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Biomedical subjects

A K Grover

Publications and source records attributed to A K Grover.

At least 19 recordsLinked to original sources
PubMed1992

Polymerase chain reaction assay of mRNA using 28S rRNA as internal standard.

A method is developed for PCR assay of mRNA using 28S rRNA as internal standard and its applicability is reported for 4 types of mRNA. It consists of reverse transcription of total RNA using primers selective for 28S rRNA and the message of interest. Routinely the 28S reaction mix is diluted 20,000-fold and the dilution error is minimized by adding 10 microCi of 3H2O before the dilution and determining the level of 3H before and after the dilution. Aliquots from the diluted 28S mix and the undiluted reverse transcripts are co-amplified by PCR in the presence of 32P-dATP. The samples are analyzed by electrophoresis, bands detected by autoradiography and in each lane the ratio is determined between cpm in the band of interest and cpm in the 28S band. The ratio is a measure of the level of mRNA in the sample and can be used to monitor changes in mRNA during treatments, development and pathogenesis.

Animals↗
PubMed1992

Calcium pump isoforms: diversity, selectivity and plasticity. Review article.

Ca2+ pumps are essential for removing cytosolic Ca2+ either across the plasma membrane (PM) or into internal organelles such as the sarcoplasmic reticulum (SR). Four genes (PMCA1, PMCA2, PMCA3 and PMCA4) have been reported to encode the PM Ca2+ pumps and three (SERCA1, SERCA2 and SERCA3) to encode the SR Ca2+ pumps. The PM Ca2+ pumps are stimulated by calmodulin, the SR Ca2+ pumps encoded by SERCA1 and SERCA2 are stimulated by phospholamban while the product of SERCA3 may be regulated directly by cAMP-dependent protein kinase. Alternative splicing of the primary transcripts of several of these genes has been reported to occur in a tissue selective manner and for others to alter during ontogeny. For the PM Ca2+ pump, alternative RNA splicing may result in isoforms with altered cyclic nucleotide dependent protein kinase sensitivity. The diversity in distribution of Ca2+ pump isoforms and their regulatory factors when coupled with different Ca2+ entry mechanisms allows for tissue selectivity and plasticity in stimulus-response coupling. The roles of various Ca2+ pump isoforms, the rationale behind their tissue selective expression and the plasticity in this expression are among the new challenges to researchers in this field.

Animals↗
PubMed1992

Peroxide inactivates calcium pumps in pig coronary artery.

To study the effects of hydrogen peroxide, pig coronary artery smooth muscle subcellular fractions enriched in plasma membrane (F2) or sarcoplasmic reticulum (F3) were incubated in various concentrations of peroxide and 5 mM azide. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ uptake was determined for F3 and phosphate-stimulated uptake for F2. Only 1.5-5 microM hydrogen peroxide was required for 50% inhibition of the Ca2+ uptake by F3, but the corresponding concentration for F2 was 10-50 microM. This effect was not prevented by superoxide dismutase. Hydrogen peroxide inhibited the Ca(2+)-dependent formation of a 115-kDa acylphosphate band in F3 and 140- and 115-kDa bands in F2. The inhibition of Ca2+ uptake in F3, however, exceeded the inhibition of the acylphosphate formation. Efflux of Ca2+ from F2 and F3 was enhanced by hydrogen peroxide but F3 was more sensitive than F2. We conclude that hydrogen peroxide has dual effect on Ca2+ dynamics in the coronary artery smooth muscle, i.e., it inactivates the Ca2+ pumps and increases membrane permeability to Ca2+. The effect is more pronounced on sarcoplasmic reticulum than on plasma membrane. Intrinsic catalase may, however, provide partial protection against such damage.

Animals↗
PubMed1992

Ca pump messenger RNA expression in pregnant rat uterus.

Rat uterus expresses transcripts for the plasma membrane Ca pump PMCA1 and for the sarcoplasmic reticulum Ca pump SERCA2. Uterus contains two mRNA isoforms for PMCA1, one encoding a cyclic nucleotide insensitive Ca pump (PMCA1a) and the other encoding a cyclic nucleotide sensitive pump (PMCA1b). The relative ratio of mRNA to 28S RNA for PMCA1a or PMCA1b and the amount of poly A+ RNA per microgram total RNA obtained from uteri of rats pregnant for 15 days and for parturient rats was similar but the ratio of SERCA2 mRNA to 28S RNA was significantly higher in the parturient rats.

Animals↗
PubMed1992

Adenosine metabolism in small coronary arteries of pig.

