM) subgroup of the Ig superfamily. This is the only known member of the CAM family on platelets. Its cell surface distribution suggests participation in cellular recognition events.

Amino Acid Sequence↗
PubMed1990

Transcription analysis of class II human leukocyte antigen genes from normal and immunodeficient B lymphocytes, using polymerase chain reaction.

The RNA transcript levels of all human leukocyte antigen class II loci were determined from class II congenital immunodeficient B cells by polymerase chain reaction amplification of cDNA. No mRNA was observed under conditions in which 0.01% normal levels could be visualized. Pre-mRNA could be amplified from normal B cells but not from immunodeficient B cells, indicating a transcription defect.

B-Lymphocytes↗
PubMed1990

Influence of ATP turnover and metabolite changes on IMP formation and glycolysis in rat skeletal muscle.

Deamination of AMP to inosine monophosphate (IMP) and NH3 is thought to be regulated by the observed increases in ADP, AMP, and H+. We have examined this hypothesis by comparing the rate of IMP accumulation in contracting and noncontracting rat skeletal muscle. The rate of IMP formation was high during ischemic contraction, and consistent with previous studies, formation of IMP was associated with high levels of muscle lactate, depletion of phosphocreatine (PCr), and increased levels of ADP and AMP. When the contraction period was followed by 5-min anoxic recovery, the metabolic changes were maintained, but no further IMP or lactate was formed. During long-term (2-4 h) anoxia, the rate of IMP formation was less than 4% of that during contraction, despite similar changes in PCr, lactate, ADP, and AMP. It is concluded that the observed changes in the intracellular chemical environment are not sufficient to explain the high rate of IMP formation during contraction but that a combination of metabolic stress and a high ATP turnover rate is required. It is suggested that a high ATP turnover rate during conditions of metabolic stress results in transient increases in ADP and AMP at the site of ATP hydrolysis and that these activate AMP deaminase and glycolysis. An alternative hypothesis is that these processes are regulated by the increase in cytosolic Ca2+ in a contracting muscle.

Adenosine Diphosphate↗
PubMed1990

Estrogen binding is a noncooperative process in primary rat uterine cells.

Estrogen binding in a primary rat uterine cell culture system did not exhibit positive cooperativity as judged by Hill coefficients. Under defined culture conditions, total specific [3H]estradiol (E2) binding in intact cells attained apparent equilibrium within 3 h after E2 had been varied from 5 to 250 pM. Thereafter, at each concentration of E2 studied, estrogen receptor (ER) levels were constant for up to 6 additional h. We have characterized a single high affinity E2 binding site with a dissociation constant (Kd alpha) of 0.01-0.05 nM E2. The binding capacity of the alpha-site is approximately 5-8 fmol/micrograms DNA, corresponding to approximately 20,000 receptors per cell. In extensive studies, we have seen no evidence for positive cooperative E2 binding. The Hill coefficient was never significantly (95% confidence level) greater than 1. Data are also presented which indicate that [3H]E2 exogenously added to the extracellular medium is in simple equilibrium with the intracellular compartment and the ER. When saturation analyses were conducted using medium that contained BSA, increased concentration of serum protein increased the apparent Kd of E2 for the receptor. No effect was observed on either the shapes of the binding curves or on the calculated Hill coefficients. Furthermore, dose correlations between E2 binding and stimulated response(s) indicated that the ER in these cells was biologically active, which was a major advantage of this culture system over freshly dispersed cells or cytosolic extracts.

Animals↗