Presence and distribution of endothelin-1 gene expression in human kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F679-F687 ◽  
Author(s):  
C. Pupilli ◽  
M. Brunori ◽  
N. Misciglia ◽  
C. Selli ◽  
L. Ianni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Renal Cortex ◽  
Cdna Probe ◽  
Renal Medulla ◽  
Human Kidney ◽  
Renal Tumors ◽  
Mrna Levels

To investigate the presence and the distribution of preproendothelin-1 (prepro-ET-1) mRNA in human kidney, eight human kidneys obtained at surgery from patients affected by localized renal tumors were studied. Northern blot analysis using a human prepro-ET-1 cDNA probe labeled with 32P showed the presence of a single band of approximately 2.3 kb that was present both in the renal cortex and medulla of all the kidneys studied. Densitometric analysis of hybridization signals demonstrated that prepro-ET-1 mRNA levels in the renal medulla were 2.2-fold higher than those in the renal cortex. The distribution of prepro-ET-1 mRNA in human kidney was investigated by in situ hybridization using a human prepro-ET-1 RNA probe labeled with 35S. The greatest density of prepro-ET-1 mRNA was observed in the renal medulla, where hybridization signal was demonstrated in vasa recta bundles and capillaries and in collecting ducts. By combining in situ hybridization with immunohistochemical detection of von Willebrand factor, we demonstrated that 93 +/- 2.5% of nontubular medullary cells containing prepro-ET-1 mRNA were endothelial cells. In the cortex, prepro-ET-1 mRNA was localized in the endothelial layer of arcuate and interlobular arteries and veins and in the endothelial cells of afferent arterioles. The results of the present study demonstrate that ET-1 gene expression is present in vascular and tubular structures of the human kidney. It is possible that ET-1 synthesized locally in the human kidney represents a local system affecting renal hemodynamics and functions through paracrine and/or autocrine actions on different renal structures.

Cell-specific metallothionein gene expression in mouse decidua and placentae

Development ◽  
1989 ◽  
Vol 107 (3) ◽  
pp. 611-621 ◽  
Author(s):  
S.K. De ◽  
M.T. McMaster ◽  
S.K. Dey ◽  
G.K. Andrews
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Embryo Implantation ◽  
Normal Pregnancy ◽  
Mrna Levels ◽  
Metallothionein Gene ◽  
Visceral Yolk Sac ◽  
Rna In Situ Hybridization ◽  
Low Levels

Oligodeoxyribonucleotide excess solution hybridization, Northern blot and in situ hybridization were used to analyze metallothionein gene expression in mouse decidua and placentae during gestation. Metallothionein (MT) -I and -II mRNA levels were constitutively elevated, 11- and 13-fold, respectively, relative to the adult liver, in the deciduum (D8), and decreased coordinately about 6-fold during the period of development when the deciduum is replaced by the developing placenta (D10-16). Coincident with this decline, levels of MT mRNA increased dramatically in the visceral yolk sac endoderm. In situ hybridization established that MT-I mRNA was present at low levels in the uterine luminal epithelium (D4), but was elevated at the site of embryo implantation exclusively in the primary decidual zone by D5, and then in the secondary decidual zone (D6-8). Although low levels of MT mRNA were detected in total placental RNA, in situ hybridization revealed constitutively high levels in the outer placental spongiotrophoblasts. Analysis of pulse-labeled proteins from decidua and placentae established that these tissues are active in the synthesis of MT. The constitutively high levels of MT mRNA in decidua were only slightly elevated following injection of cadmium (Cd) and/or zinc (Zn), whereas in placentae they increased several-fold. MT mRNA levels were equally high in decidua and experimentally induced deciduomata (D8) which establishes that decidual MT gene expression is not dependent on the presence of the embryo or some embryo-derived factor. Although the functional role of MT during development is speculative, these results establish the concept that, from the time of implantation to late in gestation, the mouse embryo is surrounded by cells, interposed between the maternal and embryonic environments, which actively express the MT genes. This suggests that MT plays an important role in the establishment and maintenance of normal pregnancy.

scholarly journals Immunohistochemistry and in situ hybridization of P-45017 alpha (17 alpha-hydroxylase/17,20-lyase).

