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R M Long

Publications and source records attributed to R M Long.

At least 37 records · Page 2Linked to original sources
PubMed1994

SIP1 is a catabolite repression-specific negative regulator of GAL gene expression.

The yeast Snf1p kinase is required for normal expression of many genes involved in utilization of non-glucose carbon. Snf1p is known to associate with several proteins. One is Sip1p, a protein that becomes phosphorylated in the presence of Snf1p and thus is a candidate Snf1p kinase substrate. We have isolated the SIP1 gene as a multicopy suppressor of the gal83-associated defect in glucose repression of GAL gene expression. Multicopy SIP1 also suppressed the gal82-associated defect in glucose repression, suggesting that SIP1, GAL83 and GAL82 function interdependently. Multicopy SIP1 gene reduces GAL1, GAL2, GAL7 and GAL10 gene expression three- to fourfold in cells grown in the presence of glucose but has no effect in cells grown on nonrepressing carbon. Sip1-deletion cells exhibited a two- to threefold increase in GAL gene expression compared to wild-type cells when grown on glucose. These studies show that SIP1 is a catabolite repression-specific negative regulator of GAL gene expression. Northern analysis revealed two SIP1 transcripts whose relative abundance changed with carbon source. Western blots revealed that Sip1p abundance is not markedly affected by carbon source, suggesting that Sip1p may be regulated post-translationally.

AMP-Activated Protein Kinases↗
PubMed1991

Cloning and sequencing of a human liver carboxylesterase isoenzyme.

A human liver lambda gt11 library was screened with antibodies raised to a purified rat liver carboxylesterase, and several clones were isolated and sequenced. The longest cDNA contained an open reading frame of 507 amino acids that represented 92% of the sequence of a mature carboxylesterase protein. This sequence possessed many structural features that are highly conserved among rabbit and rat liver carboxylesterase proteins, including Ser, His, and Asp residues that comprise the active site, two pairs of Cys residues that may participate in disulfide bond formation, and one Asn-Xxx-Thr site for N-linked carbohydrate addition. When the clone was used to probe human liver genomic DNA that had been digested with various restriction enzymes, many hybridizing bands of differing intensities were observed. The results suggest that the carboxylesterases exist as several isoenzymes in humans, and that they are encoded by multiple genes.

Amino Acid Sequence↗
PubMed1991

GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator.

The GAL4 protein of Saccharomyces cerevisiae is a DNA-binding transcriptional activator that is highly specific for the GAL genes. In vivo levels of GAL gene transcription are closely correlated with the phosphorylation state of GAL4. In vivo levels of GAL gene transcription are also affected by the activity of the GAL11 (SPT13) protein, a protein that has been implicated as a global auxiliary transcriptional factor. Here we examine the influence of GAL11 (SPT13) on the phosphorylation state of GAL4. Cells bearing a gal11 deletion mutation are defective in the production or maintenance of GAL4III, a phosphorylated form of GAL4 that is associated with higher levels of GAL gene transcription. In addition, the gal11 deletion cells are reduced in total GAL4 protein. However, the fivefold-reduced expression of the GAL1 gene observed in gal11 deletion cells cannot be due solely to reduced levels of total GAL4 protein, since gal11 deletion cells amplified for GAL4 production are still markedly reduced in GAL4 protein-dependent transcription. Thus, these data demonstrate that the GAL11 protein augments GAL4 protein-dependent transcription in a manner that is tightly coupled to the formation or maintenance of a phosphorylated form of GAL4.

Blotting, Western↗
PubMed1990

Agonist-induced down-regulation of m1 muscarinic receptors and reduction of their mRNA level in a transfected cell line.

