PubMed Health⌕ Search

Biomedical subjects

R A Shapiro

Publications and source records attributed to R A Shapiro.

At least 73 records · Page 4Linked to original sources
PubMed1989

Regulation of muscarinic acetylcholine receptor function in cardiac cells and in cells expressing cloned receptor genes.

The regulation of the number and function of the muscarinic receptors has been investigated in cultured chick cardiac cells and in cells expressing cloned genes encoding mammalian, Drosophila, and chick muscarinic receptors. A serum-free defined medium for the culture of chick embryonic heart cells has been used to study the regulation of mAChR number and function by serum lipoproteins. Addition of rooster high density lipoprotein to the culture medium results in an attenuation of muscarinic receptor-mediated inhibition of cAMP accumulation without a change in the number of receptors or inhibitory G proteins. Clones encoding the mouse m1 receptor and a homologous receptor from Drosophila have been isolated. When expressed in Y1 adrenal cells, both receptors stimulate phosphoinositide hydrolysis but do not inhibit cAMP accumulation. Deletion of 123 out of the 156 amino acids in the third cytoplasmic loop of the mouse m1 receptor does not impair its ability to stimulate phosphoinositide hydrolysis. A genomic clone encoding a muscarinic receptor expressed in chick heart has been isolated. When expressed in Y1 cells, it causes inhibition of cAMP accumulation but does not stimulate phosphoinositide hydrolysis.

Animals↗
PubMed1989

Severity of disease correlated with fever reduction in febrile infants.

A prospective study of the effects of fever reduction on the clinical appearance of infants at risk for occult bacteremia was undertaken to study the hypothesis that infants with bacteremic illness fail to improve clinically following defervescence compared with infants with benign viral illness. A total of 154 children were enrolled in the study, including 19 with bacteremia: 13 with occult Streptococcus pneumoniae bacteremia, two with occult Haemophilus influenzae, type b bacteremia, and four with Haemophilus meningitis and bacteremia. There were no differences in degree of temperature reduction with acetaminophen between the bacteremic and nonbacteremic groups of infants. Among infants with bacteremia but without meningitis, differences from nonbacteremic children were detected in clinical appearance prior to fever reduction but not following defervescence. All patients with meningitis appeared seriously ill before and after defervescence. It was concluded that clinical improvement with defervescence is not a reliable indicator of the presence of occult bacteremia. Lack of clinical improvement with defervescence may be a reliable indicator for the presence of meningitis. Because there were differences in clinical appearance prior to fever reduction, routine administration of acetaminophen may interfere with the clinical evaluation by the physician.

Acetaminophen↗
PubMed1988

Isolation, sequence, and functional expression of the mouse M1 muscarinic acetylcholine receptor gene.

A genomic clone encoding the gene for the mouse M1 muscarinic acetylcholine receptor has been isolated, placed under the control of the zinc-inducible mouse metallothionein promoter, and transfected into mouse Y1 adrenal cells. The receptor concentration was about 300 fmol/mg membrane protein in the absence of zinc and could be increased to 4000 fmol/mg membrane protein in the presence of increasing concentrations of zinc. The receptor expressed in zinc-induced cells exhibits the high affinity binding for quinuclidinyl benzilate, atropine, and pirenzepine expected of the M1 muscarinic receptor. The M1 receptor when expressed in Y1 or L cells is physiologically active, as measured by agonist-dependent stimulation of phosphatidylinositol metabolism, but does not inhibit forskolin stimulation of cAMP accumulation. In contrast, a cloned M2 muscarinic receptor when expressed in Y1 cells is able to inhibit forskolin stimulation of cAMP accumulation, but is unable to stimulate phosphatidylinositol metabolism. The stimulation of phosphatidylinositol metabolism mediated by the M1 receptor was not altered by prior treatment of Y1 cells with concentrations of islet-activating protein sufficient to eliminate M2 receptor-mediated inhibition of adenylate cyclase. The cloned M1 receptor gene thus exhibits both the pharmacological and physiological properties expected of the M1 muscarinic acetylcholine receptor. In addition, these results indicate that different subtypes of the muscarinic receptor are coupled to different physiological responses.

Adrenal Gland Neoplasms↗
PubMed1988

Regulation of mRNA stability and the poly(A) problem in Dictyostelium discoideum.

This paper reviews our studies of three aspects of post-transcriptional regulation in Dictyostelium discoideum: 1) the determinants of mRNA stability in vegetative amoebae; 2) the effects of disaggregation and cyclic AMP on the decay rates of cell-type-specific mRNAs in late developing cells; and 3) the cytoplasmic function of the 3' poly(A) tracts present on most mRNAs. We find that: 1) mRNA stability in vegetative amoebae is not dependent on mRNA size, ribosome loading, or poly(A) tract length, but may be determined by specific 3'-untranslated sequences within a given mRNA; 2) mRNA decay rates in late developing cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs; and 3) poly(A) is most likely involved in the initiation of protein synthesis via an interaction with cytoplasmic poly(A)-binding proteins.

