Search PubMed⌕ Search

Biomedical subjects

R T Miller

Publications and source records attributed to R T Miller.

At least 73 records · Page 4Linked to original sources
PubMed1994

Kinetic and mechanistic studies with bovine testicular hyaluronidase.

Bovine testicular hyaluronidase exhibits hydrolase and transglycosylase activity. To assess the magnitude of each type of reaction, the time-course of hyaluronidase catalysed hyaluronic acid degradation was followed using a sensitive and specific HPLC method. The kinetic parameters Km and Vmax were calculated for purified short chain hyaluronic acid oligomers and native hyaluronic acid based on the appearance of unreactive hyaluronic acid tetrasaccharide. For hyaluronic acid oligomers, as substrate size increased Km decreased from 2.06 to 1.09 mM while Vmax remained about the same, indicating a 5-fold increase in the enzyme-substrate association constant, k1 (kcat/Km). The values of k2 (kcat), the enzyme-substrate disassociation constant, for native hyaluronic acid and hyaluronic acid decasaccharide were similar. The value of k1 for native hyaluronic acid, however, was larger by 70-fold. Kinetic degradation mechanisms for each hyaluronic acid oligomer, using chemical-reaction kinetics, were proposed and evaluated by computer curve fitting analysis of the experimental time vs. concentration data. The derived rate constants, together with mass balance calculations, revealed that transglycosylation plays a significant role in the degradation of all hyaluronic acid oligomers studied.

Animals↗
PubMed1994

Purification and characterization of the alpha subunit of G13.

Specific antisera were produced to peptides representing the carboxyl terminus of alpha 13, a recently identified alpha subunit of the heterotrimeric guanine nucleotide-binding proteins (G proteins). Immunodetection with the antisera indicated that the 43-kDa protein is expressed ubiquitously at low levels (0.005-0.05% of membrane protein) in tissues and cultured cells. A combination of conventional and immunoaffinity chromatographic techniques was used to purify small quantities of alpha 13 from bovine brain. Quantities of protein sufficient for biochemical analysis could be produced by concurrent expression of alpha 13 with G protein beta 2 and gamma 2 subunits using a baculovirus system. The rate of dissociation of GDP from recombinant alpha 13 (r alpha 13) is slow (0.01-0.02 min-1 at 30 degrees C), and relatively high concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) are required to observe nucleotide binding. This binding was reduced significantly in the presence of 20 mM Mg2+. Rates of hydrolysis of GTP by alpha 13 were limited by nucleotide exchange; attempts to measure the intrinsic rate of hydrolysis indicate that it is greater than 0.2 min-1. Stoichiometric concentrations of beta gamma subunits inhibited binding of GTP gamma S to and hydrolysis of GTP by alpha 13. By reconstitution, the purified alpha 13 did not affect the activity of several known effector enzymes. The availability of purified r alpha 13 and knowledge of its biochemical properties will allow further characterization of its interactions with receptors and effectors.

Amino Acid Sequence↗
PubMed1994

Angiotensin II stimulation of Na-H antiporter activity is cAMP independent in OKP cells.

Angiotensin II has been reported to stimulate the proximal tubule Na-H antiporter by inhibition of adenylyl cyclase, and possibly by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism. We examined the effect of angiotensin II on Na-H antiporter activity (JNa-H) in opossum kidney (OKP) cells, a proximal tubule-like cell line, whose Na-H antiporter resembles that of the proximal tubule apical membrane. We found that angiotensin II regulates JNa-H in a concentration-dependent manner similar to the proximal tubule, with angiotensin II concentrations < 10(-8) M stimulating and > 10(-8) M inhibiting JNa-H. The stimulatory effect of angiotensin II was blocked by 10(-8) M losartan and was pertussis toxin sensitive, suggesting mediation through an angiotensin II (AT1) receptor coupled to a pertussis toxin-sensitive G protein. Acute treatment with 10(-4) M 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) inhibited JNa-H by 30% and blocked angiotensin II-induced stimulation. However, angiotensin II (10(-12)-10(-6) M) did not inhibit basal, dopamine-stimulated, or forskolin-stimulated cAMP production measured in the presence of 3-isobutyl-1-methylxanthine (IBMX). In addition, angiotensin II had no effect on cAMP levels measured in the absence of IBMX. We conclude that angiotensin II at physiological concentrations stimulates JNa-H in OKP cells via a cAMP-independent mechanism mediated by an AT1 receptor and a pertussis toxin-sensitive G protein.

