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Biomedical subjects

S R Price

Publications and source records attributed to S R Price.

At least 55 records · Page 3Linked to original sources
PubMed1996

Necessary but not sufficient: the role of glucocorticoids in the acidosis-induced increase in levels of mRNAs encoding proteins of the ATP-dependent proteolytic pathway in rat muscle.

Muscle protein degradation is accelerated by the acidosis associated with chronic renal failure. In isolated muscles from acidotic rats, a cytosolic, ATP-dependent proteolytic pathway is stimulated with a concurrent increase in the abundance of mRNAs encoding ubiquitin and subunits of the 26S proteasome complex associated with this degradative pathway. Adrenalectomy (ADX) prevents the acidosis-induced increase in muscle protein degradation unless high physiologic doses of glucocorticoids are administered to acidotic, adrenalectomized rats. We have examined the roles that acidosis and glucocorticoids have in the increase in mRNAs encoding proteins of the ATP-dependent-ubiquitin-proteasome proteolytic pathway in ADX rats. We found that ubiquitin and proteasome C2 and C9 subunit mRNA levels are increased in the white fiber, extensor digitorus longus (EDL) and mixed fiber, gastrocnemius muscles from acidotic ADX rats that received dexamethasone whereas acidosis alone or dexamethasone alone failed to increase these mRNAs. In contrast, acidosis plus dexamethasone decreased the total RNA content in both muscles. These data suggest that in muscle, the response to acidosis involves the specific activation of the ATP-ubiquitin-proteasome proteolytic pathway. Moreover, glucocorticoids are required but not directly responsible for the acidosis-induced increase in the mRNAs encoding proteins of this degradative pathway.

Acidosis↗
PubMed1995

Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies.

In vitro transcription using bacteriophage RNA polymerases and linearised plasmid or oligodeoxynucleotide templates has been used extensively to produce RNA for biochemical studies. This method is, however, not ideal for generating RNA for crystallisation because efficient synthesis requires the RNA to have a purine rich sequence at the 5' terminus, also the subsequent RNA is heterogenous in length. We have developed two methods for the large scale production of homogeneous RNA of virtually any sequence for crystallization. In the first method RNA is transcribed together with two flanking intramolecularly-, (cis-), acting ribozymes which excise the desired RNA sequence from the primary transcript, eliminating the promoter sequence and heterogeneous 3' end generated by run-off transcription. We use a combination of two hammerhead ribozymes or a hammerhead and a hairpin ribozyme. The RNA-enzyme activity generates few sequence restrictions at the 3' terminus and none at the 5' terminus, a considerable improvement on current methodologies. In the second method the BsmAI restriction endonuclease is used to linearize plasmid template DNA thereby allowing the generation of RNA with any 3' end. In combination with a 5' cis-acting hammerhead ribozyme any sequence of RNA may be generated by in vitro transcription. This has proven to be extremely useful for the synthesis of short RNAs.

Bacteriophage T7↗
PubMed1995

Mechanisms causing muscle loss in chronic renal failure.

The loss of lean body mass in uremia is associated with excessive morbidity and mortality. A potential mechanism causing protein catabolism is that uremia overcomes critical metabolic responses required to maintain protein balance whenever dietary protein is limited. These responses include reduced oxidation of essential amino acids, which improves the efficiency of protein utilization and a reduction in protein degradation. We find that metabolic acidosis stimulates both amino acid oxidation and protein degradation in muscle and thus could overcome the adaptive responses. The molecular mechanisms stimulating catabolism involve glucocorticoids and includes increased mRNAs of components of catabolic pathways. Studies in patients have confirmed that acidosis causes catabolism in chronic renal failure. Thus, we recommend that patients with metabolic acidosis receive an adequate diet and sufficient alkali to correct acidosis.

Acidosis↗
PubMed1995

Protein engineering as a tool for crystallography.

The generation of large quantities of protein by overexpression technology has enabled structural studies of many important molecules that are found in only minute quantities in the cell. An increasing number of structures of proteins overexpressed in non-native systems have been solved. Crystallographers now have an extremely powerful tool, namely protein engineering, for the generation of native and derivative crystals that diffract to high resolution. The mutation of residues or generation of compact domains through truncation has resulted in crystals with enhanced diffraction properties. Heavy atom derivative crystals isomorphous to the native protein may also be engineered either by introducing cysteines or by removing cysteines whose reaction with heavy-atom compounds results in poor crystals.

Crystallization↗
PubMed1995

Anaesthesia for a child with centronuclear myopathy.

