Biomedical subjects

J Gorski

Publications and source records attributed to J Gorski.

At least 37 records · Page 2Linked to original sources

PubMed1999

Simultaneous measurement of multiple mRNAs with a single control by quantitative competitive reverse transcriptase-polymerase chain reaction: glucose transporters Glut1 and Glut4.

Theoretical considerations for extending the application of quantitative competitive polymerase chain reaction (qc-PCR) to include the simultaneous measurement of multiple mRNAs, specifically the mammalian glucose transporters Glut1 and Glut4, are presented with experimental data in which the accuracy and flexibility of the system are examined. This method reliably measures changes in the initial concentration for each of three target DNA sequences. The reaction is not acutely sensitive to variations in either the primer sites or internal sequence, and although the initial concentrations of the target DNAs did affect the relative amplification efficiencies, the effect was limited and did not prohibit quantification. This PCR system was able to reliably detect differences as little as 50% in the initial concentration of the Glut1 target DNA sequence. Therefore, with the appropriate controls, PCR can be extended to include the simultaneous quantification of more than one target DNA with a single internal control.

Animals↗

PubMed1999

Use of antagonist peptides to inhibit in vitro T cell responses to Par j1, the major allergen of Parietaria judaica pollen.

Antigenic peptides with substituted side chains inhibit immune responses to a number of recall Ags from infectious agents in vitro. Here we show that the same strategy can be applied to peptides derived from a pollen protein, the major allergen of Parietaria judaica(Par j1), a plant responsible for most allergenic sensitization in the southern Mediterranean area. Three T cell lines responding to Par j1 protein were used to identify a stimulatory peptide. Two different monosubstituted altered peptide ligands (APL) were identified that bound to the HLA-DR of the responders, did not stimulate the T cell lines on their own, and decreased the response to subsaturating amounts of the unmodified stimulatory peptide. Most important, these APL were able to inhibit the response of these cell lines to intact Par j1 protein. A third monosubstituted peptide bound to the HLA-DR but did not show inhibitory activity. The two APL had a lower affinity than the unsubstituted peptide for the HLA-DR. The last two observations make MHC blockade an unlikely explanation for the observed effect. These results indicate the action of a specific peptide-mediated antagonism that may be useful in controlling the T cell component of an allergic response.

Allergens↗

PubMed1999

"Rate-drop response" cardiac pacing for vasovagal syncope. Rate-Drop Response Investigators Group.

Recent reports suggest that cardiac pacing incorporating a rate-drop response algorithm is associated with a reduction in the frequency of syncopal episodes in patients with apparent cardioinhibitory vasovagal syncope. The detection portion of the algorithm employs a programmable heart rate change-time duration "window" to both identify abrupt cardiac slowing suggestive of an imminent vasovagal event and trigger "high rate" pacing. The purpose of this study was to develop recommendations for programming the rate-drop response algorithm. Pacemaker programming, symptom status, and drug therapy were assessed retrospectively in 24 patients with recurrent vasovagal syncope of sufficient severity to warrant consideration of pacemaker treatment. In the 53 +/- 19 months prior to pacing, patients had experienced an approximate syncope burden of 1.2 events/month. During follow-up of 192 +/- 160 days, syncope recurred in 4 patients (approximate syncope burden, 0.3 events/month, p < 0.05 vs. pre-pacing), and pre-syncope in 5 patients. In these patients, rate-drop response parameters were initially set based on electrocardiographic and/or tilt-table recordings, and were re-programmed at least once in 14 (58%) individuals. A 20 beat/min window height (top rate minus bottom rate), a window width of 10 beats (61% of patients), and 2 or 3 confirmation beats (79% of patients) appeared to be appropriate in most patients. Treatment intervention rate was set to > 100 beats/min in 89% of patients, with a duration of 1 to 2 min in 79%. In conclusion, a narrow range of rate-drop response parameter settings appeared to be effective for most individuals in this group of highly symptomatic patients.

Adolescent↗

PubMed1999

Regulation of glucose transporters by estradiol in the immature rat uterus.