Agonist-induced reduction in both the number of m1 muscarinic receptors and the mRNA coding for the receptor protein was investigated in Chinese hamster ovary (CHO) cells which were transfected with the m1 muscarinic receptor gene. Receptor concentration was measured by the specific binding of the muscarinic ligand, [3H]quinuclidinyl benzilate ([3H]QNB), and Northern blot hybridization analysis was used to evaluate the levels of receptor mRNA. Incubation of cells with 1 mM of the muscarinic agonist, carbamylcholine (CBC), for 24 h decreased receptor density and mRNA levels in cells by 65% and 73%, respectively. These results indicate that agonist-induced down-regulation of m1 muscarinic receptors might be due to, at least in part, a decrease in receptor synthesis resulting from a reduction in the steady-state level of their mRNA.

Animals↗
PubMed1990

Impact of halogenated compounds on calcium homeostasis in hepatocytes.

Halocarbons (CCl4, 1,1-dichlorethylene) cause a wide spectrum of effects and injury in hepatocytes. One early effect of these compounds is the inhibition and destruction of the endoplasmic reticulum (ER) calcium pump. Subsequent to inhibition of this pump, the ER calcium pool is depleted and cytosolic levels of calcium are increased for a prolonged period of time. This effect of halocarbons has been characterized and is similar in vivo and in vitro. The importance of this redistribution of cell calcium in expression of halocarbon injury of hepatocytes has not been fully resolved. Several degradative enzymes (phospholipases, proteases) have been implicated as calcium-dependent mediators in toxicity. Our preliminary studies of the effect of calcium redistribution suggest that activation of a calcium-sensitive endonuclease in liver does not play a central role in initiating the lethal effect of halocarbons on hepatocytes.

Animals↗
PubMed1990

Nursing management of the pregnant hemodialysis patient.

Pregnancy is an uncommon occurrence in the end stage renal disease (ESRD) patient population and more so in the patient on chronic hemodialysis therapy. The management of the pregnant hemodialysis patient covers a wide spectrum of concerns. Many resources must be used to deliver safe, quality care to the patient. The knowledge gained from our experience in caring for a pregnant hemodialysis patient will benefit the nephrology nursing community. With the advent of erythropoietin, the number of pregnant ESRD patients may grow, and the demand for knowledge on how to care for these patients will also become increasingly evident.

Adult↗
PubMed1989

Halocarbon hepatotoxicity is not initiated by Ca2+-stimulated endonuclease activation.

Previous studies from this laboratory have demonstrated that cytosolic Ca2+ rapidly rises to supraphysiologic levels in liver cells exposed to the hepatotoxins carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE) in vivo and in vitro. The present study examines whether this increase in intracellular Ca2+ activates endonucleases that could initiate or contribute to the ensuing hepatotoxic events. Initial experiments demonstrated that there was no generalized breakdown of hepatic DNA in intact rats exposed to CCl4 and DCE, as assessed by the appearance of nucleosomal fragments in liver nuclear DNA separated on agarose gels. Nor was generalized fragmentation observed in DNA isolated from primary hepatocyte cultures exposed to halocarbons, except at very late times following loss of plasma membrane integrity. Endonuclease activation was further examined at a more sensitive level by specifically monitoring hypersensitive sites (HSS) in serum albumin gene. Actively transcribed genes, such as albumin in liver tissue, are extremely sensitive to attack by exogenous nucleolytic enzymes at discrete sites. We speculated that subtle halocarbon-induced endonuclease activation would first become evident at these sites. To locate HSS, DNA was digested with restriction enzymes Eco R1 or Hind III, electrophoresed on agarose gels, blotted onto nitrocellulose, and hybridized to a 32P-labeled 1400 bp rat albumin genomic clone. No cleavage at hypersensitive sites was detected in DNA isolated from rat liver or hepatocyte DNA at early times when elevations of Ca2+ were developing. Thus, these data indicate that endonuclease activation by intracellular Ca2+ and resultant nucleolytic destruction of DNA is not an early event in the hepatotoxicity produced by halocarbons.

Animals↗
PubMed1988

Rat liver carboxylesterase: cDNA cloning, sequencing, and evidence for a multigene family.