Centronuclear myopathy (CNM) is an inherited condition involving most muscle fibres in all the body mass, first described in 1966, which has a varying spectrum of presentations. Until recently it had not been associated with an increased risk of malignant hyperpyrexia. A seven-year-old male with CNM was admitted to our hospital for elective surgery. High dose propofol anaesthesia was used, supplemented with N2O/O2 from a new anaesthesia machine. The operation was successful with uncomplicated anaesthesia and recovery.

Anesthesia, General↗
PubMed1995

Rat muscle branched-chain ketoacid dehydrogenase activity and mRNAs increase with extracellular acidemia.

The rate-limiting enzyme in branched-chain amino acid catabolism is branched-chain ketoacid dehydrogenase (BCKAD). In rats fed NH4Cl to induce acidemia, we find increased basal BCKAD activity as well as maximal activity in skeletal muscle. Concurrently, there is a > 10-fold increase in mRNAs of BCKAD subunits in skeletal muscle plus an increase in cardiac muscle but not in liver or kidney. There was no increase in mRNA for malate dehydrogenase or for cytosolic glyceraldehyde-3-phosphate dehydrogenase. Evaluation of the translation capacity of BCKAD mRNAs in muscle of acidemic rats yielded more immunoreactive BCKAD whether the proteins were synthesized from muscle RNA using rabbit reticulocyte lysate or directly using postmitochondrial homogenates. Although the RNA from muscle of acidemic rats yielded twice as much BCKAD protein, we found no net increase in mitochondrial BCKAD protein in muscle by Western blotting. Because there is increased proteolysis in muscle of rats with acidemia, the increase in mRNA might be a mechanism to augment BCKAD synthesis and activity in muscle.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

Urea transport in initial IMCD of rats fed a low-protein diet: functional properties and mRNA abundance.

Feeding rats a low-protein (8%) diet (LPD) for 2 wk induces a facilitated urea transporter in rat initial inner medullary collecting ducts (IMCDs). To determine whether this is preceded by an increase in mRNA abundance, we designed degenerate polymerase chain reaction primers to the rabbit facilitated urea transporter (UT2; G. You, C. P. Smith, Y. Kanai, W.-S. Lee, M. Stelzner, and M. A. Hediger. Nature Lond. 365: 844-847, 1993) and amplified a 716-bp cDNA fragment to perform Northern analysis of the base or tip of rat inner medulla. In the base, the predominant transcript was a 2.9-kb band, which increased 55% after 1 wk on an LPD; there was no change in a 4-kb band. In the tip, the 4-kb band predominated, but neither band varied with an LPD. Next, we functionally characterized the induced urea transporter using microperfused initial IMCDs from rats fed an LPD for 2 wk. First, 100 pM arginine vasopressin (AVP) stimulated urea permeability (Purea); 10 nM AVP increased Purea further. Second, raising perfusate and bath osmolality to 690 mosmol/kgH2O (NaCl added) stimulated Purea; adding AVP (10 nM) increased Purea further. Third, thiourea reversibly inhibited AVP-stimulated Purea.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Acidosis and glucocorticoids interact to provoke muscle protein and amino acid catabolism.

Malnutrition and a loss of lean body mass frequently complicate chronic renal failure. Muscle wasting in uremia is caused by increased protein degradation, decreased protein synthesis and increased branched-chain amino acid oxidation. Acidosis and glucocorticoids are pivotal in these pathophysiologic aberrations. When the acidosis of chronic renal failure is corrected by feeding bicarbonate, protein degradation and amino acid oxidation normalize. Likewise, if patients and animals with normal renal function are made acidotic, protein degradation and amino acid oxidation increase. In adrenalectomized, acidotic rats, proteolysis increases only when they are supplemented with physiologic concentrations of glucocorticoids, suggesting that glucocorticoids are necessary for increased proteolysis. Acidosis stimulates the ATP-dependent proteolytic process involving ubiquitin and the 26S proteasome. Thus, acidosis evokes a glucocorticoid-dependent catabolic response in muscle that can account for the protein wasting associated with uremia.

Acidosis, Renal Tubular↗
PubMed1995

Regulation of aldose reductase, sorbitol dehydrogenase, and taurine cotransporter mRNA in rat medulla.