A series of metabolic changes within the immature rat uterus begins minutes after the administration of microgram quantities of estradiol (E2). One of the earliest effects that has been measured is an increase in the rate at which the uterus takes up glucose. To characterize the effect of E2 on glucose transport stimulation, whole protein preparations were examined for the presence of mammalian glucose transport proteins Glut1 through Glut5. E2-stimulated changes in the steady state levels of messenger RNA (mRNA) and protein were measured for Glut1 and Glut4 by quantitative competitive RT-PCR and Western blots. Both Glut1 mRNA and protein increased approximately 3- to 4-fold within 4-8 h. This increase in Glut1 mRNA and protein agrees with the maximal stimulation of the glucose transport rate that was observed. No translocation of either Glut1 or Glut4 was observed 2 h after E2 injection, indicating that translocation is not the mechanism responsible for the initial E2-stimulated increase in glucose transport observed in immature rat uterus. These data support the conclusion that the prolonged increase in glucose transport rate is due to either the transcriptional activation of Glut1 and/or the increased Glut1 mRNA half-life.

Animals↗

PubMed1999

Evaluating the effects of endocrine disruptors on endocrine function during development.

The major concerns with endocrine disruptors in the environment are based mostly on effects that have been observed on the developing embryo and fetus. The focus of the present manuscript is on disruption of three hormonal systems: estrogens, androgens, and thyroid hormones. These three hormonal systems have been well characterized with regard to their roles in normal development, and their actions during development are known to be perturbed by endocrine-disrupting chemicals. During development, organs are especially sensitive to low concentrations of the sex steroids and thyroid hormones. Changes induced by exposure to these hormones during development are often irreversible, in contrast with the reversible changes induced by transient hormone exposure in the adult. Although it is known that there are differences in embryonic/fetal/neonatal versus adult endocrine responses, minimal experimental information is available to aid in characterizing the risk of endocrine disruptors with regard to a number of issues. Issues discussed here include the hypothesis of greater sensitivity of embryos/fetuses to endocrine disruptors, irreversible consequences of exposure before maturation of homeostatic systems and during periods of genetic imprinting, and quantitative information related to the shape of the dose-response curve for specific developmental phenomena.

Adult↗

PubMed1998

Estrogen receptor alpha requires no accessory factors for high-affinity binding to a consensus response element.

Estrogen receptor (ER) alpha is commonly thought to bind to a consensus estrogen response element (ERE) as a homodimer, but previous experiments have not ruled out the presence of other proteins in the ERalpha/ERE complex. To characterize this interaction in more detail, we overexpressed mouse (m) ERalpha in a baculovirus system, using the selective advantage of the apoptosis inhibitor p35. Recombinant mERalpha possesses the predicted molecular weight and binds 17beta-estradiol and an oligonucleotide containing a consensus vitellogenin ERE with high affinity. Over a wide concentration range of mERalpha protein (0.1-50 nM), only one complex was detected between mERalpha and vitellogenin ERE in gel shift assays. The ratio of E2:vitellogenin ERE bound by mERalpha was close to 2:1, and each complex contained only one ERE. The molecular weight of the complex was determined to be 160 000, very close to that predicted for two mERalpha proteins and one ERE oligonucleotide, therefore providing strong evidence that no other proteins were present. Recombinant mERalpha was purified such that it was the only protein observable by silver stain. Purified mERalpha and mERalpha in a nuclear extract behaved identically in Ferguson analysis, providing more evidence that only mERalpha was binding to the ERE. Purified mERalpha bound vitellogenin ERE with high affinity (Kd = 0. 92 +/- 0.20 nM), indicating that no other proteins are necessary for high-affinity mERalpha interaction with a consensus ERE. To determine whether ERalpha in an estrogen-responsive mammalian tissue behaves the same as the overexpressed mERalpha, we tested rat uterine cytosol by Ferguson analysis. ERalpha in rat uterine cytosol behaved identically to overexpressed mERalpha, suggesting that ERalpha in the uterine extract also binds to DNA predominantly as a homodimer with no additional proteins.

Animals↗

PubMed1998

Estrogen-induced rat pituitary tumor is associated with loss of retinoblastoma susceptibility gene product.

Chronic treatment of rats with the estrogens 17beta-estradiol or diethylstilbestrol (DES) induces pituitary tumors in Fischer 344 but not Brown-Norway or Sprague-Dawley rats. Functional loss of retinoblastoma susceptibility gene product (pRb), a major regulatory protein for the G1 to S transition of the cell cycle, has been shown in several tumors. Here we report a decreased level of pRb in pituitary tumors of the Fischer 344 rat as compared with resistant Sprague Dawley and Brown-Norway strains. pRb protein levels decreased 70% in Fischer 344 rats that were treated with diethylstilbestrol for 10 weeks as compared with tumor resistant control animals. Interestingly, the F1 hybrid (Fischer 344 x Norway) showed an intermediate range of pRb protein expression as compared with those of the parental strains. pRb expression levels in nonhemorrhagic F2 (F1 x F1) rats correlated with the size of the tumors. One week withdrawal of DES increased pRb levels as compared with continuously treated rats. Also, there was a decreased association of cyclin D and cyclin dependent kinase in susceptible tumors, supporting the hypothesis of a physical and possibly functional loss of pRb in the diethylstilbestrol-induced pituitary tumor. These results suggest that the difference in pRb regulation, whether it is a direct or indirect effect of estrogen, is related to tumor resistance or susceptibility in these two rat strains.