Adenosine preferentially relaxes small coronary arteries over large ones, and small bovine coronary arteries are also known to have a higher density of adenosine receptors. Here we report a possible role of adenosine metabolism in this process. Subcellular fractions, from right coronary artery (lumen diameter of 2-3 mm) of pig designated as large coronary artery and its subsequent branches (lumen diameter of 0.5-1 mm) as small coronary arteries, were prepared and characterized. In comparison to the various large artery subcellular fractions, the corresponding small artery fractions were richer in 5'-nucleotidase but poorer in adenosine deaminase. Thus a cascade of events may promote adenosine relaxation in small coronary arteries: higher activity of 5'-nucleotidase leads to production of more adenosine, larger number of receptors allows greater reactivity to adenosine, and lower adenosine deaminase level promotes prolonged action of adenosine.

5'-Nucleotidase↗
PubMed1991

Expression of cyclic-nucleotide-sensitive and -insensitive isoforms of the plasma membrane Ca2+ pump in smooth muscle and other tissues.

cDNA clones encoding the plasma membrane Ca2+ pump isoform PMCA1 were obtained from rabbit stomach smooth muscle. The PMCA1 gene has a 154 base exon which can be alternatively spliced. In splices containing 0, 87 or 114 bases of this exon, the mRNA downstream from this position encodes a protein containing the peptide sequence Lys-Arg-Asn-Ser-Ser (KRNSS), which can be phosphorylated by cyclic-nucleotide-sensitive protein kinase. However, in those splices containing 154 bases, the mRNA encodes a protein that does not contain this sequence. The cDNA clone obtained in this study did not contain the latter exon, and thus it coded for KRNSS. The presence of the various splices of PMCA1 was determined in stomach smooth muscle and other tissues by reverse transcription followed by a polymerase chain reaction. Percentage of transcripts encoding the potentially cyclic-nucleotide-sensitive isoform in various tissues were as follows: liver, 100%; stomach mucosa, 100%; heart, 100%; stomach smooth muscle, 86%; aorta, 83%; brain, 55%. Thus brain was the only tissue which expressed a very high proportion of the isoform of PMCA1 that is insensitive to cyclic-nucleotide-dependent protein kinases.

Amino Acid Sequence↗
PubMed1991

Expression of isoforms of internal Ca2+ pump in cardiac, smooth muscle and non-muscle tissues.

Smooth muscle and several non-muscle tissues contain mRNA for an alternative splice of the mRNA for the cardiac sarcoplasmic reticulum (SR) Ca,Mg-ATPase. Based on amino acid composition deduced from cDNA sequences the cardiac isoform (Ic) is 110 kDa while the smooth muscle and the non-muscle isoform (Is) is 115 kDa. This prediction in their molecular masses was tested at the protein level in rabbit stomach, aorta, uterus and vas deferens smooth muscles; stomach mucosa, brain, liver, kidney and heart. The major species of the acylphosphates formed in the presence of Ca2+ and electrophoresed in acid SDS-acrylamide gels were 5 kDa smaller for the heart (Ic) than those for all the other tissues (Is). The size difference was also confirmed in Western blots using a monoclonal antibody which binds to both Is and Ic. Thus consistent with the mRNA splices for the internal Ca2+ pumps previously reported to be present in these tissues, rabbit heart expresses predominantly the Ca2+ pump protein Ic and the various smooth muscle, mucosa, brain, liver and kidney express mainly the isoform Is.

Animals↗
PubMed1991

Serum beta-2 microglobulin and C-reactive protein levels in acute adenovirus conjunctivitis.

Serum beta-2 microglobulin and C-reactive protein levels were measured in 52 patients with acute adenovirus conjunctivitis and 58 healthy subjects, by ELISA technique. Serum beta-2 microglobulin levels were found to be significantly increased while there was no change in C-reactive protein levels. Serum beta-2 microglobulin levels closely paralleled the severity of the disease. The increased beta-2 microglobulin level in serum showed a significant fall to normal level on clinical improvement. There was no difference in the serum beta-2 microglobulin levels in virus-isolation positive and negative patients.

Acute Disease↗
PubMed1991

Serum alpha-1 acid glycoprotein levels in acute idiopathic anterior uveitis.

Serum alpha-1 acid glycoprotein levels were measured in 56 patients with acute idiopathic anterior uveitis and 42 healthy subjects by a radial immunodiffusion method. The levels were found to be increased significantly in moderate and severe types of uveitis; they were normal in mild type. Serum alpha-1 acid glycoprotein levels closely paralleled the severity of the disease. The serum levels of this protein returned to normal after clinical improvement.

Acute Disease↗
PubMed1990

Abundance of sarcoplasmic reticulum calcium pump isoforms in stomach and cardiac muscles.