The regulation of mRNA for aldose reductase, sorbitol dehydrogenase, and the Na+/Cl-/taurine cotransporter was studied with three in vivo models in which urinary concentration is reduced: Sprague-Dawley rats undergoing a water diuresis or fed a low-protein diet or Brattleboro rats. In Sprague-Dawley rats, 3 days of water diuresis reduced inner medullary aldose reductase mRNA abundance 6.5-fold compared with untreated rats, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. When water diuretic rats were acutely deprived of water, urine osmolality increased significantly after 4 h but aldose reductase mRNA did not increase until 12 h. Heat shock protein-70 mRNA was not increased by water deprivation. Second, in rats fed a low-protein diet for 3 wk, aldose reductase mRNA increased two-fold, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. Finally, in Brattleboro rats, urine osmolality and levels of aldose reductase and taurine cotransporter mRNA increased in response to 1 day of water deprivation, whereas sorbitol dehydrogenase mRNA was unchanged. Administering vasopressin (1 U/day) to Brattleboro rats for 8 days also increased urine osmolality and aldose reductase mRNA but did not alter sorbitol dehydrogenase or taurine cotransporter mRNA. This result is consistent with the hypothesis that changes in urine osmolality induce changes in aldose reductase mRNA abundance that are independent of vasopressin. It was concluded that, in rat inner medulla: (1) aldose reductase mRNA abundance varies with changes in water balance or dietary protein, whereas sorbitol dehydrogenase and taurine cotransporter mRNA do not; and (2) heat shock protein-70 mRNA abundance is not increased during acute osmotic stress.

Aldehyde Reductase↗
PubMed1994

Metabolic consequences of uremia: extending the concept of adaptive responses to protein metabolism.

An early response to metabolic acidosis is an increase in the degradation of muscle protein to provide the nitrogen needed to increase glutamine production so the kidney can excrete acid. In patients with renal insufficiency, this process may represent an example of a trade-off adaptation to uremia. It requires a hormone (glucocorticoids) and the metabolic response is maladaptive because the inability of the damaged kidney to maintain acid-base balance results in loss of muscle protein. Studies of cultured cells and rats and humans with normal kidneys demonstrate that acidosis stimulates the degradation of both amino acids and protein, which would block the normal adaptive responses to a low-protein diet (ie, to reduce the degradation of essential amino acids and protein). Evidence from studies in rats and humans with chronic uremia show that acidosis is a major stimulus for catabolism. The mechanism includes stimulation of specific pathways for the degradation of protein and amino acids. Since other catabolic conditions (eg, starvation) appear to stimulate the same pathways, understanding the mechanism in acidosis could be applicable to other conditions. Thus, the loss of lean body mass in uremia appears to be a consequence of a normal metabolic response that persists until acidosis is corrected.

Acidosis↗
PubMed1994

Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle.

In rat muscle metabolic acidosis increases ATP-dependent protein degradation and levels of mRNAs for ubiquitin (Ub) and proteasome subunits. Because adrenalectomy (ADX) abolishes the proteolytic response to acidosis in muscle, we examined whether glucocorticoids (GCs) are necessary for acidosis-induced changes in Ub and proteasome mRNAs in muscles. Total RNA content of the white fiber extensor digitorum longus or mixed fiber gastrocnemius muscles were lowest in muscles of ADX rats given acid plus GCs. In contrast, the abundance of Ub and C2 and C9 proteasome subunits mRNAs were increased in muscles from this group compared with untreated ADX rats or ADX rats given acid or GCs alone. Because total RNA is reduced, the increase in these mRNAs in muscles of ADX rats receiving acid plus GCs provides evidence for a specific activation of the ATP-dependent-Ub-proteasome pathway. Thus, GCs are required but not sufficient to produce the coordinated increase in mRNAs encoding ubiquitin and proteasome subunits occurring in muscles of acidotic rats.

Acidosis↗
PubMed1994

Na pump defects in chronic uremia cannot be attributed to changes in Na-K-ATPase mRNA or protein.

We have found abnormalities in Na-K-adenosine-triphosphatase (Na-K-ATPase) function in different tissues of rats with chronic renal failure (CRF). A potential mechanism for these findings is a change in Na-K-ATPase alpha- and/or beta-gene expression. To evaluate this possibility, we compared CRF with pair-fed, sham-operated rats to determine whether chronic uremia changes the expression of Na-K-ATPase alpha 1-, alpha 2-, beta 1-, and beta 2-isoform mRNAs or protein in different types of skeletal muscle, heart, liver, adipose, and kidney tissue. In CRF rats, alpha 1-mRNA in heart tended to be higher and beta 2-mRNA was lower in fat and kidney. There were no other statistically significant differences in isoform mRNAs in tissues of CRF compared with the control rats. Western blot analysis revealed a 38% increase in alpha 1-protein in adipocytes and a 61% decrease in kidney of CRF rats but no significant differences in the amounts of isoform protein in other tissues. Thus, in uremia, posttranslational events or inhibitors of the enzyme are more likely causes of defects in Na-K-ATPase than changes in mRNA or protein abundance.

Animals↗
PubMed1994

Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes.

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway.

Acidosis↗
PubMed1992

Regulation of ADP-ribosylation factor (ARF) expression. Cross-species conservation of the developmental and tissue-specific alternative polyadenylation of ARF 4 mRNA.