Animals↗

PubMed1998

A class I MHC-restricted recall response to a viral peptide is highly polyclonal despite stringent CDR3 selection: implications for establishing memory T cell repertoires in "real-world" conditions.

In this study, we analyze the recall response to influenza A matrix peptide M1(58-66) restricted by HLA-A2 in one individual and find a strict CDR3 selection as well as a high degree of polyclonality. The TCR beta-chain repertoire of memory T cells specific for this Ag system has been shown previously to be constrained by the use of the BV17 family and the I/sRS(A)/S amino acid motif in the CDR3 region. Our sequence analysis of BV17 TCR from a CTL line showed the repertoire to be highly polyclonal, as 95 distinct CDR3 sequences (clonotypes) were identified expressing this CDR3 motif. The clonotype frequencies showed a power law distribution with an extensive low-frequency tail. The clonotypes present in the high-frequency component of the distribution could be measured directly in the PBMC. This measurement showed that the relative frequencies of these clonotypes before stimulation were similar to their frequencies after culturing. Analysis of short-term cultures showed that the responding clonotypes have a similar ability to proliferate, which is independent of TCR beta-chain CDR3 sequence or precursor frequency. These data indicate that the memory T cell repertoire is composed of a surprisingly diverse set of T cell clonotypes with a limited potential for expansion. We propose that the high-frequency component represents T cells that have existed the longest. In keeping with this hypothesis, these clonotypes were measured over a 2-year period, during which their precursor frequency did not change.

Hematopoietic Stem Cells↗

PubMed1998

Differential regulation by estrogens of growth and prolactin synthesis in pituitary cells suggests that only a small pool of estrogen receptors is required for growth.

PR1 cells are a prolactin (PRL)-secreting cell line derived from a pituitary lactotroph tumor found in 17beta-estradiol-treated Fischer 344 rats. We examined the effect of estrogen on cell proliferation and PRL synthesis under various culture conditions. Estrogen, at extremely low concentrations, induces cell proliferation in this cell line, whereas antiestrogen inhibits proliferation. Interestingly, the proliferation response is much more sensitive than the PRL response because 0.01 pM estradiol or diethylstilbestrol induces half-maximal growth induction [ approximately 0.1% estrogen receptor (ER) occupancy is required], whereas 0.01 nM concentration is required for half-maximal PRL induction ( approximately 50% ER occupancy is required). The proliferation response is not as sensitive to antiestrogen as the PRL response, because 10 nM concentration of the pure antiestrogen ICI 182,780 could not inhibit 1 nM estradiol- or diethylstilbestrol-induced proliferation. The same concentration of ICI 182,780 decreased PRL secretion to 1% of estradiol- or diethylstilbestrol-induced prolactin secretion suggesting a possible dichotomy of ER control of proliferation and PRL synthesis. The Kd of ER binding in these cells is about 3 x 10(-11) M. These results with the PR1 cells extend previous studies in other estrogen- regulated systems and suggest that only a small pool of ER is required for cell proliferation in contrast with the regulation of expression of specific genes. They also raise questions as to how a dimeric receptor functions when only one ligand site is occupied or when both an estrogen and an antiestrogen occupy one dimer.

Animals↗

PubMed1998

Estrogen receptor down-regulation is regulated noncooperatively by estrogen at the transcription level.

We examined noncooperativity of estrogen action by studying the transcription of estrogen receptor (ER) down-regulation in an ER stably transfected cell line (Rat1 + ER). The time-course of the ER transcription rate following 17beta-estradiol (E2) (10 nM) treatment was measured by nuclear run-on assays. ER transcription rates decreased to 40 +/- 3% within 1 h as compared with no treatment and stayed suppressed to 24 h of E2 treatment. The ER transcription rate decrease was noncooperative when measured at 24 h of E2 treatment. This suggested that initial E2 binding to the ER is also a noncooperative process. The Hill coefficients of E2 binding to ER were near unity with an equilibrium dissociation constant of 1-3 nM when measured after a 1 h incubation. These results show that E2 noncooperatively binds ER and down-regulates transcription of ER noncooperatively in intact Ratl + ER cells.