1992 ◽  
Vol 40 (7) ◽  
pp. 903-908 ◽  
Author(s):  
T Suzuki ◽  
H Sasano ◽  
T Sawai ◽  
J I Mason ◽  
H Nagura
Keyword(s):  
In Situ Hybridization ◽  
Leydig Cells ◽  
Cellular Localization ◽  
Cdna Probe ◽  
Simultaneous Detection ◽  
Seminiferous Tubules ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Theca Interna

Cytochrome P-45017 alpha catalyzes both 17 alpha-hydroxylation and 17,20-side-chain cleavage in steroidogenesis and lies at a key branch point in the pathways of steroid hormone biosynthesis. To obtain information on the precise localization of P-45017 alpha in swine testis, ovary, and adrenal, we undertook the simultaneous detection of P-45017 alpha mRNA and protein by combining immunohistochemistry with in situ hybridization. In situ hybridization was performed on 4% paraformaldehyde-fixed, paraffin-embedded sections by employing either a 39-base oligomer or a cDNA insert (1.7 KB) of porcine testis P-45017 alpha as DNA probe. Immunohistochemical study was performed by employing anti-P-45017 alpha. Hybridization signals were obtained in Leydig cells of the testis, theca interna of the ovarian follicle, and zona fasciculata reticularis cells of the adrenal cortex. Oligonucleotide probing yielded lower background signal than the cDNA probe. No specific signals were obtained in seminiferous tubules of the testis, medulla, and zona glomerulosa of the adrenal, and in membrana granulosa and interstitial cells of the ovary. Hybridization signals were obtained in the cells where immunoreactivity of the enzyme was observed by immunohistochemistry, except for some Leydig cells of the testis and theca interna cells of the ovary in which only immunoreactivity but not hybridization signal was observed. The present study provided detailed information about the precise cellular localization of P-45017 alpha expression at both the protein and mRNA levels in swine adrenal glands and gonads. This approach of simultaneous immunohistochemistry and in situ hybridization analysis of steroidogenic enzymes can be applied in the future to tissues exhibiting abnormal steroid metabolism and should contribute to a better understanding of steroidogenesis.

scholarly journals Localization of protamine 1 mRNA in different stages of the cycle of the rat seminiferous epithelium.

1988 ◽  
Vol 107 (2) ◽  
pp. 407-412 ◽  
Author(s):  
P Mali ◽  
M Sandberg ◽  
E Vuorio ◽  
P C Yelick ◽  
N B Hecht ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Northern Blot ◽  
Northern Blot Analysis ◽  
Cdna Probe ◽  
Seminiferous Epithelium ◽  
Seminiferous Tubules ◽  
Mrna Levels ◽  
Low Levels ◽  
Protamine 1

A mouse protamine 1 cDNA probe was used to study P1 protamine gene expression during the cycle of the seminiferous epithelium in the rat. In situ hybridization experiments showed that transcription of the P1 protamine mRNA starts in the middle of step 7 of spermiogenesis during substage VIIc. The mRNA levels stay high in steps 7-14 spermatids but decrease during steps 15-16 and are virtually undetectable in steps 17-19 spermatids. Northern blot analyses of RNAs isolated from microdissected pools of seminiferous tubules show high P1 protamine mRNA concentrations during stages VIIc-XIV-III of the cycle and lower levels during stages IV-VIIb. Owing to a post-transcriptional shortening of the poly(A) tail by 130 bases, a decrease in the size of protamine 1 mRNA from approximately 580 to 450 nucleotides was observed in stages XIII-XIV suggesting an initiation of protamine 1 synthesis in step 13-14 spermatids. In stages II-VI (steps 16-18 spermatids), only the smaller size protamine 1 mRNA was detectable. The expression of protamine 1 mRNAs has been localized in the very last phase of the haploid gene activity. Although the in situ hybridization suggests a disappearance of protamine 1 mRNA after step 16 of spermiogenesis, Northern blot analysis shows that low levels of mRNA are present during the period of final condensation of the chromatin, reflecting the association of protamine with DNA.

Quantitative in-situ hybridization histochemistry in the rat pituitary gland: effect of bromocriptine on prolactin and pro-opiomelanocortin gene expression

1988 ◽  
Vol 118 (2) ◽  
pp. 205-NP ◽  
Author(s):  
A. Levy ◽  
S. L. Lightman
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Messenger Rna ◽  
Cdna Probe ◽  
Intermediate Lobe ◽  
Hybridization Histochemistry ◽  
In Situ Hybridization Histochemistry ◽  
Pomc Mrna ◽  
Prolactin Mrna

