Nongastric H-K-ATPase in rodent prostate: lobe-specific expression and apical localization

2002 ◽  
Vol 282 (4) ◽  
pp. C907-C916 ◽  
Author(s):  
Nikolay B. Pestov ◽  
Tatyana V. Korneenko ◽  
Gail Adams ◽  
Manoranjani Tillekeratne ◽  
Mikhail I. Shakhparonov ◽  
...  
Keyword(s):  
Lateral Lobe ◽  
Distal Colon ◽  
Pcr Analysis ◽  
Protein Abundance ◽  
Ventral Lobe ◽  
Rt Pcr ◽  
Specific Expression ◽  
Α Subunit ◽  
Apical Membranes ◽  
Coagulating Gland

The molecular basis of active ion transport in secretory glands such as the prostate is not well characterized. Rat nongastric H-K-ATPase is expressed at high levels in distal colon surface cell apical membranes and thus is referred to as “colonic.” Here we show that the ATPase is expressed in rodent prostate complex in a lobe-specific manner. RT-PCR and Western blot analyses indicate that rat nongastric H-K-ATPase α-subunit (αng) mRNA and protein are present in coagulating gland (anterior prostate) and lateral and dorsal prostate and absent from ventral lobe, whereas Na-K-ATPase α-subunit is present in all lobes. RT-PCR analysis shows that Na-K-ATPase α4 and α3 and gastric H-K-ATPase α-subunit are not present in significant amounts in all prostate lobes. Relatively low levels of Na-K-ATPase α2were found in lateral, dorsal, and anterior lobes. αngprotein expression is anteriodorsolateral: highest in coagulating gland, somewhat lower in dorsal lobe, and even lower in lateral lobe. Na-K-ATPase protein abundance has the reverse order: expression in ventral lobe is higher than in coagulating gland. αngprotein abundance is higher in coagulating gland than distal colon membranes. Immunohistochemistry shows that in rat and mouse coagulating gland epithelium αng protein has an apical polarization and Na-K-ATPase α1 is localized in basolateral membranes. The presence of nongastric H-K-ATPase in rodent prostate apical membranes may indicate its involvement in potassium concentration regulation in secretions of these glands.

Identification of a gene cluster for cell-surface genes of the SRS superfamily inNeospora caninumand characterization of the novelSRS9gene

Parasitology ◽  
2011 ◽  
Vol 138 (14) ◽  
pp. 1832-1842 ◽  
Author(s):  
V. RISCO-CASTILLO ◽  
V. MARUGÁN-HERNÁNDEZ ◽  
A. FERNÁNDEZ-GARCÍA ◽  
A. AGUADO-MARTÍNEZ ◽  
E. JIMÉNEZ-RUIZ ◽  
...  
Keyword(s):  
Western Blot ◽  
Gene Cluster ◽  
Neospora Caninum ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Initial Characterization ◽  
Hydrophilic Region

SUMMARYHere we present the detection of a gene cluster forNeospora caninumsurface genes, similar to theToxoplasma gondiiSRS9 locus, and the cloning and characterization of the NcSRS9gene. PCR genome walking, using NcBSR4gene as a framework, allows the identification, upstream NcBSR4, of 2 sequences homologous to theSRS5and the Ubiquinol-cytochrome C reductase genes and, downstream NcBSR4, of an ORF of 1191 bp coding for a 396-amino acid polypeptide with 59% similarity to the TgSRS9 antigen. A putative 39-residue signal peptide was found at the NH2-terminus followed by a hydrophilic region, and a potential site for a glycosylphosphatidylinositol anchor at the COOH-terminus. A recombinant NcSRS9 protein was produced and was recognized on a Western blot by a low proportion of sera from a panel of naturally infected cows and calves. In addition, Western blot analysis using polyclonal anti-rNcSRS9 revealed stage-specific expression of NcSRS9 in bradyzoites but not in tachyzoites, and immunohistochemistry on brain from a congenitally infected calf showed NcSRS9 recognition in bradyzoites contained in tissue cysts. However, bradyzoite-specific expression of NcSRS9 could not be proven by immunofluorescence on bradyzoites obtainedin vitroand RT-PCR analysis showed no significant variations of NcSRS9transcripts duringin vitrotachyzoite-bradyzoite switch, probably due to incomplete maturity ofin vitrobradyzoites. Initial characterization of NcSRS9 in this study may lead to further studies for a better understanding ofN. caninumpersistence.

Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells

1998 ◽  
Vol 274 (2) ◽  
pp. G424-G429 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
Satish K. Singh ◽  
John Geibel ◽  
Henry J. Binder
Keyword(s):  
Spatial Distribution ◽  
Atpase Activity ◽  
Distal Colon ◽  
Compelling Evidence ◽  
Α Subunit ◽  
Colonic Crypts ◽  
Rat Distal Colon ◽  
Acid Load ◽  
Apical Membranes ◽  
Crypt Cells

Two distinct colonic H+-K+-adenosinetriphosphatase (H+-K+-ATPase) isoforms can be identified in part on the basis of their sensitivity to ouabain. The colonic H+-K+-ATPase α-subunit (HKcα) was recently cloned, and its message and protein are present in surface (and the upper 20% of crypt) cells in the rat distal colon. These studies were performed to establish the spatial distribution of the ouabain-sensitive and ouabain-insensitive components of both H+-K+-ATPase activity in apical membranes prepared from surface and crypt cells and K+-dependent intracellular pH (pHi) recovery from an acid load both in isolated perfused colonic crypts and in surface epithelial cells. Whereas H+-K+-ATPase activity in apical membranes from surface cells was 46% ouabain sensitive, its activity in crypt apical membranes was 96% ouabain sensitive. Similarly, K+-dependent pHi recovery in isolated crypts was completely ouabain sensitive, whereas in surface cells K+-dependent pHi recovery was insensitive to ouabain. These studies provide compelling evidence that HKcα encodes the colonic ouabain-insensitive H+-K+-ATPase and that a colonic ouabain-sensitive H+-K+-ATPase isoform is present in colonic crypts and remains to be cloned and identified.

scholarly journals The Stimulatory G Protein α-Subunit Gsα Is Imprinted in Human Thyroid Glands: Implications for Thyroid Function in Pseudohypoparathyroidism Types 1A and 1B

2003 ◽  
Vol 88 (9) ◽  
pp. 4336-4341 ◽  
Author(s):  
Jie Liu ◽  
Beth Erlichman ◽  
Lee S. Weinstein
Keyword(s):  
G Protein ◽  
Human Thyroid ◽  
Pcr Analysis ◽  
Specific Expression ◽  
Α Subunit ◽  
Albright Hereditary Osteodystrophy ◽  
Camp Generation ◽  
Gsα Gene ◽  
Stimulatory G Protein ◽  
G Protein Α Subunit

The stimulatory G protein α-subunit Gsα couples receptors to adenylyl cyclase and is required for hormone-stimulated cAMP generation. In Albright hereditary osteodystrophy, heterozygous Gsα null mutations only lead to PTH, TSH, and gonadotropin resistance when inherited maternally [pseudohypoparathyroidism type 1A; (PHP1A)]. Maternal-specific expression of Gsα in specific hormone targets could explain this observation. Using hot-stop PCR analysis on total RNA from six normal human thyroid specimens, we showed that the majority of the Gsα mRNA (72 ± 3%) was derived from the maternal allele. This is consistent with the presence of TSH resistance in patients with maternal Gsα null mutations (PHP1A) and the absence of TSH resistance in patients with paternal Gsα mutations (pseudopseudohypoparathyroidism). Patients with PTH resistance in the absence of Albright hereditary osteodystrophy (PHP1B) have an imprinting defect of the Gsα gene resulting in both alleles having a paternal epigenotype, which would lead to a more moderate level of thyroid-specific Gsα deficiency. We found evidence of borderline TSH resistance in 10 of 22 PHP1B patients. This study provides further evidence for tissue-specific imprinting of Gsα in humans and provides a potential mechanism for mild to moderate TSH resistance in PHP1A and borderline resistance in some patients with PHP1B.

