Search PubMed⌕ Search

Biomedical subjects

J Gorski

Publications and source records attributed to J Gorski.

At least 91 records · Page 5Linked to original sources
PubMed1994

Adenosine deaminase activity in gastric cancer.

Adenosine deaminase activity, the key enzyme of adenosine inactivation, was studied in slices taken endoscopically from gastric cancer and macroscopically unchanged gastric mucosa surrounding the cancer. The activity of the enzyme was measured in mucosa homogenates by determination of ammonia liberated from substrate. It was found that adenosine deaminase activity in neoplastic lesions did not differ significantly from normal mucosa and that the gastric region studied (antrum, corpus) did not have an impact. A significant difference in enzyme activity was noticed between intestinal and diffuse-type gastric carcinoma (according to Lauren's classification); the intestinal type was characterized by lower adenosine deaminase activity than was the diffuse type. Since the activity of adenosine deaminase in gastric cancer did not exhibit significant differences from normal mucosa the diagnostic value of its determination is of less importance.

Adenosine Deaminase↗
PubMed1994

Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analyzed by CDR3 size spectratyping. Correlation with immune status.

The analysis of the T cell repertoires involved in local or systemic immune responses is beginning to play an important role in many clinical situations. These include autoimmunity, response to viral or bacterial superantigens, alloimmunity including allograft rejection, and tumor immunity. Here we analyze circulating T cell repertoires by determining TCR beta-chain gene complexity using a modification of V beta family-specific PCR. This approach, called CDR3 size spectratyping, uses the size heterogeneity of the CDR3 as a further source of specificity in TCR analysis. It has been used here to analyze the complexity and stability of circulating T cell repertoires in normal adults, including bone marrow donors, and bone marrow transplant recipients. Normal spectratypes are both complex and stable. The repertoire complexity of marrow recipients correlates with their state of immune function. Contractions and gaps in repertoires are revealed in individuals suffering from recurrent infections associated with T cell impairment. Spectratype analysis is applicable to other studies of specific repertoire skewing such as may be associated with immunodeficiency or found at sites of immune activity.

Adult↗
PubMed1994

The non-B-DNA structure of d(CA/TG)n differs from that of Z-DNA.

Chemical probing of two predominantly alternating purine-pyrimidine d(CA/TG)n repeats led us to propose previously that in supercoiled plasmids these elements adopt a non-B-DNA structure distinct from that of Z-DNA formed by d(CG)n sequences. Here, we present further evidence supporting this contention. Reactivity with the conformation-sensitive reagent chloroacetaldehyde, which reacts with unpaired adenines and cytosines, was confined strictly to adenines in the d(CA/TG)n repeat. In contrast, only bases outside the d(CG)n repeat exhibited chloroacetaldehyde reactivity. Two-dimensional gel analysis of topoisomers containing d(CA/TG)n tracts with bases out of strict purine-pyrimidine alteration revealed multiple superhelical-dependent transitions to an alternative left-handed structure. Within individual plasmid molecules, these multiple transitions resulted from the stepwise conversion of contiguous segments of alternating purine-pyrimidine sequence, which are delimited by bases out of alternation, to the full-length alternative conformation. When the left-handed helices increased in length to include more bases out of alternation, the average helical pitch changed substantially to produce a less tightly wound left-handed helix. Overall, these data indicate that d(CA/TG)n tracts adopt a left-handed conformation significantly different from that of the canonical Z-DNA structure of d(CG)n sequences.

Acetaldehyde↗
PubMed1994

Structural and functional analysis of HLA-DR beta-promoter polymorphism and isomorphism.

Evolutionary relatedness among the highly polymorphic DR beta genes has been established based on shared nucleotide sequences and structural organization of DR beta loci. The evolution of promoter regions of the B1*0701, B1*0101, B1*1501, B5*0101 genes was analyzed by cloning and sequencing. This shows that the polymorphism and isomorphism of HLA DR beta genes extend into the 5' flanking promoter region of the genes and that evolutionary relatedness also exists among the DR beta gene promoters. This suggests that DR beta gene promoters and coding regions coevolved. The effect of the naturally occurring nucleotide substitutions in the polymorphic and isomorphic DR beta promoters on transcriptional activity has been determined in a transient expression system. The transcriptional activity of two polymorphic DR beta promoters, B1*1501 and B1*0701, and two isomorphic DR2 promoters, B1*1501 and B5*0101, is the same for these promoters. Together these data suggest that naturally occurring substitutions do not significantly affect the transcriptional activity of these promoters.

