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Biomedical subjects

J Gorski

Publications and source records attributed to J Gorski.

At least 19 recordsLinked to original sources
PubMed1992

A molecular model of RGD ligands. Antibody D gene segments that direct specificity for the integrin alpha IIb beta 3.

Following an ill-defined activation event, the Arg-Gly-Asp (RGD) recognition site of the platelet integrin, glycoprotein IIb-IIIa (alpha IIb beta 3), can bind to fluid-phase, RGD-containing protein ligands, such as fibrinogen, or to the murine monoclonal IgM, PAC-1, which contains the sequence Arg-Tyr-Asp (RYD) within the third complementarity-determining region of its heavy chain (H3). PAC-1 has thus become a widely exploited marker of platelet alpha IIb beta 3 activation. In this report, we compare PAC-1 with two murine IgG, OP-G2 (IgG1 kappa) and LJ-CP3 (IgG1 kappa), that also contain the sequence RYD in H3 but bind to alpha IIb beta 3 without prior activation. Each antibody can inhibit the binding of the other two to intact platelets or to purified IIb-IIIa, the binding of each antibody is completely inhibited by peptides containing RGD, and H3 of each antibody uses the germline D-gene DSP 2.10 (CTATAGGTACGAC) which includes the sequence RYD. Two other murine IgG, HP20 and PCG1-1, cloned and sequenced by other laboratories, also utilize the DSP 2.10 sequence, but neither antibody binds to alpha IIb beta 3. From a comparison of the H3 sequences of these antibodies, we have developed a molecular model of the H3 loop region which can explain these differences in specificity. This model predicts that both the ability to bind to alpha IIb beta 3 and the activation dependence of that binding are a function of the orientation and, therefore, accessibility of the RYD sequence. This model and refinements thereof can be exploited to study the molecular basis for specificity and affinity of RGD-containing ligands for integrins.

Amino Acid Sequence↗
PubMed1992

A ligand-induced conformational change in the estrogen receptor is localized in the steroid binding domain.

Upon binding estrogen, the estrogen receptor (ER) is proposed to undergo some form of conformational transition leading to increased transcription from estrogen-responsive genes. In vitro methods used to study the transition often do not separate heat-induced effects on the ER from estrogen-induced effects. The technique of affinity partitioning with PEG-palmitate was used to study the change in the hydrophobic surface properties of the ER upon binding ligand with and without in vitro heating. Upon binding estradiol (E2), the full-length rat uterine cytosolic ER undergoes a dramatic decrease in surface hydrophobicity. The binding of the anti-estrogen 4-hydroxytamoxifen (4-OHT) results in a similar decrease in surface hydrophobicity. These effects are independent of any conformational changes induced by heating the ER to 30 degrees C for 45 min. The use of the human ER steroid binding domain overproduced in Escherichia coli (ER-C) and the trypsin-generated steroid binding domain from rat uterine cytosolic ER demonstrates that the decrease in surface hydrophobicity upon binding E2 or 4-OHT is localized to the steroid binding domain. Gel filtration analysis indicates that the change in surface hydrophobicity upon binding ligand is an inherent property of the steroid binding domain and not due to a ligand-induced change in the oligomeric state of the receptor. The decrease in surface hydrophobicity of the steroid binding domain of the ER upon binding E2 or 4-OHT represents an early and possibly a necessary event in estrogen action and may be important for "tight" binding of the ER in the nucleus.

Animals↗
PubMed1992

DNA allosterically modulates the steroid binding domain of the estrogen receptor.

The ability of DNA to allosterically alter the conformation of the estrogen receptor's (ER) steroid binding domain was investigated. Using dissociation kinetics we observed that when DNA was bound to the DNA binding domain of the rat uterine ER the rate of estrogen dissociation from the steroid binding domain increased almost 2-fold. This change in the rate of estrogen dissociation depended on the concentration of DNA used and correlated with the thermodynamic binding affinities (Kd) of the ER for two different DNA sequences. We were unable to detect a DNA-induced change in the trypsin cleavage pattern of the amino terminal end of the ER. Using a whole cell dissociation kinetic assay with MCF-7 breast cancer cells we observed a 7-fold slower rate of estrogen dissociation from the ER within the cell than from the ER in vitro. This suggests that additional factors, other than DNA binding, may modify the steroid binding domain within the cell. We conclude that DNA can allosterically modulate the structure of the steroid binding domain of the ER, and we hypothesize that this conformational change may be necessary for the full transcriptional activity of the ER.

Allosteric Regulation↗
PubMed1992

The predictive value of HLA-DR oligotyping for MLC responses.

Comparison of HLA proteins between a patient and potential unrelated marrow donors is difficult because many similar, but not identical, HLA proteins are expressed in the human population. A reliable and practical method to detect these subtle differences is provided by oligotyping, a new technique that identifies polymorphic sequences in the genes encoding the HLA proteins. Oligotyping was used to compare polymorphic HLA-DR sequences in 286 pairs of samples from patients and potential unrelated donors who were serologically matched for HLA-DR specificities. Oligotyping detected HLA-DR differences in 53% of these pairs and all mismatched pairs were reactive in primary mixed lymphocyte cultures. Where HLA-DR disparity was not detected by oligotyping, 37% of the pairs were nonreactive in MLC. The remaining 63% often contained an allele associated with the HLA-DRw11 serological specificity. In the absence of HLA-DRB1*11, oligotyping was predictive of MLC reactivity for samples with HLA-DR2, -DR4, and DRw52. In clinical settings, the ability to predict MLC reactivity on the basis of precise HLA typing provides an alternative to MLC. Further, the relationship between specific polymorphic sequences and reactivity in MLC may lead to more fundamental insights into the mechanisms involved in alloreactive responses.