Animals↗

PubMed1998

Estrogen causes cell death of estrogen receptor stably transfected cells via apoptosis.

We examined the effect of estrogen on the growth of estrogen receptor (ER) stably transfected cells (Rat1 + ER). 17-Beta-estradiol (E2, 10 nM) inhibited approximately 35-50% of Rat1 + ER growth after 3 d of treatment. The half-maximal growth inhibition occurred at 0.5-0.75 nM of E2 concentration and was saturated above 10 nM. This E2-induced antiproliferative effect was mediated through the ER since E2 did not cause any change in ER-negative parental Rat1 cells. Cells started to detach from plates and the adherent cells exhibited nuclear condensation. Apoptotic cell populations showed a 25% increase at 2 d of E2 treatment over controls that were quantified by fluorescence-activated cell sorter analysis. This indicates that E2 induced apoptosis in Rat1 + ER cells.

Animals↗

PubMed1998

A gene involved in XY sex reversal is located on chromosome 9, distal to marker D9S1779.

The genetic mechanisms involved in sex differentiation are poorly understood, and progress in identification of the genes involved has been slow. The fortuitous finding of chromosomal rearrangements in association with a sex-reversed phenotype has led to the isolation of SRY and SOX9, both shown to be involved in the sex-determining pathway. In addition, duplications of the X chromosome, deletions of chromosomes 9 and 10, and translocations involving chromosome 17 have been reported to be associated with abnormal testicular differentiation, leading to male-to-female sex reversal in 46,XY individuals. We present the cytogenetic and molecular analyses of four sex-reversed XY females, each with gonadal dysgenesis and other variable malformations, and with terminal deletions of distal chromosome 9p, resulting from unbalanced autosomal translocations. PCR amplification and DNA sequence analysis of SRY revealed no mutations in the high-mobility-group domain (i.e., HMG box) in any of the four patients. Conventional and molecular cytogenetic analyses of metaphase chromosomes from each patient suggest that the smallest region of overlap (SRO) of deletions involves a very small region of distal band 9p24. Loss-of-heterozygosity studies using 17 highly polymorphic microsatellite markers, as well as FISH using YAC clones corresponding to the most distal markers on 9p, showed that the SRO lies distal to marker D9S1779. These results significantly narrow the putative sex-determining gene to the very terminal region of the short arm of chromosome 9.

Child, Preschool↗

PubMed1998

T cell immunobiology of alloimmune thrombocytopenias.

Allorecognition and generation of alloantisera depend on T cell help. Here we summarize our recent work on identifying the T cells that recognize a specific platelet alloantigen generated by a Pro to Leu polymorphism in beta-chain of the platelet integrin alphaIIb beta3. The ability to generate alloantibodies is restricted by HLA-DRB3*0101. By measuring peptide binding to HLA-DRB3*0101, we have shown that the polymorphism controls the generation of the T cell epitope. This is not a result of the polymorphism changing a T cell contact residue, but rather by its generating a peptide anchor. The result is a directional antibody response in which only the beta-chain that is antigenic is the one that produces a peptide that binds to the HLA-DR. This mechanism of generating alloantibodies may be a paradigm for a whole class of responses.

Animals↗

PubMed1997

Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy.

Interaction of estrogen receptor (ER) with DNA sequences known as estrogen response elements (ERE) is required for estrogen regulation of the expression of target genes. To characterize the affinity and specificity of ER interaction with ERE sequences in vitro under equilibrium conditions, fluorescence anisotropy assays were performed using recombinant, purified ER and a fluorescein-labeled 35-base pair oligonucleotide bearing an idealized palindromic ERE. In buffer containing 100 mM KCl, the baculovirus-expressed, purified human ER bound with similar affinity to the consensus ERE and a mutant ERE with a single base pair change per half-site. Above 225 mM KCl, ER exhibited discrimination between the consensus and mutated ERE targets. Between 225 and 275 mM KCl, binding to the consensus ERE was independent of salt concentration and occurred with an equilibrium dissociation constant (Kd) of 1.8 +/- 0.6 nM, whereas binding to the mutant ERE was not detected at ER concentrations below 100 nM under the same conditions. At 300 mM KCl, the Kd for the consensus ERE increased approximately 25-fold, suggesting complex salt concentration dependence. Both estrogen-occupied and unoccupied ER bound to the consensus ERE sequence with similar affinity, indicating that estrogen affects ER activity at a step other than DNA binding. Unlike the full-length ER, the recombinant DNA binding domain of ER did not discriminate between the consensus and mutated ERE sequences even at buffer salt concentrations greater than 200 mM NaCl, suggesting that ER sequences outside the DNA binding domain may be important in promoting specific binding.