Amino Acid Sequence↗
PubMed1994

DNA topology regulates rat prolactin gene transcription.

A cell-free transcription system was used to examine the effects of DNA negative supercoiling on transcription from rat prolactin promoters. The mRNA was faithfully transcribed from prolactin promoters that contained the DNA fragment spanning from -1960 to -10 base pairs upstream of the prolactin transcription start site. Prolactin promoters in supercoiled form, prepared from Escherichia coli, have a higher transcriptional efficiency than the promoter in the relaxed form. Deletion of DNA sequences containing the estrogen response element and putative non-B form DNA elements from the upstream region of the prolactin gene did not alter this effect of DNA topology on prolactin transcription. We further separated transcription into preinitiation and elongation steps by using sarkosyl and found that more preinitiation complexes were assembled on the supercoiled prolactin promoters than on the relaxed promoters. DNA supercoiling therefore plays an important role in controlling prolactin gene expression by facilitating the formation of preinitiation complexes on the promoters.

Animals↗
PubMed1994

The proximal promoter of the bovine luteinizing hormone beta-subunit gene confers gonadotrope-specific expression and regulation by gonadotropin-releasing hormone, testosterone, and 17 beta-estradiol in transgenic mice.

Transient transfection studies have proven useful in unraveling the molecular mechanisms underlying gonadotrope-specific expression and hormonal regulation of the gene encoding the alpha-subunit of the glycoprotein hormones. In contrast, similar studies performed with the LH beta gene have been less informative. When assayed by transient transfection into alpha T3-1 cells, activity of a 776-basepair bovine LH beta promoter-chloramphenicol acetyltransferase fusion gene (bLH beta CAT) was no greater than that of a promoterless control. To determine whether limited activity in vitro reflected the absence of critical regulatory elements, we examined activity of bovine LH beta fusion genes after stable integration in transgenic mice. In contrast to transient transfection studies, the LH beta promoter targeted high levels of CAT expression specifically to the pituitary. In addition, a bLH beta TK fusion gene was active only in gonadotropes. The bLH beta CAT transgene was also evaluated for responsiveness to gonadal steroids and GnRH. Testosterone and 17 beta-estradiol were capable of suppressing activity 70-80% in castrated males, despite the absence of high affinity binding sites for androgen or estrogen receptors. This suggests that feedback inhibition of LH beta CAT transgene expression by gonadal steroids may occur through an indirect mechanism, possibly at the level of the hypothalamus. To address whether the bLH beta CAT transgene could be regulated by GnRH, we treated ovariectomized females with antide, a GnRH antagonist. Antide suppressed transgene activity by 60%. Thus, the proximal promoter of the bovine LH beta subunit gene directs appropriate patterns of cell-specific expression and retains responsiveness to gonadal steroids and GnRH. In light of the robust activity of the LH beta promoter in transgenic mice, we suggest that this animal model can be exploited further to dissect the complex mechanisms that underlie gonadotrope-specific expression and hormonal regulation of the LH beta gene.

Animals↗
PubMed1993

Structural characterization of the trypsinized estrogen receptor.

Structural differences between the unoccupied and ligand-occupied rat uterine estrogen receptors (ERs) were investigated using partial proteolysis followed by immunoblotting, affinity labeling, and gel filtration chromatography. Trypsin digestion of the unoccupied ER at 4 degrees C resulted in retention of 70-80% of high-affinity [3H]estradiol binding. Only two fragments of the rat ER were detected after prolonged trypsin treatment of the unoccupied ER followed by affinity labeling with [3H]tamoxifen aziridine. One fragment represents the intact steroid binding domain (28 kDa), and the other fragment is about 10 kDa. The small 10-kDa fragment of the ER detected by denaturing gel electrophoresis is shown to be held in a large oligomeric complex in solution using gel filtration chromatography. This oligomeric complex probably represents the steroid binding domain, which has its tertiary structure maintained predominantly by noncovalent interactions between the trypsin-generated fragments. The estrogen, anti-estrogen, and unoccupied trypsinized ERs all result in similar patterns of fragments after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detection by immunoblotting. Although no new trypsin cleavage sites were exposed, the sensitivity of the available trypsin sites was altered by heating the ER and, to a lesser extent, by hormone treatment. Gel filtration chromatography of the trypsinized estradiol- and 4-hydroxytamoxifen-occupied ERs demonstrates similar, diffuse peaks centered at about the correct size for the intact steroid binding domain (28 kDa), whereas the trypsinized unoccupied ER results in a sharp, discrete peak centered at about 80 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Mucosal adenosine deaminase activity and gastric ulcer healing.