Amino Acid Sequence↗
PubMed1992

Muscle triglyceride metabolism during exercise.

Skeletal muscle cell contains a considerable amount of triglycerides. The amount stored depends on the animal species as well as on muscle fiber composition. It is well documented that triglycerides in the fast-twitch red muscle and to a lesser extent in the slow-twitch muscle, but not those in the fast-twitch white muscle, are mobilized during prolonged exercise. Yet, little is known about the regulation of the metabolism of muscle triglycerides either at rest or during exercise. This is well reflected by the fact that an enzyme responsible for the hydrolysis of muscle triglycerides has not been identified. Mobilization of muscle triglycerides during exercise seems to be under both adrenergic and noradrenergic control. Accumulation of lactic acid and reduction in muscle pH are likely to be strong inhibitors of muscle triglyceride lipolysis. Reduction of carbohydrate availability accelerates mobilization of muscle triglycerides during exercise. The relationship between the plasma free fatty acids and muscle triglyceride metabolism seems to be complex. It has been proposed that most free fatty acids entering the muscle cell is esterified before being oxidized, but this is arguable for contracting skeletal muscles. It is suggested that most free fatty acids entering contracting high oxidative myocytes are transported directly to the mitochondria. A much lesser portion is likely esterified. It is proposed that triglycerides stored in the contracting muscle cell are mobilized when the delivery of the blood-borne-free fatty acids to the mitochondria is insufficient.

Animals↗
PubMed1992

Progress towards the eradication of dracunculiasis.

In 1980 the World Health Organization instituted the Decade of Safe Water and Sanitation. As part of that initiative new impetus was given to programmes which dealt with the eradication of dracunculiasis. This paper, based on the review of several studies, discusses the nature of the disease, its impact upon the affected population, the possible solutions, and the progress accomplished thus far in several African nations. The use of health education is advocated as a major contributor towards the eradication of dracunculiasis.

Communicable Disease Control↗
PubMed1992

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

Immunodetection methods were used to detect estrogen receptors (ER) in male reproductive tracts on fetal days 13, 15, and 17 and on the day of birth. Immunocytochemistry revealed that most of the cells of the gonad and associated Wolffian duct stained for ER on fetal day 13. During the next 6 days, ER distribution changed, and by the day of birth, ERs were observed only in epithelial cells of the epididymis (derived from the Wolffian duct) and in a portion of cells from the testis. Immunoblots confirmed that a band the size of the ER stained in reproductive tracts for all ages studied. Similar to the fetal female, ERs are present throughout the early development of the fetal male reproductive tract. However, in contrast to the female, ERs appear to decrease in the male fetal reproductive tract at the time of birth.

Animals↗
PubMed1992

Overproduction of full-length and truncated human estrogen receptors in Escherichia coli.

The full-length human estrogen receptor (hER) as well as two overlapping peptides of hER were overproduced in Escherichia coli JM109 cells, using the inducible pIC vector system. The N-terminal receptor peptide contains the DNA-binding domain as well as the hinge region, whereas the C-terminal peptide contains the same hinge region and the hormone-binding domain. Typically, 1-6 mg of estrogen receptor (ER) peptides can be recovered from 1 L E. coli cell cultures. The majority of the overexpressed proteins are found in inclusion bodies, which allow the isolation of ER peptides in high yields and of 50-80% purity. Induction for short time periods at 10 microM inducer yielded up to 50% of the ER peptides in soluble form with full biological activity. Both the intact receptor and the C-terminal fragment specifically bound estrogens and antiestrogens, whereas ER peptides that contained the DNA-binding domain were retained on a DNA-agarose resin.

Cloning, Molecular↗
PubMed1992

Polyclonal antibodies from rabbits and chickens against the estrogen receptor and related peptides. Use in the affinity isolation of estrogen receptors and the retrieval of chromatin fragments associating with estrogen receptors.

Polyclonal antibodies from chickens and rabbits have been prepared against polypeptides representing two regions of the human estrogen receptor (hER). The estrogen receptor (ER) peptides used as antigens were overproduced in Escherichia coli. When indicated, the antibodies were affinity purified using resins to which the antigens contained in bacterial inclusion bodies had been coupled in high yield to epoxy-activated agarose. The antibodies recognize denatured human, bovine, rat, and rabbit ER in immunoblotting experiments. Immuno-precipitation of native ER protein was readily accomplished using rabbit antisera and immobilized protein A. The chicken antibodies, available in larger quantities, were also useful for immunoisolation after coupling to agarose. With the use of these reagents, the selective retrieval of chromatin fragments from MCF-7 cells that interact with ER has been achieved.

Animals↗
PubMed1991

Marked effects of salt on estrogen receptor binding to DNA: biologically relevant discrimination between DNA sequences.

Avidin-biotin complexed with DNA (ABCD) assays were employed to determine the binding affinity of estrogen receptor (ER) to DNA under various salt conditions. Type and concentration of salt in the reaction buffer dramatically affected the ability of the ER to discriminate between DNA sequences. Under appropriate salt conditions, ER was able to bind to the estrogen response element from the Xenopus vitellogenin A2 gene with at least 3 orders of magnitude greater affinity than a two base pair mutant sequence, and 5 orders of magnitude greater affinity than plasmid DNA. In these studies, the best discrimination was observed under conditions of salt type and concentration that more closely approximated intracellular conditions, i.e., 100-150 mM potassium salts. Analysis of the binding affinities for ER to all three types of DNA over a range of KCl concentrations indicated that the ionic interactions upon ER binding were the same for the three DNA molecules tested. Therefore, the additional stability of ER binding to target DNA sequences was contributed by nonionic interactions.

Animals↗
PubMed1991

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.

Amino Acid Sequence↗
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↗