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Z Krozowski

Publications and source records attributed to Z Krozowski.

At least 37 records · Page 2Linked to original sources
PubMed1995

The human 11 beta-hydroxysteroid dehydrogenase type II enzyme: comparisons with other species and localization to the distal nephron.

Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralocorticoid target cells. The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) inactivates glucocorticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11 beta-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11 beta-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Furthermore, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11 beta-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11 beta-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactive was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11 beta-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Localization of the gene for human 11 beta-hydroxysteroid dehydrogenase type 2 (HSD11B2) to chromosome band 16q22.

The enzyme 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD2) is an NAD-dependent, high-affinity isoform that potently inactivates glucocorticoids. In the present study we have used fluorescence in situ hybridization and an 11 beta HSD2 cDNA isolated from human kidney as probe to localize the gene encoding 11 beta HSD2. The gene, which has been given the symbol HSD11B2, maps to human chromosome band 16q22.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1995

Immunohistochemical localization of the 11 beta-hydroxysteroid dehydrogenase type II enzyme in human kidney and placenta.

It has been proposed that the inactivation of glucocorticoids by the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) is an obligatory step in the kidney, permitting binding of aldosterone to the mineralocorticoid receptor, and in the placenta, protecting the fetus from high circulating levels of maternal glucocorticoids. Both low and high affinity isoforms of 11 beta HSD are known to exist, with evidence accumulating that the former species (11 beta HSD1) does not fulfill criteria that would allow it to perform these physiological functions. We have recently cloned a high affinity isoform of the enzyme (11 beta HSD2) from a human kidney library and have shown this species to possess all of the characteristics predicted from whole cell studies. In the present study we have raised a polyclonal antibody (HUH23) to a synthetic peptide deduced from the carboxy-terminus of the protein. The immunopurified antibody recognized a single band at 41,000 daltons on Western blots of mammalian cells transfected with an expression plasmid containing 11 beta HSD2, slightly smaller than the predicted 44,140 daltons protein. A single band of identical size was also seen in blots of human kidney and placenta, suggesting post-translational processing of the enzyme. Immunohistochemical studies on frozen sections of human kidney showed strong 11 beta HSD2 immunoreactivity in the cortical distal convoluted tubules and collecting ducts. Strong staining was also observed in medullary tubules, which had the appearance of collecting ducts and the thick ascending limb of Henle's loop. Staining of medium intensity was observed in vascular smooth muscle cells. Epithelial cells of glomeruli showed weak but detectable reactivity with HUH23. In the placenta, HUH23 antibody immunoreactivity was restricted to syncytial trophoblast cells in which strong staining was observed. These results suggest that the 11 beta HSD2 enzyme colocalizes with the mineralocorticoid receptor in the distal nephron where it allows aldosterone to occupy its physiological receptor. Furthermore, 11 beta HSD2 is also ideally situated in the placenta to protect the fetus from high circulating levels of maternal glucocorticoids.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1994

Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form.

Pseudohypoaldosteronism is a syndrome characterized by salt wasting and a failure to thrive due to the resistance towards the action of aldosterone. Aldosterone levels and plasma renin activity are extremely elevated and aldosterone binding sites in peripheral mononuclear leukocytes have regularly shown to be reduced or absent. Sporadic as well as familial cases have been identified and an autosomal dominant as well as an autosomal recessive mode of inheritance has been described. A defect in the aldosterone receptor has been postulated, however, molecular genetic analysis in selected patients has not revealed a mutation in the sequence of the coding region of the cDNA of the mineralocorticoid receptor gene. In the present study we have used a fluorescence-labeled antibody to detect possible receptor expression in monocytes from patients with various clinical forms of pseudohypoaldosteronism. Patients with the sporadic as well as with the autosomal dominant form were clearly immunopositive despite being negative in terms of aldosterone receptor binding. In contrast in two patients with the autosomal recessive form there was no detectable receptor protein, consistent with the results obtained in the aldosterone binding studies. These results suggest that the pathogenesis of pseudohypoaldosteronism is heterogeneous not only regarding the mode of inheritance but also in terms of receptor binding. Thus, in a subgroup of patients the inability of the receptor to bind ligand may be due to a defect involving other, probably cellular factors rather than a deficiency or a defect in the mineralocorticoid receptor system itself.