A cDNA clone was isolated from a rat liver lambda gt11 expression library by screening with polyclonal antibodies raised against a rat liver microsomal carboxylesterase. This clone of 1.8 kb contained an open reading frame encoding a mature protein of 531 amino acids with a predicted molecular weight of 58,084. The 5' portion of the clone coded for 9 amino acids of a putative signal peptide. The 3' end of the clone included an untranslated region and a poly (A) tail. Carboxylesterase active site regions, five potential N-linked glycosylation sites, and 2 postulated cystine disulfide bridges were found in the cDNA-deduced amino acid sequence. Sequences obtained from tryptic peptides and the NH2-terminus of the purified native carboxylesterase were aligned with the deduced amino acid sequence, and the overall identity was 84%. Southern blot analysis suggested the presence of multiple genes. Thus it is concluded that we have cloned a rat liver carboxylesterase, and that this enzyme is a member of a multigene family.

Amino Acid Sequence↗
PubMed1988

Evaluation of the calcium mobilizing action of acetaminophen and bromobenzene in rat hepatocyte cultures.

Acetaminophen (APAP) and bromobenzene (BrB) are reported to selectively inhibit plasma membrane (PM) but not endoplasmic reticulum (ER) Ca2+ transport in rat liver (1). The ability of these hepatotoxicants to increase cytoplasmic Ca2+ levels as a result of disrupted Ca2+ pumping was determined in cultured rat hepatocytes by monitoring the activity of glycogen phosphorylase alpha, a Ca2+ -sensitive (via phosphorylase kinase) enzyme. Following exposure to 2.5 to 10 mM APAP for five minutes, dose-dependent increases in phosphorylase alpha activity were observed (58 to 190 U/g protein). Endoplasmic reticulum Ca2+ pump activity was not inhibited after any dose of APAP (56 nmol Ca2+ per milligram protein per 30 minutes). Phosphorylase alpha activity remained elevated for 60 minutes after exposure to APAP (124 microliters/g protein). Following exposure to 0.5 to 2 mM BrB for five minutes, phosphorylase alpha activity also increased (58 to 229 U/g protein) in a dose-related manner. Endoplasmic reticulum Ca2+ pump activity was inhibited after BrB exposure (from 58 to 16 nmol Ca2+ per milligram protein per 30 minutes). Phosphorylase alpha activity remained elevated for 60 minutes after exposure to BrB (147 U/g protein). Evidence of elevated cytoplasmic Ca2+ is consistent with the inhibition of Ca2+ -extruding/sequestering mechanisms at hepatocyte PM and/or ER. Prolonged elevation of cytosolic Ca2+ levels could overstimulate Ca2+ -sensitive processes within liver cells and thus initiate or contribute to hepatotoxic injury.

Acetaminophen↗
PubMed1988

Biochemical evaluation of rat hepatocyte primary cultures as a model for carbon tetrachloride hepatotoxicity: comparative studies in vivo and in vitro.

In order to evaluate how well the development of CCl4 hepatotoxicity in vivo can be modeled in primary cultures of rat hepatocytes, biochemical alterations were determined in liver samples from rats given CCl4 and in liver cells cultured for 18 hr then exposed to CCl4. Soluble thiol levels matched closely between tissue and hepatocytes (11 vs 12 micrograms-SH/mg protein) prior to exposure. Comparable concentrations of CCl4 were measured in blood (0.30 mM at 30 min) and in culture medium (0.49 mM at 5 min). Simultaneous inhibition of the endoplasmic reticulum calcium pump and stimulation of phosphorylase a activity occurred at early times in vivo (30 min) and in vitro (5 min). Glucose-6-phosphatase was inhibited next in liver (120 min) and in cells (20 min). 5'-Nucleotidase was not affected at any time points examined in either system. Leakage of glutamic-pyruvic transaminase and depletion of glycogen were maximal at later times in vivo (greater than or equal to 8 hr) and in cells (30 min). Total calcium content was increased severalfold in liver tissue (24 hr), but was not elevated in hepatocytes. This lack of calcium accumulation in cells appeared to result from impaired mitochondrial calcium uptake. Thus CCl4-induced biochemical changes followed nearly the same continuum in both models, although the progression was much more rapid in vitro than in vivo.