Expression of HKα2 protein is increased selectively in renal medulla by chronic hypokalemia

1998 ◽  
Vol 275 (3) ◽  
pp. F433-F440 ◽  
Author(s):  
Juan Codina ◽  
Juan T. Delmas-Mata ◽  
Thomas D. DuBose
Keyword(s):  
Plasma Membranes ◽  
Renal Cortex ◽  
Renal Medulla ◽  
Distal Colon ◽  
Northern Analysis ◽  
Protein Abundance ◽  
Α Subunit ◽  
Chronic Metabolic Acidosis ◽  
Rat Distal Colon ◽  
Selective Increase

Our laboratory has demonstrated by Northern analysis that chronic hypokalemia increases HKα2 (i.e., α-subunit of the colonic H+-K+-ATPase) mRNA abundance in the rat. To determine whether the increase in mRNA correlated with an increase in HKα2 protein, an antibody was raised against a synthetic peptide derived from amino acids 686–698 of the HKα2sequence. The anti-HKα2 antibody hybridized to rat distal colon membranes which migrated at ∼100 kDa (expected mobility of HKα2). HKα2 protein was not detected in plasma membranes from rat whole kidney or stomach (100 μg) derived from control animals. The antibody was then used to investigate changes in expression of HKα2 in renal cortex, renal medulla, and distal colon in two pathophysiological conditions: 1) chronic hypokalemia (LK) and 2) chronic metabolic acidosis (CMA). In LK rats there was a marked, but selective, increase in the abundance of HKα2 protein in membranes prepared from renal medulla. Nevertheless, a corresponding increase in HKα2 protein abundance was not observed in membranes prepared from the distal colon of LK rats. HKα2 protein abundance in CMA was indistinguishable from controls. Moreover, chronic hypokalemia had no effect on expression of α1-Na+-K+-ATPase or HKα1 in kidney or distal colon under any experimental condition. Therefore, HKα2 protein is tissue- and site-specifically upregulated in response to chronic hypokalemia but not by CMA. Furthermore, this regulatory response is localized to the renal medulla.

scholarly journals Allele-specific expression patterns of interleukin-2 and Pax-5 revealed by a sensitive single-cell RT-PCR analysis

2000 ◽  
Vol 10 (13) ◽  
pp. 789-792 ◽  
Author(s):  
Kristina L. Rhoades ◽  
Nandita Singh ◽  
Itamar Simon ◽  
Barbara Glidden ◽  
Howard Cedar ◽  
...  
Keyword(s):  
Single Cell ◽  
Expression Patterns ◽  
Interleukin 2 ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Specific Expression ◽  
Allele Specific Expression ◽  
Allele Specific

scholarly journals Detrimental Effects of Hypoxia-Specific Expression of Uracil DNA Glycosylase (Ung) in Mycobacterium smegmatis

2010 ◽  
Vol 192 (24) ◽  
pp. 6439-6446 ◽  
Author(s):  
Krishna Kurthkoti ◽  
Umesh Varshney
Keyword(s):  
Dna Repair ◽  
Excision Repair ◽  
Dna Glycosylase ◽  
Pcr Analysis ◽  
Bacterial Survival ◽  
Rt Pcr ◽  
Repair Gene ◽  
Specific Expression ◽  
Uracil Dna Glycosylase ◽  
Oxygen Levels