Adenosine deaminase activity was studied in gastric corpus mucosa close to an ulcer crater. It was found that 6 weeks of therapy with ranitidine was accompanied by a decrease in enzyme activity in the mucosa around healed ulcers and an increase around those which failed to heal. The different activities of adenosine deaminase in the vicinity of healed and unhealed ulcers may indicate its possible role in peptic ulcer healing.

Adenosine Deaminase↗
PubMed1993

Estrogen receptor and progesterone receptor genes are expressed differentially in mouse embryos during preimplantation development.

Estrogen and progesterone play an important role in the development and implantation of preimplantation embryos. However, it is controversial whether these hormones act directly on the embryos. The effects of these hormones depend on the existence of their specific receptors. To determine whether estrogen receptor (ER) and progesterone receptor genes are expressed in mouse preimplantation embryos, we examined RNA from embryos at different stages of preimplantation development by reverse transcription-polymerase chain reaction techniques. ER mRNA was found in oocytes and fertilized eggs. The message level began to decline at the two-cell stage and reached its lowest level at the five- to eight-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 ER and progesterone receptor at this stage of development. These results provide a basis for determining the direct role of estrogen and progesterone in preimplantation embryos.

Age Factors↗
PubMed1993

Activation of the silent progesterone receptor gene by ectopic expression of estrogen receptors in a rat fibroblast cell line.

We describe the construction and characterization of a novel estrogen (E2)-responsive cell line, Rat1+ER, which ectopically expresses estrogen receptor (ER). Human ER cDNA was introduced by retrovirus-mediated gene transfer into the Rat1 cell line, which does not express functional ER endogenously. Rat1+ER cells express functional ER based on radioreceptor assays, immunoblotting, and transient transfection experiments using E2-responsive reporter plasmids. The effects of this ectopic ER expression were studied on three endogenous E2-responsive genes, prolactin (PRL), progesterone receptor (PR), and epidermal growth factor receptor (EGFR). PRL, usually expressed in the lactotrophs of the pituitary, is not expressed at all in Rat1+ER cells, with or without E2 addition, and appears to require other factors for expression. In contrast, although PR is not expressed in Rat1 cells, it is induced in Rat1+ER cells upon the addition of E2. This induction appears to occur at the transcriptional level and is insensitive to cycloheximide treatment. This is one of the few examples where the expression of one gene activates an otherwise silent gene. Another contrasting observation is that, although EGFR is basally expressed in Rat1+ER cells, the addition of E2 has no effect. Our studies paint a complicated picture of E2 regulation of endogenous genes: the activation of the PR gene may only require the presence of E2 and ER, whereas EGFR and PRL genes require factors in addition to ER for basal as well as E2-regulated expression.

Animals↗
PubMed1993

High affinity binding of the estrogen receptor to a DNA response element does not require homodimer formation or estrogen.

We have developed an antibody-based DNA binding assay to study the effects of the absence or presence of an estrogen receptor agonist and two antagonists on the thermodynamic binding parameters for estrogen receptor binding to a consensus estrogen response element in vitro. We first demonstrate that antibodies raised against a region of the estrogen receptor directly adjacent to the DNA-binding domain do not interfere with the receptor's ability to preferentially bind to the vitellogenin estrogen response element in plasmid DNA. Exploiting this property to develop a quantitative DNA binding assay, we demonstrate that estrogen is not required for high affinity binding of the receptor for an oligonucleotide containing this element, nor do antiestrogens alter this interaction. In addition, we find that 1 mol of estrogen receptor is complexed with high affinity to 1 mol of vitellogenin estrogen response element, instead of two estrogen receptors/response element as would be predicted if estrogen receptor homodimer formation was required for high affinity DNA binding. Our data best fit a model in which the active estrogen receptor form is a monomer or heterodimer, but not a homodimer, and the regulatory role of estrogen on the estrogen receptor is the induction of protein-protein, but not protein-DNA, interactions.