Adult↗
PubMed1994

The short-chain alcohol dehydrogenase superfamily: variations on a common theme.

Multiple alignment of members of the short-chain alcohol dehydrogenase (SCAD) superfamily, according to the conserved domains A-F, has revealed a number of important relationships. It can be shown that the 17 beta-hydroxysteroid dehydrogenase type 2 enzyme is more closely related to D-beta-hydroxybutyrate dehydrogenase than it is to 17 beta-hydroxysteroid dehydrogenase type 1. Carbonyl reductase, previously considered to be a member of the aldo-keto reductase superfamily, displayed high homology in the conserved domains and is clearly part of the SCAD superfamily despite the insertion of a large peptide between conserved domains. Alignment of the product of the Leishmania methotrexate resistance gene HMTX showed that an internal, highly conserved domain can be substituted by an unrelated sequence without loss of biological activity. Furthermore, comparisons of the chimeric trifunctional enzyme enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-hydroxyacyl-CoA epimerase with other family members suggests that the region between the conserved B and C domains is the last to diverge between closely related enzymes and that the F domain appears to evolve with a different evolutionary clock to the rest of the protein. Finally, a highly conserved pattern of serine and threonine residues in the active site of SCAD enzymes indicates that these residues may play an important role in catalysis. These observations should facilitate alignment of future members of the SCAD superfamily.

17-Hydroxysteroid Dehydrogenases↗
PubMed1993

Characterization of 11 beta-HSD1B gene expression and enzymatic activity.

Nuclease protection analysis has been used to study the distribution of an mRNA that has been predicted to give rise to a truncated form of the enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1B). The 11 beta-HSD1B mRNA was found exclusively in the kidney, predominantly localized within the medulla with low amounts of the message in the cortex. Neither the renal papilla nor a range of peripheral and central tissues showed evidence of 11 beta-HSD1B mRNA. Expression of the whole (11 beta-HSD1A) and truncated enzymes in COS cells resulted in immunoreactive 34 kDa and 26 kDa proteins respectively, while kidney homogenates showed 34 kDa and 40 kDa species. The 11 beta-HSD1A enzyme converted corticosterone and cortisol to their respective 11-dehydro metabolites with an absolute dependence on NADP as co-factor, while the 26 kDa 11 beta-HSD1B protein was unable to metabolize either steroid. These results suggest that transcription of the 11 beta-HSD1B mRNA is associated with a mechanism down-regulating production of 11 beta-HSD1 activity.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1993

Retinoic acid receptor, cytosolic retinol-binding and retinoic acid-binding protein mRNA transcripts and proteins in rat insulin-secreting cells.