Animals↗

PubMed1997

Concurrent or sequential delta and beta TCR gene rearrangement during thymocyte development: individual thymi follow distinct pathways.

Analysis of TCR rearrangement profiles of well-defined thymocyte populations in a number of individual thymi provides evidence for a new pathway of lineage commitment. In all of the thymi analyzed, alphabeta thymocytes have rearrangements in the delta locus that are enhanced for out-of-frame rearrangements. Thus, not only did alphabeta thymocytes pass through a stage in differentiation that included delta rearrangement, but they also constitute a population that was relatively unsuccessful at these rearrangements. Interestingly, in some thymi, gammadelta thymocytes have out-of-frame beta rearrangements. This represents a novel pathway in which delta and beta rearrangement happens concurrently. In this pathway, selection favors whichever gene is in frame, thus improving the chances of generating useful T cells. In other thymi, gammadelta cells showed no obvious beta gene rearrangement, indicating a sequential rearrangement. Thus, alphabeta/gammadelta lineage commitment can follow at least two distinct pathways in different individuals.

Cell Differentiation↗

PubMed1997

Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03). The majority of unoccupied cytosolic ER is in the T population. Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population. The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain. Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains). The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.

Animals↗

PubMed1997

An integrin polymorphism that defines reactivity with alloantibodies generates an anchor for MHC class II peptide binding: a model for unidirectional alloimmune responses.

Polymorphic proteins are often the targets of T and B lymphocytes in alloimmune responses. The polymorphic residue 33 of integrin beta3 is responsible for both a B cell response and a T cell response. Leu at position 33 controls the epitopes recognized by alloantibodies generated in individuals homozygous for Pro at 33. The alloantibody response shows a tight class II MHC restriction, and T cells have been identified that respond to the alloantigen. As part of the T cell response, the alloantigen could be recognized as foreign, and the autoantigen would be ignored due to self-tolerance. Alternatively, the polymorphism could change the ability of the peptide to bind. We tested these two possibilities using synthetic peptides corresponding to the polymorphic region and insect cell-derived class II MHC. The Leu33-containing peptide bound to the restricting MHC allele, whereas the Pro33 peptide did not. Thus, the presence of Leu at position 33 generates an anchor specific for the restricting MHC allele and helps define the peptide binding motif of this allele, HLA-DRB3*0101 (DR52a). These data indicate that certain alloresponses can be viewed entirely as foreign because the polymorphism generates a functional anchor residue in a peptide that otherwise would not bind. On the basis of these results, we would predict a unidirectional alloantibody response as the lack of binding of the Pro33 peptide would preclude T cell help. This could explain the observation that alloantibodies to the Pro33 allele are very rare.

Antigens, CD↗

PubMed1997

DRB4 promoter polymorphism in DR7 individuals: correlation with DRB4 pre-mRNA and mRNA levels.

DRB genes encode proteins which play an important role in the immune response, and variation in their expression levels may be of functional immunological significance. To date, HLA class II gene promoter polymorphism has been analyzed almost exclusively in Epstein-Barr virus (EBV)-transformed B-cell lines. While previous studies have established important paradigms with regard to the mechanisms in DRB gene expression, the role of DRB4 promoter polymorphism in the variation of DRB4 expression levels, if any, is still unclear. We analyzed the effects of a previously described polymorphism in the promoter and 5' untranslated region of the DRB4 locus in peripheral blood mononuclear cells (PBMCs) from nine healthy DR7 individuals using the RNase protection assay. The RNase protection analyses showed that the location of transcription initiation is not affected. The DRB4 alleles show a threefold difference in pre-mRNA and a corresponding fivefold difference in mRNA levels in PBMCs. These data show that the DRB4 promoter polymorphism is associated with differential gene expression of this locus. The difference in pre-mRNA levels is directly affected by this DRB4 promoter polymorphism. The additional difference in mRNA levels is the result of post-transcriptional regulation. Therefore, the DRB4 promoter polymorphism may explain the previously observed variation in HLA class II DRB4 gene expression levels among different healthy DR7 individuals.

Alleles↗