ABSTRACT The temporal effect of orally administered bromocriptine on pro-opiomelanocortin (POMC) and prolactin gene expression in male Sprague–Dawley rats was examined using in-situ hybridization histochemistry. Messenger RNA in the anterior and intermediate lobes could be clearly delineated in each section. Administration of bromocriptine resulted in a reduction in hybridization of 35S-labelled cDNA probe to prolactin mRNA from 0·69 × 1012 to 0·29 × 1012 copies bound/g after 150 h. POMC mRNA in the anterior lobe remained unchanged with 0·08 × 1012 copies of probe bound/g for the duration of the experiment, while in the intermediate lobe it decreased from 2·44 × 1012 to 0·44 × 1012 copies of probe bound/g at 150 h. The rate of reduction in intermediate lobe POMC mRNA was similar to that of prolactin mRNA for the first 24 h but was subsequently more rapid and more profound, falling to 20% of the control value at 84 h and to 18% at 150 h. J. Endocr. (1988) 118, 205–210

Expression of Tissue Kallikrein in Human Kidney

1994 ◽  
Vol 87 (1) ◽  
pp. 5-11 ◽  
Author(s):  
Allan D. Cumming ◽  
Timothy Walsh ◽  
Davina Wojtacha ◽  
Stewart Fleming ◽  
David Thomson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nephrotic Syndrome ◽  
In Situ Hybridization ◽  
Blood Vessels ◽  
Human Tissue ◽  
Human Kidney ◽  
Distal Tubule ◽  
Tissue Kallikrein ◽  
Peripolar Cells

1. We studied the distribution of human tissue kallikrein mRNA in normal and diseased kidney, using in situ hybridization, together with immunohistochemical localization of renal kallikrein protein. Materials studied were (a) normal tissue from kidneys removed because of localized renal carcinoma, (b) kidneys removed because of post-traumatic haemorrhage and (c) renal biopsy specimens from patients with membranous glomerulonephritis and nephrotic syndrome. 2. A 1.35 kb EcoRI fragment of human tissue kallikrein cDNA was labelled with [32P]dCTP using the random-primer technique, and used for in situ hybridization. A specific rabbit antibody to active human urinary kallikrein was employed for immunocytochemistry, using a peroxidase-antiperoxidase method. 3. By in situ hybridization, no tissue kallikrein gene expression was seen in the carcinoma nephrectomy specimens. Positive expression was seen in the trauma nephrectomy tissue, and in four of five nephrotic syndrome biopsies. In all kidneys, expression was confined to the renal cortex. The dominant site of gene expression was the distal tubule. Apart from one area of positive signal related to an epithelial cell of Bowman's capsule, expression was not observed in glomeruli. Expression was also seen in the walls of large- and medium-sized blood vessels. 4. By immunohistochemistry, the dominant site of immunoreactivity was the distal tubule. Dense staining was also seen in granular peripolar cells and in isolated parietal epithelial cells close to the vascular pole. Isolated immunoreactive cells were seen in the media of large- and medium-sized arteries. 5. The tissue kallikrein gene in the kidney may not be constitutively expressed, but is expressed in response to physiological or pathological stimuli. The dominant site of gene expression and tissue kallikrein localization is the distal tubule. Although kallikrein is present in granular peripolar cells in human kidney, mRNA was not located at this site. It is possible that kallikrein in these cells derives from absorption from glomerular filtrate. Tissue kallikrein mRNA and protein are present in the walls of blood vessels in human kidney. The significance of vascular kallikrein expression requires further investigation.

Relaxin gene expression in the porcine follicle during preovulatory development induced by gonadotrophins

1990 ◽  
Vol 5 (3) ◽  
pp. 211-219 ◽  
Author(s):  
C. A. Bagnell ◽  
W. Tsark ◽  
L. Tashima ◽  
B. R. Downey ◽  
B. K. Tsang ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Ovarian Function ◽  
Relative Concentration ◽  
Molecular Size ◽  
Northern Analysis ◽  
Mrna Levels ◽  
In Vitro Production

ABSTRACT Northern analysis was used to identify relaxin gene expression in ovaries of prepubertal pigs primed with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG). The cellular distribution of relaxin transcript in the developing follicle was localized by in-situ hybridization histochemistry. Three probes complementary to non-overlapping regions of the porcine prorelaxin molecule were used to identify relaxin gene expression in ovarian follicular tissue collected 0, 48, 60, 72 and 84 h after treatment with PMSG/hCG. A 1 kb transcript was detected in ovarian extracts of prepubertal gilts from 48 to 84 h after PMSG stimulation. This corresponds to the molecular size of the relaxin transcript reported in the pregnant sow ovary. Relaxin mRNA levels increased in ovaries from animals 48 through 84 h after PMSG. In-situ hybridization showed that the site of relaxin synthesis was the theca interna layer of the developing follicle. Relaxin mRNA was not observed in other follicular cell types, in small or atretic follicles or in follicles from unstimulated animals. The distribution and relative concentration of relaxin mRNA showed a good correlation with in-vitro production and immunohistochemical localization of relaxin previously reported in the developing pig follicle. The presence of both protein and mRNA for relaxin in the growing follicle supports a role for relaxin as a local regulator of ovarian function.