To define the mechanism of vitamin A action at the beta-cell level, we tested for the presence of messenger RNA for retinoic acid receptors alpha, beta, and gamma; cytosolic retinol-binding protein; and cytosolic retinoic acid-binding protein in RINm5F cells, an insulin-secreting cell line, and determined whether cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein are present in isolated purified normal rat beta-cells. Northern blot analyses showed two transcripts of retinoic acid receptor alpha messenger RNA (3.8 and 2.4 kb), one transcript of retinoic acid receptor messenger RNA (3.8 kb), and one transcript of cytosolic retinol-binding protein (0.9 kb) in RINm5F cells. Ribonuclease protection assays also showed the presence of cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein in RINm5F cells. Quantitatively, cytosolic retinol-binding protein levels were 0.10 +/- 0.02 pg/micrograms total RNA. Using specific radioimmunoassays, normal isolated purified rat beta-cells contained CRBP (19.2 +/- 2.38) and cytosolic retinoic acid-binding protein (16 +/- 0.53 ng/10(6) cells). The presence of message for retinoic acid receptors alpha and gamma, cytosolic retinol-binding protein, cytosolic retinoic acid-binding protein, and the gene products of cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein in insulin-secreting cells support a mechanism of vitamin A action and role for cytosolic and nuclear receptors at the beta-cell level similar to that suggested in nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Tissue-specific expression of an 11 beta-hydroxysteroid dehydrogenase with a truncated N-terminal domain. A potential mechanism for differential intracellular localization within mineralocorticoid target cells.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD) is thought to confer specificity on the nonselective Type I adrenocorticoid receptor by converting glucocorticoids to receptor-inactive metabolites in mineralocorticoid target tissues. S1 nuclease analyses using a rat liver 11-HSD probe demonstrated tissue-specific expression of the 5' region of the 11-HSD gene in the liver, lung, and kidney not evident in previous studies. Renal tissue contained a unique protected species which mapped to a position within the coding region, consistent with a divergence in liver and kidney protein sequences. Screening of a rat kidney cDNA library resulted in the isolation of several clones (11-HSD1B) noncolinear in their 5' regions with the liver sequence (11-HSD1A). Nucleic acid sequence analysis showed that the divergent clones code for a protein lacking a 26-amino acid NH2-terminal putative membrane-spanning signal peptide. The deletion of the leader sequence from the microsomal 11-HSD1A protein may result in a nuclear localization of the 11-HSD1B isoform. The renal 11-HSD1A and 11-HSD1B species increased coordinately during ontogeny and in parallel with the developmental surge in glucocorticoids. At least three alternate sites of polyadenylation were found to be utilized by the 11-HSD gene. Southern blot analysis showed the presence of a single gene in the rat. This study shows the expression of a kidney-specific 11-HSD isoform which may protect the Type I adrenocorticoid receptor from occupation by glucocorticoids in the nucleus of a mineralocorticoid target cell.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

Type I corticosteroid receptor-like immunoreactivity in the rat salivary glands and distal colon: modulation by corticosteroids.

A 167 amino acid fragment of the N-terminal domain of the human type I corticosteroid (mineralocorticoid) receptor was fused to the glutathione S-transferase gene using the Gex expression plasmid and the fusion protein used to raise the monospecific polyclonal antibody, MINREC4. Immunostaining experiments showed that MINREC4 specifically bound type I receptor in the distal tubule of the kidney, the ductal elements of the salivary glands and the epithelium of the distal colon in the rat. Adrenalectomy abolished staining in the parotid and colon, and reduced immunoreactivity in the submandibular gland. Administration of corticosterone or aldosterone resulted in partial restoration of immunostaining in the parotid, and a complete restoration of staining to intact levels in the submandibular gland and colon. These results suggest that adrenocorticoid binding to the type I receptor may result in tissue specific conformational changes in the binding protein and that the MINREC4 antibody may be used to study the effects.

Aldosterone↗
PubMed1992

Postnatal development of corticosteroid receptor immunoreactivity in the rat cerebellum and brain stem.

The postnatal development of corticosteroid receptor immunoreactivity in the rat cerebellum and related brainstem nuclei was studied using a type I receptor antiserum, MINREC4, and a type II receptor monoclonal antibody, BUGR2. Type I receptor immunoreactive (ir) Purkinje cells were first observed at postnatal day 5 (P5), and increased to adult levels by P20. Type I-ir cells, presumably migrating granule cells, were observed in the developing molecular layer of the cerebellum at P5. By P30, the density of type I-ir cells in the definitive molecular and granular layers was still less than adult levels. In contrast, type II-ir Purkinje cells were first observed at P15 and increased to adult levels by P20. No type II-ir cells were observed in the proliferative and migratory zones of the molecular layer. By P30, the density of type II-ir cells in the molecular and granular layers was far less than adult levels. In the deep cerebellar nuclei and most brain stem nuclei type I-ir was observed at P5 and developed to adult levels by P30. Type II-ir was observed in the deep cerebellar nuclei, red and medial vestibular nuclei by P15. The pontine and inferior olivary nuclei showed type II-ir cells by P10. Type II-ir in these regions developed to adult levels by P30. The earlier development of type I-ir suggests that the type I receptor may mediate the actions of corticosteroids in the cerebellum and related brain stem nuclei during early postnatal life.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Type I corticosteroid receptor-like immunoreactivity in the rat CNS: distribution and regulation by corticosteroids.