Dictyostelium↗
PubMed1988

Isolation of a cDNA for rat brain glutaminase.

A single phage was isolated from a lambda gt11 rat brain cDNA library by screening with antibodies prepared against rat renal glutaminase. Partial proteolysis of the fusion protein produced by a lysogen of the isolated phage generated a series of immunoreactive peptides that co-migrated with those derived from the purified brain glutaminase. The cDNA has a single open reading frame which encodes 326 amino acids that are in frame with beta-galactosidase. A 72-kDa protein, corresponding in size to the precursor of mitochondrial glutaminase, was immunoprecipitated from the translation products of rat renal mRNA that selectively hybridized to the cDNA. A probe made from the glutaminase cDNA detected an mRNA about 6 kb in length. This mRNA was present in rat brain and normal kidney RNA, increased 6-fold in acidotic kidney RNA, but was not detectable in liver RNA.

Amino Acid Sequence↗
PubMed1988

Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

DNA↗
PubMed1987

Changes in the levels of translatable glutaminase mRNA during onset and recovery from metabolic acidosis.

The amount of the mitochondrial glutaminase present within rat kidney is increased 5-fold during chronic metabolic acidosis. This adaptive response is due to a corresponding increase in the relative rate of glutaminase synthesis. Poly(A+) RNA was purified from the kidneys of control, 7-day acidotic, and 2-day recovered rats and then fractionated by electrophoresis on a low melting temperature agarose gel. Translation of the fractionated RNA in a rabbit reticulocyte lysate yields a 72,000-dalton protein that is specifically precipitated by anti-glutaminase IgG. The level of this protein is at least 3-fold greater in the translation products of the fractionated poly(A+) RNA derived from the acidotic vs. control or recovered rats. Therefore, the 72,000-dalton product of translation is the apparent precursor to the 68,000- and 65,000-dalton proteins that are contained in the mitochondrial glutaminase. From its relative electrophoretic mobility, the size of the glutaminase mRNA was estimated to be approximately 6.5 kilobases. The relative levels of translatable glutaminase mRNA were determined by using unfractionated poly(A+) RNA prepared from rats at various times following onset and recovery from acidosis. The observed increase occurred gradually, requiring 7 days to reach a maximal induction of 4.2-fold. The increase could be due to the increased transcription of a stable mRNA (t1/2 approximately 3 days). However, 2 days of recovery was sufficient to return the level of translatable glutaminase mRNA to normal. Thus, the selective inactivation or the altered stability of the glutaminase mRNA must also contribute to the regulation of the glutaminase gene expression.

Acidosis↗
PubMed1987

Immunoblot analysis of glutaminase peptides in intact and solubilized mitochondria isolated from various rat tissues.

Antibodies were prepared against isolated rat renal glutaminase and affinity-purified against the 65 kDa peptide contained in the purified rat brain glutaminase. The affinity-purified IgGs were then used to compare the glutaminase immunoreactive peptides contained in samples that had been subjected to SDS/polyacrylamide-gel electrophoresis and transferred to nitrocellulose. The purified brain glutaminase and isolated brain mitochondria contain 68 and 65 kDa peptides that exhibit nearly equivalent immunostaining. Partial proteolysis of the isolated 68 and 65 kDa peptides with Staphylococcus aureus V8 proteinase produced an identical pattern of immunoreactive proteolytic fragments. However, digestion of the two peptides with chymotrypsin resulted in similar, but slightly different, patterns. The pattern of immunostaining was unaltered even when the brain mitochondria were solubilized with Triton X-100 and stored for 2 days at 4 degrees C. A very similar pattern was observed when intact renal mitochondria were subjected to immunoblot analysis. However, when renal mitochondria were solubilized, the 68 kDa peptide was rapidly degraded to the 65 kDa form. At 4 degrees C this reaction occurs with apparent first-order kinetics and a t1/2 of 35 min. Degradation of the 65 kDa form of the renal glutaminase occurs with much slower kinetics, but is nearly complete after 24 h. Solubilization of mitochondria isolated from various zones of the kidney indicated that the responsible endogenous proteinase was localized primarily in the cortex. Mitochondria isolated from intestinal or renal papillary tissue contain four glutaminase immunoreactive peptides (Mr 68,000, 65,000, 61,000 and 58,000). The smallest of these peptides is identical in size with the single immunoreactive peptide observed in liver tissue.

Animals↗
PubMed1985

The orientation of phosphate-dependent glutaminase on the inner membrane of rat renal mitochondria.

