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J Gorski

Publications and source records attributed to J Gorski.

At least 73 records · Page 4Linked to original sources
PubMed1996

Triacylglycerol synthesis in the different skeletal muscle fiber sections of the rat.

Triacylglycerol (TG) synthesis rates of low-oxidative fast-twitch white and high-oxidative fast- and slow-twitch red skeletal muscle fiber sections of adult rats were measured by the incorporation of perfusate-derived palmitate into the neutral lipid fraction by use of a perfused hindquarter preparation under high-flow conditions. The perfusion medium consisted of 95% O2-5% CO2 Krebs-Henseleit buffer, pH 7.4, containing 5 g/100 ml bovine serum albumin, 100 microU/ml insulin, 5 mmol/l glucose, amino acids, and added fatty acids (FA), including 0.1 microCi/ml [3H]palmitate. FA incorporation was linear with time. TG synthesis rates correlated (r > or = 0.90) with the oxidative capacity of each of the different fiber type sections and increased in proportion to the perfusate FA concentration (0.25, 0.5, 1.0, 1.5, and 2.0 mmol/l). TG synthesis rates among different muscle fiber sections were related (r > or = 0.90) to perfusate flow during high-flow conditions; however, this was not causal, because TG synthesis rates within each muscle fiber section were independent of flow rate when experimentally varied over an approximately threefold range. Thus the relatively high TG synthesis rates observed in the high-oxidative muscle sections are not uniquely related to their high-flow capacities but are inherent to the TG synthesis process, probably events associated with FA uptake and/or capacity of the TG synthesis pathway.

Animals↗
PubMed1995

The role of estrogen receptor in modulation of chromatin conformation in the 5' flanking region of the rat prolactin gene.

To determine whether the estrogen receptor (ER) has a role in the modification of chromatin structure, we developed cell lines to model discrete stages in the estrogen response. Each cell line carries a population of stably expressed papillomavirus-based minichromosomes containing the 5' flanking region of the rat prolactin gene. We examined ER effects at the distal enhancer domain of the rat prolactin promoter, using DNaseI to probe for alterations of the nucleoprotein complex. Within 1 h after the start of estrogen treatment, modifications in the chromatin state of the distal enhancer region were detected in a pituitary-derived, permissive cell line (GH3G1J). In rat-1 fibroblast cell lines that maintain the same stably expressed papillomavirus-based minichromosomes in the absence of ER or pituitary-specific transcription factors (Rat-1.2A2; non-permissive), no estrogen-induced modifications in the chromatin state were detected at 1 or 24 h. In rat-1 fibroblast cell lines that also contained ectopically expressed, functional ER (Rat-1 + ER.8A1), no estrogen-induced modifications in the chromatin state were detected at 1 h, but a 24 h a specific modification in the local structure was induced. These data support a model in which the ER interacts with chromatin to modify local structure in such a way as to induce a permissive state for interactions of transcription factors necessary for hormone-induced activation of gene transcription.

Animals↗
PubMed1995

Restricted and conserved T-cell repertoires involved in allorecognition of class II major histocompatibility complex.

The nature of the alloreactive T-cell response is not yet clearly understood. These strong cellular responses are thought to be the basis of allograft rejection and graft-vs.-host disease. The question of the extent of responding T-cell repertoires has so far been addressed by cellular cloning, often combined with molecular T-cell receptor (TCR) analysis. Here we present a broad repertoire analysis of primed responder cells from mixed lymphocyte cultures in which two different DR1/3 responders were stimulated with DR3/4 cells. Repertoire analysis was performed by TCR spectratyping, a method by which T cells are analyzed on the basis of the complementarity-determining region 3 length of different variable region (V) families. Strikingly, both responders showed very similar repertoires when the TCR V beta was used as a lineage marker. This was not seen when TCR V alpha was analyzed. A different pattern of TCR V beta was observed if the stimulating alloantigen was changed. This finding indicates that alloreactive T cells form a specific repertoire for each alloantigen. Since conservation appears to be linked to TCR V beta, the question of different roles of alpha and beta chains in allorecognition is raised.