Previous maps of Type I corticosteroid receptor binding in the rat central nervous system (CNS) revealed a restricted distribution of the receptor in limbic regions, hypothalamus, and circumventricular organs. More recent studies have shown a more widespread expression of the receptor, with high levels of Type I receptor mRNA in limbic, motor, and sensory systems. We have used two antisera against peptide sequences derived from the cDNA of the human Type I corticosteroid receptor to map the regional distribution and corticosteroid regulation of the intracellular location of Type I corticosteroid receptor-like immunoreactivity (Type I-ir) in the rat CNS. Neurons showing Type I-ir were observed at all levels of the CNS. Highest densities of immunoreactive neurons were observed in limbic regions, isocortex, and some thalamic nuclei. Motor, sensory, and visceral systems often showed moderate densities of immunoreactive neurons. Type I-ir glia were observed in some fiber systems, e.g., corpus callosum, medial lemniscus, cerebral peduncles, spinal trigeminal tract, and funiculi of the spinal cord. In the majority of neurons and in glia, Type I-ir showed a diffusely nuclear and cytoplasmic location. Long-term adrenalectomy reduced immunoreactivity in most neurons and glia. Neuronal Type I-ir was localized mainly in the cytoplasm after long-term adrenalectomy. Nuclear immunoreactivity was retained in some neurons in the globus pallidus, motor trigeminal nucleus, and laminae 8 and 9 of the spinal cord. Acute treatment with corticosterone or aldosterone restored neuronal and glial Type I-ir to densities below that seen in intact rats.

Adrenal Cortex Hormones↗
PubMed1991

Postnatal development of corticosteroid receptor immunoreactivity in the rat hippocampus.

Postnatal changes in corticosteroid receptor immunoreactivity in the rat hippocampus were examined using an antiserum against a fusion protein containing an N-terminal peptide of the Type I receptor, and a monoclonal antibody against the rat liver Type II-receptor. Age-related regional differences were observed. In the pyramidal cell layer of Ammon's horn, and granule cell layer of the dentate gyrus (DG), the percentage of Type I receptor immunoreactive (Type I-ir) and Type II receptor immunoreactive (Type II-ir) cells was high perinatally, declined sharply by postnatal day 10 (P10), and showed a variable increase to adult levels subsequently. The pyramidal cells of CA1-CA2, subiculum and DG showed a selective increase in Type II-ir in late postnatal life into adulthood, while most other regions showed higher Type I-ir in both early and late postnatal life, suggesting different roles for these receptors during development. Type II-ir was predominantly nuclear in most neurons, except for a transient appearance of cytoplasmic Type II-ir in neurons of the stratum oriens and molecular layers of Ammon's horn and dentate gyrus of P20-P30. Type I-ir was diffusely nuclear and cytoplasmic at all developmental ages. This is suggestive of differential genomic and extragenomic roles for these receptors during postnatal development of the hippocampus.

Animals↗
PubMed1991

Glial cells express both mineralocorticoid and glucocorticoid receptors.

In the brain, the action of glucocorticoid steroids is mediated via two intracellular receptors, the mineralocorticoid (MR), or type I receptor, and the glucocorticoid (GR), or type II receptor. These receptors are expressed in many types of neurons and are co-expressed in some neurons such as the hippocampal pyramidal cells. Although glucocorticoids are known to affect gliogenesis and glial cell differentiation, the expression of the GR in different types of glial cells throughout the brain has not been thoroughly studied and the expression of the MR in glia not previously reported. Here we review studies suggesting that both receptors are expressed in astrocytes and oligodendrocytes.

Animals↗
PubMed1991

The biphasic modulation of inhibin mRNA levels and secretion by PMSG in rat granulosa cells in vitro.