ABSTRACT Mycobacterium tuberculosis is known to reside latently in a significant fraction of the human population. Although the bacterium possesses an aerobic mode of metabolism, it adapts to persistence under hypoxic conditions such as those encountered in granulomas. While in mammalian systems hypoxia is a recognized DNA-damaging stress, aspects of DNA repair in mycobacteria under such conditions have not been studied. We subjected M ycobacterium smegmatis, a model organism, to the Wayne's protocol of hypoxia. Analysis of the mRNA of a key DNA repair enzyme, uracil DNA glycosylase (Ung), by real-time reverse transcriptase PCR (RT-PCR) revealed its downregulation during hypoxia. However, within an hour of recovery of the culture under normal oxygen levels, the Ung mRNA was restored. Analysis of Ung by immunoblotting and enzyme assays supported the RNA analysis results. To understand its physiological significance, we misexpressed Ung in M. smegmatis by using a hypoxia-responsive promoter of narK2 from M. tuberculosis. Although the misexpression of Ung during hypoxia decreased C-to-T mutations, it compromised bacterial survival upon recovery at normal oxygen levels. RT-PCR analysis of other base excision repair gene transcripts (UdgB and Fpg) suggested that these DNA repair functions also share with Ung the phenomenon of downregulation during hypoxia and recovery with return to normal oxygen conditions. We discuss the potential utility of this phenomenon in developing attenuated strains of mycobacteria.

Identification of the β-subunit for nongastric H-K-ATPase in rat anterior prostate

2004 ◽  
Vol 286 (6) ◽  
pp. C1229-C1237 ◽  
Author(s):  
Nikolay B. Pestov ◽  
Tatyana V. Korneenko ◽  
Rossen Radkov ◽  
Hao Zhao ◽  
Mikhail I. Shakhparonov ◽  
...  
Keyword(s):  
Structural Organization ◽  
Anterior Lobe ◽  
Β Subunit ◽  
Rt Pcr ◽  
Α Subunit ◽  
Direct Demonstration ◽  
Subunit Isoforms ◽  
Anterior Prostate ◽  
Apical Membranes

The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase α-subunit (αng) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to α1-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase β-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of β-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three β-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is β1 > β3 ≫ β2. Immunohistochemical experiments demonstrate that β1 is present predominantly in apical membranes, coinciding with αng, whereas β3 is localized in the basolateral compartment, coinciding with α1. This is the first direct demonstration of the αng-β1 colocalization in situ indicating that, in rat AP, αng associates only with β1. The existence of αng-β1 complex has been confirmed by immunoprecipitation experiments. These results indicate that β1-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.

Renal principal cell-specific expression of green fluorescent protein in transgenic mice

2002 ◽  
Vol 283 (6) ◽  
pp. F1351-F1364 ◽  
Author(s):  
Ludmilla Zharkikh ◽  
Xiaohong Zhu ◽  
Peter K. Stricklett ◽  
Donald E. Kohan ◽  
Greg Chipman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Intercalated Cells ◽  
Rt Pcr ◽  
Specific Expression ◽  
Principal Cells ◽  
Green Fluorescent

The purpose of this study is to develop transgenic mice with principal cell-specific expression of green fluorescent protein (GFP). After the cloning and sequencing of the mouse aquaporin-2 (AQP2) gene, 9.5 kb of the promoter were used to drive expression of GFP in transgenic mice. In transgenic mice, GFP was selectively expressed in principal cells of the renal collecting duct and not in intercalated cells. Expression was increased by dehydration of mice. AQP2 and GFP expression was maintained in primary cultures of renal medulla that were stimulated with cAMP or vasopressin analogs. GFP-expressing cells were then isolated by fluorescence-activated cell sorting. RT-PCR analysis showed expression of AQP2, AQP3, AQP4, vasopressin type 2 receptor, and cAMP response element binding protein but not H+-ATPase B1 subunit or anion exchanger 1. After expansion of these cells in culture, RT-PCR analysis showed continued expression of the same genes. This pattern of gene expression is that of principal cells rather than intercalated cells. This transgenic mouse model can be used in future studies of gene expression during the development, differentiation, and maturation of renal principal cells.

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