Alanine Transaminase↗
PubMed1987

Cytosolic calcium after carbon tetrachloride, 1,1-dichloroethylene, and phenylephrine exposure. Studies in rat hepatocytes with phosphorylase a and quin2.

Carbon tetrachloride (CCl4) and 1,1-dichloroethylene (DCE), both hepatotoxins, inhibit sequestration of Ca2+ by rat liver endoplasmic reticulum (ER) both in vivo and in vitro. It is possible that, as a result, cytosolic Ca2+ concentrations rise in liver cells. In experiments presented here, isolated hepatocytes were exposed to CCl4, DCE, and phenylephrine (PE), a non-hepatotoxic alpha 1-adrenergic agent that mobilizes Ca2+. Cytoplasmic Ca2+ concentrations were evaluated by two methods: indirectly by assaying the activity of glycogen phosphorylase a, and directly by monitoring the fluorescence of quin2. In primary hepatocyte cultures, CCl4, DCE, and PE exposure increased the activity of phosphorylase a at 5 min from 39 +/- 2 to 130 +/- 12, 80 +/- 13, and 97 +/- 10 nmoles PO4(3-)/mg protein/min respectively. In rat hepatocyte suspensions loaded with quin2 and exposed to CCl4, DCE, or PE, cytosolic Ca2+ concentrations were elevated within 20 sec to 0.83 +/- 0.13, 0.59 +/- 0.06 and 0.99 +/- 0.14 microM Ca2+ respectively. Basal Ca2+ levels in these cells averaged 0.25 +/- 0.03 microM. Thus, CCl4 and PE apparently increased cytosolic Ca2+ levels to approximately the same extent, whereas DCE was somewhat less effective. The durations of the effects of CCl4 and PE were examined further by determining their time courses of elevated phosphorylase a activity. In hepatocyte cultures, increased phosphorylase a activity persisted through at least 60 min following CCl4 exposure. In contrast, phosphorylase a activity returned to basal levels by 20 min after PE. Increases in cytoplasmic Ca2+ levels that are sustained rather than transient may distinguish these hepatotoxic chlorinated aliphatic hydrocarbons from non-toxic hormonal agents.

Aminoquinolines↗
PubMed1986

Inhibition of liver endoplasmic reticulum calcium pump by CCl4 and release of a sequestered calcium pool.

One of the earliest effects observed in rat liver after CCl4 administration is inhibition of an ATP-dependent calcium pump found at the endoplasmic reticulum. This report confirms that the amount of calcium associated with the microsomal fraction is reduced after CCl4 administration and, for the first time, demonstrates time-, dose-, and metabolism-dependent relationships between inhibition of the liver microsomal calcium pump and the amount of calcium found in the microsomal fraction. Furthermore, release of calcium from the endoplasmic reticulum is shown to cause activation of a cytoplasmic enzyme that responds to increases of ionized calcium, glycogen phosphorylase. This suggests that the endoplasmic reticulum calcium pump sequesters an intracellular pool of calcium within the endoplasmic reticulum. This pool of calcium may be released into the cytoplasm as a consequence of inhibition of the calcium pump by CCl4.

Animals↗
PubMed1986

Elevated cytosolic calcium in rat hepatocytes exposed to carbon tetrachloride.