Granulosa cell cultures derived from diethylstilboestrol-treated immature rats were used to study the in vitro effect of pregnant mare serum gonadotrophin (PMSG) on steady state mRNA levels for the inhibin alpha and beta A subunits and the secretion of immunoreactive inhibin and progesterone. After 48 h treatment the dose-response curve of PMSG revealed a maximum stimulation (2.5-3.5 fold) of cytosolic alpha and beta A mRNAs over the range of 1 to 10 mU PMSG mL-1, with corresponding stimulation of inhibin secretion. A high dose of PMSG (160-500 mU mL-1) clearly suppressed inhibin alpha mRNA levels as well as inhibin secretion, whereas progesterone (P) was maximally stimulated (up to 600 fold). Although the level of cytosolic inhibin beta A subunit mRNA was also down-regulated by a high concentration of PMSG in the culture medium, the doses required to suppress its mRNA level to less than those of the control varied. These data demonstrate that low doses of follicle stimulating hormone/luteinizing hormone (FSH/LH)-like (PMSG) activity enhances and high doses decrease the steady-state mRNA levels of inhibin in rat granulosa cells in vitro; this biphasic regulation in vitro reflects the differential regulation of inhibin secretion observed during the rat oestrous cycle.

Animals↗
PubMed1990

Characterization of multiple mRNAs originating from the chicken progesterone receptor gene. Evidence for a specific transcript encoding form A.

The structure of the 42-kilobase (kb) long chicken progesterone receptor (cPR) gene and of all six transcripts that are detectable on Northern blots was determined. The first of 8 exons encodes the N-terminal region A/B which is highly divergent among different species and contains a constitutive transcription activation function. The DNA (DBD)- and hormone-binding domains (HBD) are assembled from 2 and 5 exons, respectively, with the individual "zinc fingers" of the DBD encoded by separate exons. In addition to the previously described 4.5-kb cPR mRNA species, alternative polyadenylation, splicing variation, and "5'-truncation" lead to the generation of 5 further mRNAs. Most importantly, this 5'-truncation produces, by an as yet unidentified mechanism, an abundant transcript which encodes form A but not form B of cPR. Lack of splicing at the exon 2 splice-donor and polyadenylation due to a signal site in the second intron generates a previously undetected 3.4-kb mRNA species. The corresponding cDNA was sequenced in its entirety and shown to encode only region A/B and the N-terminal "finger" of the DBD. Alternative polyadenylation upstream of the signal site for the 4.5-kb mRNA is responsible for the appearance of a 3.3-kb mRNA. The longest cPR mRNA (8.2 kb) originates from a transcription termination point more than 3 kb downstream of the 4.5-kb mRNA 3'-end. Finally, the primary sequence of more than 2 kb upstream sequences of the cPR gene, containing several consensus hexamer progestin/glucocorticoid receptor-binding sites (PRE/GRE and putative Sp1 binding motifs, is discussed.

Amino Acid Sequence↗
PubMed1990

Characterization of 11 beta-hydroxysteroid dehydrogenase gene expression: identification of multiple unique forms of messenger ribonucleic acid in the rat kidney.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11-HSD) appears to be involved in mediating aldosterone specificity of otherwise nonselective type I receptors in mineralocorticoid target tissues. In the present study gene expression of 11-HSD was characterized in various tissues of the rat by use of a complementary DNA probe coding for the rat liver 11-HSD. In the liver, lung, testis, colon, heart, hippocampus, and kidney papilla a single message was observed of length approximately 1700 nucleotides (nt). In the kidney cortex/medulla, however, messenger RNA (mRNA) species were observed at 1900 nt, 1600 nt and 1500 nt, and deadenylation studies showed that the renal 1900 nt species was heterogeneous. Northern blot analysis of 11-HSD mRNA showed low levels of expression in the kidney of the neonate and much higher levels in liver and lung with expression increasing markedly in all three tissues over development. In mature rats, a low salt diet significantly elevated 11-HSD mRNA in the liver but not in other tissues. We interpret these data as evidence for the existence of a family of 11-HSD genes, and consistent with the possibility that the hepatic species may modulate occupancy of type II (classical) glucocorticoid rather than type I receptors.

11-beta-Hydroxysteroid Dehydrogenases↗