Rabbit stomach smooth muscle contains mRNA for an internal Ca2+ pump identical in sequence to that reported for rabbit uterus [Lytton, Zarain-Herzberg, Periasamy & MacLennan (198) J. Biol. Chem. 264, 7059-7065]. This is an alternatively spliced form (Is) of the cardiac muscle sarcoplasmic reticulum Ca2+ pump (Ic). The splicing results in replacement of the last 4 amino acids (Ala-Ile-Leu-Glu) present in Ic by 49 amino acids and by a different 3'-non-coding region. Using cDNA probes against the conserved and the alternatively spliced regions, we determined that poly(A+) RNA isolated from rabbit stomach smooth muscle did not contain any transcripts for Ic. The poly(A+) RNA from cardiac muscle contained transcripts mostly for Ic, but also some for Is. The abundance of the Ca2(+)-pump transcripts as measured by the binding of a cDNA probe against the conserved region to poly(A+) RNA was 6-8 times higher in cardiac than in smooth muscle. The amount of the corresponding pump protein, measured using two antibodies, was 60-80 times higher in cardiac membranes than in smooth muscle membranes. Thus the protein-to-transcript level was approx. 10-fold higher in the cardiac muscle. We conclude that the regulation of the abundance of this protein occurs at steps leading to the formation of the mature mRNA for the two splices, which may differ in their translation efficiency.

Animals↗
PubMed1990

Splenic abscess.

We report a splenic abscess in an elderly diabetic female. Awareness of the disease is emphasised.

Abscess↗
PubMed1989

Protection of Ca pump of coronary artery against inactivation by superoxide radical.

Superoxide radicals inactivate endoplasmic reticular (ER) Ca2+ pump in membranes isolated from smooth muscle of pig right coronary artery [Am. J. Physiol. 255 (Cell Physiol. 24): C297-C303, 1988]. We report on protective mechanisms against such inactivation. This tissue contained superoxide dismutase (SOD) and catalase. SOD was distributed primarily in cytosolic fraction, was cyanide sensitive, and was also present in mitochondrial fraction, and approximately 25% of this was cyanide insensitive. Catalase was distributed mainly in mitochondrial fraction and did not protect against inactivation of ER Ca2+ pump by superoxide radicals generated using xanthine plus xanthine oxidase. However, cytosolic fraction protected against this inactivation by two mechanisms: 1) DTT carried over from homogenization medium and 2) its intrinsic SOD content. Soluble fraction was concentrated, dialyzed to remove 1,4-dithiothreitol (DTT), lyophilized, and suspended in a small volume of DTT-free buffer. It still protected against superoxide inactivation of Ca2+ pump. On Sephacryl-300 gel chromatography, protecting activity comigrated with SOD. DTT protected against inactivation, but glutathione and cysteine protected only partially. Neither sulfhydryl agents nor SOD could reverse the inactivation process. Ca2+ pump activity was abolished by dithionitrobenzoate and p-chloromercuric benzoate. Superoxide may inactivate ER Ca2+ pump by irreversibly modifying key sulfhydryl group(s) on pump molecule and SOD in coronary artery smooth muscle may partially protect against this inactivation.

Animals↗
PubMed1988

Monoclonal antibody against an epitope on the cytoplasmic aspect of the plasma membrane calcium pump.

Monoclonal antibody PM4A2B was prepared by immunizing mice with calmodulin affinity purified Ca2+-Mg2+-adenosine triphosphatase from rabbit erythrocytes and screening the clones with a plasma membrane-enriched fraction (F1) from rabbit stomach smooth muscle. On Western blots, PM4A2B reacted with F1 and with ghosts, right-side-out vesicles, and inside-out vesicles prepared from erythrocytes giving one major band at 130 kDa and minor lower molecular weight bands whose intensity increased on freezing and thawing the membranes. On enzyme-linked immunosorbent assay, PM4A2B reacted with inside-out vesicles, but not with the right-side-out vesicles or ghosts prepared from erythrocytes. It activated the ATP-dependent Ca2+ uptake by F1 and by the inside-out vesicles prepared from the erythrocytes. PM4A2B should be useful in determining membrane sidedness as well as in investigating the mechanism of the sarcolemmal Ca2+ pump.

Animals↗
PubMed1988

Evidence for a Na+/H+ antiport in stomach smooth muscle cells.

Single smooth muscle cells were isolated from circular muscle of the canine gastric corpus by collagenase incubation. Cytoplasmic pH (pHi) of these cells was measured fluorometrically using the trapped dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Cells were examined for their Na+/H+ exchange activity after intracellular acidification. Cells acid-loaded by propionate exposure, the NH4+ prepulse technique or suspension in a Na+-depleted medium regained almost normal pHi upon exposure to a Na+ medium. The Na+-dependent alkalinization was amiloride sensitive. As well, addition of amiloride to cells suspended in a Na+ medium caused a concurrent decrease in pHi. The study indicates that a Na+/H+ antiport is present in these smooth muscle cells.

Amiloride↗