CCl4 rapidly and severely inhibits hepatic endoplasmic reticulum calcium (Ca++) sequestration in rats exposed to this hepatotoxin in vivo. As a consequence, it is possible that cytosolic Ca++ concentrations become elevated in liver cells. In this study, the authors examined intracellular Ca++ concentrations in cultured rat hepatocytes exposed to CCl4 by monitoring the activity of phosphorylase a. Glycogen phosphorylase is converted to its a form in response to increases in cytoplasmic Ca++. Elevated phosphorylase a activity was observed within 2.5 min and was maintained for at least 30 min after exposure of hepatocytes to CCl4. Endoplasmic reticulum Ca++ pump activity decreased in a parallel manner. Phosphorylase activation was cyclic AMP independent and did not require extracellular Ca++. Cytoplasmic enzyme was released from hepatocytes within 30 min after CCl4 addition. Thus it was confirmed that exposure of hepatocytes to CCl4 causes release of Ca++ from an intracellular store (likely endoplasmic reticulum) and resultant activation of a Ca++-responsive cytosolic enzyme. From a calibration curve, it was estimated that cytosolic Ca++ is elevated up to 100-fold in rat hepatocytes exposed to the model hepatotoxin CCl4. It is postulated that prolonged elevation of intracellular Ca++ concentrations may trigger excessive stimulation of Ca++-sensitive enzymes capable of initiating irreversible liver cell injury.

Alanine Transaminase↗
PubMed1984

Hepatic macromolecular covalent binding of mononitrotoluenes in Fischer-344 rats.

The mononitrotoluenes are important industrial chemicals which display isomeric specificity in their ability to induce hepatic DNA excision repair in Fischer-344 rats. Covalent binding of the structurally related hepatocarcinogen, 2,6-dinitrotoluene, to hepatic DNA is markedly decreased by prior administration of the sulfotransferase inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP). The objectives of this study were to determine whether hepatic macromolecular covalent binding of the mononitrotoluene isomers differed and to determine whether covalent binding of the mononitrotoluenes to hepatic DNA in vivo was decreased by inhibitors of sulfotransferase. Male Fischer-344 rats were given a single oral dose of [ring-U-14C]-2-, 3- or 4-nitrotoluene (2-, 3- or 4-NT) and killed at various times thereafter. Livers were removed and analyzed for total and covalently bound radiolabel. Maximal concentrations of total radiolabel were observed between 3 and 12 h after the dose, and there were no large differences among the 3 isomers in peak concentrations achieved. Covalent binding to hepatic macromolecules was maximal 12 h after administration for all three isomers. Thereafter, concentrations of covalently bound 2-NT-derived material were always 2-6 times higher than those of 3- or 4-NT-derived material. When DNA was isolated from livers of rats given the mononitrotoluenes 12 h previously, only 2-NT was observed to covalently bind at concentrations above the limits of detection of the assay. The covalent binding of 2-NT, but not that of 3- or 4-NT, to both total hepatic macromolecules and DNA was markedly decreased by prior administration of either PCP or DCNP. Covalent binding to hepatic DNA was decreased by greater than 96%. The results of this study correlate well with studies which have demonstrated that 2-NT, but not 3- or 4-NT, induces DNA excision repair. Furthermore, they suggest that 2-NT, like the hepatocarcinogen 2,6-dinitrotoluene, requires the action of sulfotransferase for its conversion to a species capable of covalently binding to hepatic DNA.

Animals↗
PubMedDate not supplied

Ureteral endometriosis and ovarian mucinous cystadenoma presenting with acute pyonephrosis.

BACKGROUND: Endometriosis is a common disease, but ureteral involvement is rare. Nonspecific clinical presentations of ureteral endometriosis may result in diagnostic difficulty. AIM: To discuss the diagnosis and management of such a case. METHODS: To report a case of ureteral involvement with endometriosis and review the literature. RESULTS: The case presented with right lower quadrant pain giving rise to initial diagnostic possibility of acute appendicitis. Subsequent evaluation revealed the diagnosis of right pyonephrosis due to midureteral endometriosis with right ovarian mucinous cystadenoma. CONCLUSION: The diagnosis of ureteral endometriosis requires a high index of clinical suspicion. The importance of ultrasound in the evaluation of acute abdomen in women can not be overemphasised.

Abdominal Pain↗