Sucrase-isomaltase gene expression along crypt-villus axis of human small intestine is regulated at level of mRNA abundance

1992 ◽  
Vol 262 (1) ◽  
pp. G123-G130 ◽  
Author(s):  
P. G. Traber ◽  
L. Yu ◽  
G. D. Wu ◽  
T. A. Judge
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Stem Cell Population ◽  
Specific Gene ◽  
Mrna Levels ◽  
Human Small Intestine ◽  
Crypt Cells

The mucosal lining of the small intestine is a complex epithelium that is continually renewed by division of a stem cell population located in intestinal crypts, migration of daughter cells along the villus, and, finally, extrusion of senescent cells into the lumen. The majority of cells in both crypt and villus cell compartments are enterocytes that acquire differentiated functions as they migrate out of the crypt. Sucrase-isomaltase (SI) is an enterocyte-specific, brush-border enzyme that has little activity in crypt cells and maximal activity in low and mid villus cells. The mechanism by which enterocytes acquire SI enzymatic activity as they move from crypt to villus is controversial. In this study we examined the distribution of SI mRNA along the crypt-villus axis of human small intestine using isolated epithelial cells and in situ hybridization. A complementary DNA to the 5' portion of the human SI mRNA was amplified and cloned using the polymerase chain reaction. Hybridization analysis of RNA extracted from human intestinal epithelial cells showed that the cloned cDNA recognized a single 6.5-kb mRNA. In situ hybridization of duodenal biopsy specimens was performed using a single-stranded RNA probe derived from this cDNA. This analysis showed that there was little SI mRNA in crypt cells and appearance of mRNA in enterocytes located at the crypt-villus junction. The mRNA levels were maximal in lower and mid villus cells with decreased levels noted in villus tip cells. These results are identical to those previously described in rat intestine and suggest that expression of the SI gene as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation. Study of SI gene regulation may provide a useful model to investigate the mechanisms that regulate enterocyte-specific gene expression and intestinal differentiation.

scholarly journals Cellular analysis by in situ hybridization and immunoperoxidase of alpha-fetoprotein and albumin gene expression in rat liver during the perinatal period.

1986 ◽  
Vol 103 (3) ◽  
pp. 777-786 ◽  
Author(s):  
A M Poliard ◽  
D Bernuau ◽  
I Tournier ◽  
L G Legrès ◽  
D Schoevaert ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Situ Hybridization ◽  
Rat Liver ◽  
Cdna Probe ◽  
Relative Number ◽  
Perinatal Period ◽  
Alpha Fetoprotein ◽  
Cellular Analysis ◽  
Afp Mrna

To analyze at the cellular level the decrease in alpha-fetoprotein (AFP) gene expression during the early postnatal growth, we searched for AFP gene transcripts by in situ hybridization using a specific cDNA probe, and for the corresponding protein by immunocytochemistry, on rat liver sections at various times of the perinatal period. The relative number of mRNA sequences was evaluated by Northern blot analysis. Albumin (ALB) gene expression was studied simultaneously with the same techniques. In 17-19-d-old fetuses all hepatocytes express simultaneously, for both genes, the mRNAs and the corresponding proteins. During the first postnatal weeks, at a time when the global number of AFP mRNA molecules decreases, all hepatocytes still contain cytoplasmic transcripts and protein. A zonal heterogeneity in the level of AFP gene expression develops around the first week, a higher number of gene products being detected in perivenous than in periportal hepatocytes. This heterogeneity persists until the fourth week when AFP mRNA sequences and protein are barely detectable. All hepatocytes express the ALB gene after birth, but at around the second week, a periportal intensification of the in situ hybridization signal and immunostaining becomes apparent. Our data indicate that co-expression of the AFP and ALB genes by all hepatocytes is a normal step in liver ontogeny; the diminution of AFP gene expression after birth is not the result of the disappearance of specialized cell clones; and zonal quantitative differences in the level of AFP and ALB gene expression are observed within the maturing liver lobule.

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