Clone Cells↗
PubMed1995

Estrogen receptor binding to DNA: affinity for nonpalindromic elements from the rat prolactin gene.

The estrogen receptor (ER) binds with high affinity to the nonclassical estrogen response elements (ERE) found in the rat prolactin gene. There are two putative EREs in this gene; at -1582 and -1722 upstream of the transcriptional start site. We used DNA binding assays based on the immunoprecipitation of receptor with bound DNA to quantitate the binding of ER to these two elements. ER bound each element with significantly higher affinity than it bound to nonspecific DNA, but with 10-100-fold less affinity than that for the classical ERE sequence derived from the vitellogenin A2 gene. These comparisons rank the prolactin gene sequences as weak EREs. We also observed a 1:1 ratio of ER to ERE in the bound complexes, indicating that these high-affinity interactions were not dependent upon an ER homodimer. These data support the role of these sequences in mediating estrogen regulation of the prolactin gene.

Animals↗
PubMed1995

Molecular analysis of T cell repertoires. Spectratypes generated by multiplex polymerase chain reaction and evaluated by radioactivity or fluorescence.

The analysis of T-cell repertoires has been facilitated by the introduction of methods in which the length heterogeneity of the third complementarity region (CDR3) is used to further refine V-family-specific PCR. We call our implementation of this technique T-cell spectratyping. This method is especially important in analysis of specific expansion or retention of T cells in human immune system function. Current methodologies are cumbersome in the number of PCR reactions and gels needed for complete analysis of TCR BV repertoires. We describe here the optimized conditions for using 11 TCR BV primer pairs in multiplex PCR which allow for a more compact analysis. In addition, the two primers act as controls for each other in the PCR. The use of these primers is shown using either fluorescent or radiolabeled constant primers. The two labeling methods give comparable results. Fluorescent primers avoid the difficulties associated with use of radioactivity. Autoradiography with 32P-labeled primers is simpler, requiring less instrumentation.

Autoradiography↗
PubMed1995

Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor.

Estrogen antagonists such as ICI164,384 do not inhibit 17 beta-estradiol (E2)-dependent gene activity in yeast expressing the mammalian estrogen receptor although these compounds bind to receptors isolated from these cells. Various explanations have been offered for antiestrogen resistance in yeast systems including differences in cell-specific components and lack of permeability of the yeast cell wall to these compounds. We have used a strain of Saccharomyces cerevisiae transformed with the human estrogen receptor gene, and two estrogen response elements linked to a lacZ reporter gene to study the pharmacology of estrogen agonists and antagonists. The rank order of potency of estrogen agonists in this strain (CY525) is similar to that in estrogen-dependent mammalian cells: DES > or = E2 > E1 > E3 = zeranol. Competitive binding with 3H-E2 by these compounds in cell-free extracts of CY525 results in a similar order of potency with a reverse order for E1 and E3. The pure estrogen antagonist ICI164,384 also binds to the receptor from cell-free extracts of CY525 with an IC50 of approximately 14nM. As in mammalian cells ICI164,384 does not induce E2-dependent gene activity. However, unlike mammalian cells, E2-induced gene activity in CY525 is not inhibited by ICI164,384. Intact CY525 cells incubated with 3H-17 beta estradiol were found to specifically bind the labeled ligand since excess unlabeled E2 effectively competed for binding. Unlabeled DES and E1 were also found to compete, however, excess unlabeled ICI164,384, E3 and the second generation antagonist ICI182,720 were unable to displace 3H-E2 binding in intact cells. These results indicate that certain compounds enter the intact yeast cell more readily than others and offer an explanation for antagonist resistance in these organisms.

Base Sequence↗
PubMed1995

Embryonic estrogen receptors: do they have a physiological function?