Phosphate-dependent glutaminase is associated with the inner membrane of rat renal mitochondria. The orientation of this enzyme was characterized by comparing its sensitivity in isolated mitochondria and in mitoplasts to two membrane impermeable inhibitors. Mitoplasts were prepared by repeated swelling of mitochondria in a hypotonic phosphate solution. This procedure released greater than 70% of the adenylate kinase from the intermembrane space, but less than 10 and 25% of the marker activities characteristic of the inner membrane and matrix compartments, respectively. The addition of 20 microM p-chloromercuriphenylsulfonate (pCMPS) caused a rapid inactivation of the purified glutaminase. In contrast, the glutaminase contained in isolated mitochondria and mitoplasts was only slightly affected by the addition of up to 2 mM pCMPS. Similarly, the activity in mitochondria and mitoplasts was not inhibited by the addition of an excess of inactivating Fab antibodies. However, a similar extent of inactivation occurred when either membrane fraction was incubated with concentrations of octylglucoside greater than 0.35%. Mitochondria were also treated with increasing concentrations of digitonin. At 0.4 mg digitonin/mg protein, all of the adenylate kinase was released but the glutaminase activity was either slightly inhibited or unaffected by the addition of pCMPS or the Fab antibodies, respectively. These studies establish that the glutaminase is localized on the inner surface of the inner membrane. Therefore, mitochondrial catabolism of glutamine must occur only within the matrix compartment.

4-Chloromercuribenzenesulfonate↗
PubMed1985

Comparison of the phosphate-dependent glutaminase obtained from rat brain and kidney.

A phosphate-dependent glutaminase was purified 1200-fold from rat brain. In the absence of a polyvalent anion, the glutaminase exists as an inactive protomer which has an estimated Mr of 126000. The addition of 100mM-phosphate causes maximal activation and a dimerization (Mr 249000) of the glutaminase. The phosphate activation is sigmoidal, with a K0.5 of 25mM and a Hill coefficient (h) of 1.5 Glutamate inhibition is competitive with respect to glutamine and is decreased by increasing the concentration of phosphate. Phosphate also decreases the Km for glutamine. The purified glutaminase contains a predominant peptide (Mr 65000) and a minor peptide (Mr 68000) that are present in an approximate ratio of 4:1 respectively. The glutaminase immunoprecipitated from freshly solubilized brain tissue or from synaptosomal and non-synaptosomal brain mitochondria contains the same distribution of the two peptides. In contrast, the glutaminase purified from rat kidney contains five to seven peptides that range in Mr value from 59000 to 48000, and immunoprecipitates derived from freshly solubilized renal tissue contain only the Mr-65000 peptide. Partial proteolysis and size fractionation of the three immunoprecipitated peptides indicate that they are structurally related. The series of peptides characteristic of the purified renal glutaminase is generated on storage of the solubilized extract of kidney tissue. The glutaminase contained in the solubilized brain extract is not degraded unless a renal extract is added. Thus the difference in the pattern of peptides associated with the two purified enzymes is due to an endogenous renal proteinase that is not present in brain.

Animals↗
PubMed1984

Renal tumors associated with tuberous sclerosis: the case for aggressive surgical management.

Tuberous sclerosis is associated commonly with renal angiomyolipoma. However, the radiographic differentiation of angiomyolipoma and renal carcinoma can be difficult, and the natural history of angiomyolipoma may be associated with serious complications. We present a case of bilateral renal cell carcinoma associated with tuberous sclerosis. Aggressive but conserving renal surgery should be considered in patients with tuberous sclerosis and enlarging renal masses.

Adult↗
PubMed1982

Inhibition by glutamate of phosphate-dependent glutaminase of rat kidney.

A membrane-associated form of phosphate-dependent glutaminase was derived from sonicated mitochondria and purified essentially free of gamma-glutamyl transpeptidase activity. Increasing concentrations of phosphate cause a sigmoidal activation of the membrane-bound glutaminase. Phosphate also causes a similar effect on the rate of glutaminase inactivation by the two affinity labels, L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo-L-norleucine, as observed previously for the solubilized and purified enzyme. Therefore the two forms of glutaminase undergo similar phosphate-induced changes in conformation. A sensitive radioactive assay was developed and used to determine the kinetics of glutamate inhibition of the membrane-associated glutaminase. The Km for glutamine decreases from 36 to 4 mM when the phosphate concentration is increased from 5 to 100 mM. Glutamate is a competitive inhibitor with respect to glutamine at both high and low concentrations of phosphate. However, the Ki for glutamate is increased from 5 to 52 mM with increasing phosphate concentration. Therefore glutamine and glutamate interact with the same site on the glutaminase, but the specificity of the site is determined by the available phosphate concentration.

Animals↗