Animals↗
PubMed1993

Multiple transitions to non-B-DNA structures occur in the distal regulatory region of the rat prolactin gene.

The developmentally regulated rat prolactin (rPRL) gene presents a promising model system toward understanding the biological role of non-B-DNA structural elements. Two predominantly alternating purine-pyrimidine (APP) (dA-dC)n.(dG-dT)n repeats of 58 and 178 base-pairs flank the (A + T)-rich distal regulatory region. We have characterized several transitions to non-B-DNA structures within this region in negatively supercoiled plasmids by utilizing high resolution chemical probing. Each repeat undergoes a full-length conversion to a novel left-handed helical structure via the stepwise nucleation and propagation of discrete "segments". These segments are delimited by out-of-alternation bases that are susceptible to attack by potassium permanganate and thus appear to be significantly unstacked within the left-handed helices. Moreover, the spatial order of successive right- to left-handed DNA transitions within each repeat exhibits a clear polarity toward the distal regulatory region of the rPRL gene. An additional transition involving the long-range unpairing of (A + T)-rich sequences establishes a directional propagation toward the regulatory region. These data demonstrate a complex series of quasi-independent transitions to non-B-DNA structures that impinge upon a known regulatory control region.

Animals↗
PubMed1993

Analysis of transcripts of genes located within the HLA-D region in B cells from an HLA-severe combined immunodeficiency individual.

The mRNA levels of LMP7, HLA-DMA, and HLA-DMB genes, all located within the HLA-D region, were analyzed in the "bare lymphocyte" line SJO, which had been shown to be characterized by a transcription defect of class II genes (DP, DQ, and DR). This was done using semiquantitative PCR amplification of cDNA generated from total RNA. The results show that the transcription of the LMP7 gene in SJO cells is equivalent to that in the control B-cell line Raji. In contrast, the HLA-DM gene transcripts were not present in the SJO cell line. This observation indicates that the promoter similarity between the classic class II genes and HLA-DM is sufficient for the SJO defect to extend to the DM loci.

Base Sequence↗
PubMed1993

Perturbations in the model of estrogen receptor regulation of gene expression.

Estrogen receptors are key elements in the mechanisms of action of estrogenic hormones. Models of how estrogens and their receptors interact and subsequently modify gene expression should be reevaluated to explain new data currently available. The following review discusses nuclear localization, DNA binding, and protein structural changes of the estrogen receptor induced by estrogen binding. We also discuss how these phenomena relate to the induction of changes in gene expression.

Animals↗
PubMed1993

Ontogeny of the epidermal growth factor receptor during development of the fetal bovine mesonephros and associated organs of the urogenital tract.

A primary cell culture system was developed to study the epidermal growth factor (EGF) receptor in the fetal bovine mesonephros and its urogenital derivatives. Radioreceptor assays demonstrated EGF binding as early as Day 37 in mesonephric cells and in cells derived from the fetal reproductive ducts, gonads, and metanephros--all mesonephric derivatives. Immunocytochemical studies revealed that EGF receptors were localized in the ductal and tubular epithelium of these urogenital organs. EGF induced DNA synthesis and tyrosine phosphorylation in the bovine mesonephric cells, suggesting that EGF receptors detected in these cells were functional. In addition, transforming growth factor alpha, the putative fetal ligand for the EGF receptor, was found to specifically compete for EGF binding to the receptor, as well as to induce DNA synthesis in a manner similar to that of EGF. Estrogen did not regulate EGF receptors or specifically bind to bovine mesonephric cells. The development of estrogen receptors in the bovine species occurs markedly later than that of the EGF receptor, in contrast to the mouse, where both receptors are observed at very early stages of reproductive tract development.

Animals↗