In normal estrogen target tissues, estrogen action is mediated through a specific nuclear transcription factor, the estrogen receptor (ER). The site of estrogen action in the developing organism is therefore determined by cells that contain ER and other necessary tissue and gene-specific components for estrogen-mediated transcription. Immunocytochemical methods were used to determine the cellular localization and tissue distribution of ERs in reproductive tracts of mouse fetuses. Nuclear staining for ER was observed in reproductive tracts at fetal days 13 to 15. ERs were present in the precursors of both male and female reproductive tracts at these early developmental stages, which may be attributable to their similar embryonic origins. However, as the tissues undergo sexual differentiation at later fetal and early neonatal ages, ER increases in the female reproductive tracts as compared with the male. ER was detected by immunoblotting on fetal day 10 (before sexual differentiation) in extracts of whole mouse embryos. To determine whether ER and progesterone receptor genes are expressed earlier in development, we examined RNA from preimplantation mouse embryos using reverse transcriptase-polymerase chain reaction techniques. ER mRNA was found in oocytes and fertilized eggs. Message concentration declined at the 2-cell stage and reached its lowest level at the 5- to 8-cell stage. ER mRNA was not detectable at the morula stage but reappeared at the blastocyst stage. Progesterone receptor mRNA was not detectable until the blastocyst stage. The embryonic expression of ER and progesterone receptor genes in the blastocyst suggests a possible functional requirement for estrogen and progesterone receptors in preimplantation embryos.

Animals↗
PubMed1995

Central adrenergic system does not contribute to morphine stimulated pineal secretion of melatonin.

The effect of long-term morphine treatment of rats subjected to central chemical sympathectomy (CCS) was studied. The experiment was performed on 7-week-old male Wistar rats. CCS was performed with 6-hydroxydopamine injected into the lateral ventricle of the brain. The serum concentration of melatonin was assayed by specific RIA. The study showed that morphine alone significantly increased melatonin concentrations in serum. CCS alone significantly decreased melatonin concentrations in serum at the time of highest secretory activity of the pineal gland. Long-term morphine treatment of rats subjected to CCS significantly increased the serum concentration of melatonin. Therefore, it may be concluded that the central adrenergic system does not take part in the morphine-stimulated secretion of melatonin.

Journal Article↗
PubMed1995

Selection of an RNA domain that binds Zn2+.

We have selected an RNA that depends on zinc for affinity to a column, starting from a pool of ribooligonucleotides with 50 randomized positions. This RNA's chemical sensitivities, calculated folding thermodynamics, and activity when fragmented suggest that an ion binding site lies within a complex 21-nt hairpin loop, near the junction with an imperfect helical stem. This RNA site has an unselected selectivity among divalents, preferring nickel, cobalt, and cadmium to calcium, magnesium, and manganese, as expected for a simple site of chelation. A moderate zinc-dependent change in loop structure accompanies divalent binding and can be detected by chemical probing and zinc-dependent UV-induced crosslinking. The latter also demonstrates the apposition of loop sequences to make a structure that may be related to the E-loop motif found in a number of other RNA molecules; the E-loop motif, accordingly, may be a divalent site.

Base Sequence↗
PubMed1995

Analysis of reconstituting T cell receptor repertoires in bone marrow transplant recipients.

Allogeneic bone marrow transplantation (BMT) has become the treatment of choice for a variety of hematopoietic disorders. An important factor limiting the use of allogeneic BMT is delayed restoration of immune function. A quantitative understanding of immune reconstitution of the T cell compartment based on an efficient method of analysis would be of benefit. Distinct lineages of T cells which have resulted from previous and ongoing clonal expansion can be identified by their unique T cell receptors (TCR). Thus, TCR complexity can be used as a measure of repertoire complexity. Here we use a polymerase chain reaction-based approach which visualizes the size heterogeneity of the third complementarity determining region (CDR3) to study T cell reconstitution in adult bone marrow transplant recipients. We find that repertoire complexity, as determined by the number of bands of different length for each V family, reflects the general immune status of individuals tested. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. This approach provides a new tool in the analysis of reconstitution of alpha/beta T cell repertoires and it can be also be applied to B cells and gamma/delta T cells.

Bone Marrow↗