ADP-ribosylation factors (ARFs), approximately 20-kDa guanine nucleotide-binding proteins, are involved in protein trafficking and enhance cholera toxin ADP-ribosyltransferase activity. Expression of six ARF genes was examined in mammalian tissues; only ARF 4 mRNA was detected in rat testis in forms considerably shorter than those in other tissues. Testis-specific expression of short forms of ARF 4 mRNA was observed in several mammalian species. On Northern analysis of the developmental expression of rat ARF 4 mRNA, appearance of the shorter species was consistent with its involvement in a late stage of spermatogenesis. Sequences of products of rapid amplification of cDNA ends (RACE-polymerase chain reaction) of rat ARF 4 mRNA revealed that different mRNAs resulted from the use of three polyadenylation signals, one AUUAAA and two AAUAAA. Sequences of 3'-untranslated regions of rat and human ARF 4 mRNA were very similar with identical polyadenylation signals at similar positions. Of the ARF 4 mRNAs identified by RACE-PCR, with sizes of 1.1, 1.3, and 1.8 kb, the 1.1-kb mRNA was predominant in adult testis. By in situ hybridization, the 1.1-kb mRNA was identified primarily in mature sperm, consistent with the developmental studies. Shorter mRNAs, thought to be more stable, may compensate for cessation of transcription at late stages of spermatogenesis.

ADP-Ribosylation Factors↗
PubMed1992

Effects of phospholipid and GTP on recombinant ADP-ribosylation factors (ARFs). Molecular basis for differences in requirements for activity of mammalian ARFs.

ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that were first identified based on their ability to stimulate the cholera toxin-catalyzed ADP-ribosylation of Gs alpha and thus activate adenylyl cyclase. Proteins with ARF activity have been characterized from different mammalian tissues and exhibited different requirements for activity, stability, and phospholipid. Based on molecular cloning and mRNA distribution, at least six mammalian ARFs, which fall into three classes, have been identified. To test whether individual ARFs might have different requirements for optimal activity, as judged by their ability to enhance cholera toxin ADP-ribosyltransferase activity, four ARFs from classes I, II, and III were produced as recombinant proteins in Escherichia coli and characterized. Recombinant bovine ARF 2 (rARF 2) and human ARF 3 (rARF 3) (class I), human ARF 5 (rARF 5, class II), and human ARF 6 (rARF 6, class III) differed in the effects of phospholipid and detergent on their ability to enhance cholera toxin activity; rARFs 2, 3, and 5 required dimyristoylphosphatidylcholine (DMPC) and cholate, whereas rARF 6 did not require phospholipid/detergent for activity. Further characterization of two of the more divergent ARFs (ARFs 2 and 6) showed that both exhibited guanosine 5'-O-(3-thio)triphosphate binding which was enhanced by DMPC/cholate. In the transferase assay, rARF 2 required approximately 4 microM GTP for half-maximal stimulation of toxin activity, whereas rARF 6 required 0.05 microM GTP. rARF 6 exhibited a delay in activation of toxin not detected with rARF 2 that may be related to a requirement for guanine nucleotide exchange and/or GTP binding. These findings are consistent with the conclusion that the highly conserved members of the ARF family have different requirements for optimal activity.

ADP-Ribosylation Factor 6↗
PubMed1992

Conservation of a 23-kDa human transplantation antigen in mammalian species.

A group of transplantation antigens, referred to as tum- antigens, were identified in mouse tumor cells that had been mutagenized to produce variant cells and were recognized by clonal cytolytic T lymphocytes (CTL). Alterations in these variant cells that were recognized by CTL resulted from point mutations in the genes of specific proteins. We have isolated human and bovine cDNA clones that encode the homologs of the mouse tum- antigen P198. This 23.6-kDa protein is highly basic with a predicted pI of 11.55. p23/P198 is highly conserved across mammalian species, with > 94% identity (97% including conservative substitutions) among the human, bovine, and mouse deduced amino acid sequences. The nucleotide sequences of both the coding and 5'- and 3'-untranslated regions from human, bovine, and mouse are also highly conserved with > 88% identity in the coding regions. Hybridization of poly(A)+ RNA from various mammalian sources with cDNA and oligonucleotides specific for the coding region identified two mRNAs of 1.2 and 0.8 kb, whereas probes specific for the 3'-untranslated region between two consensus polyadenylation signals hybridized with the 1.2-kb, but not the 0.8-kb, mRNA. The abundance of the 1.2-kb mRNA relative to that of the 0.8-kb species varied depending upon the cell type. A single predominant transcription initiation site was mapped by primer extension. These studies indicate that this highly basic 23.6-kDa protein is encoded by two major mRNA species that differ only in the length of their 3'-untranslated regions and that the mechanism that gives rise to these two mRNAs, utilization of alternative polyadenylation sites, is conserved across species.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