Adenylate Cyclase Toxin↗
PubMed1994

Regulation of hormone-sensitive calcium influx by the adenylyl cyclase system in renal epithelial cells.

To study signaling pathways regulated by alpha s and alpha i1 in renal epithelial cells, we expressed mutant, activated forms of alpha s and alpha i1 in a continuous proximal tubule cell line (MCT cells). alpha sQ227L increased cAMP production, and alpha ilQ204L reduced forskolin-sensitive cAMP production. alpha ilQ204L increased and alpha sQ227L decreased bradykinin-induced Ca influx across the cell membrane, but neither mutant affected bradykinin-stimulated intracellular Ca release or basal Ca influx. Bradykinin-stimulated Ca influx was reduced by dibutyryl cAMP, isoproterenol, and forskolin. Expression of a mutant regulatory type I subunit for cAMP-dependent protein kinase with reduced affinity for cAMP and treatment with KT-5720, a specific cAMP-dependent protein kinase inhibitor, enhanced Ca influx to a degree similar to that in cells expressing alpha ilQ204L. Bradykinin-stimulated c-fos mRNA expression is partially dependent on extracellular Ca. alpha sQ227L reduced and alpha ilQ204L enhanced bradykinin-stimulated c-fos expression. Consequently, in bradykinin-stimulated cells, the adenylyl cyclase system regulates Ca influx through cAMP-dependent protein kinase, but not intracellular Ca release. Furthermore, the Ca influx mechanism acts as an integrator of two signaling pathways such that Ca-dependent signals are damped by activators of adenylyl cyclase and enhanced by inhibitors of adenylyl cyclase.

Adenylyl Cyclases↗
PubMed1994

Acid activation of immediate early genes in renal epithelial cells.

These studies examined the effect of acidosis on immediate early (IE) gene expression in renal tubule cells. In MCT cells, an SV40 transformed mouse proximal tubule cell line, incubation in acid media led to transient increases in c-fos, c-jun, junB, and egr-1 mRNA abundance, peaking at 30 min to 1 h. In vivo metabolic acidosis caused more prolonged increases in these mRNA species in renal cortex. Nuclear runon studies demonstrated increased rates of transcription for these IE genes. In addition, pretreatment of cells with cycloheximide caused superinduction of these mRNA by acid incubation. These responses are similar to those elicited by growth factors. Inhibition of tyrosine kinase pathways prevented IE gene activation by acid, while inhibition of protein kinase C and/or increases in cell calcium had no effect. In 3T3 cells, acid activated IE genes by a different mechanism in that the increase in mRNA did not include c-jun, was more prolonged, and was blocked by cycloheximide. In summary, incubation of renal cells in acid media leads to activation of IE genes that is similar to growth factor-induced IE gene activation, and is likely mediated by tyrosine kinase pathways.

3T3 Cells↗
PubMed1993

Primary right atrial chondrosarcoma in a dog.

Primary right atrial chondrosarcoma was diagnosed in a dog that had clinical signs of right-sided heart failure. The tumor was attached to the intra-atrial septum, dorsal to the septal leaf of the tricuspid valve. Tumor cells were not identified elsewhere.

Animals↗
PubMed1993

Effect of regeneration and hyperplasia on levels of DNA base oxidation in rat liver.

Elevations of oxidatively modified DNA bases have been associated with a variety of carcinogens and tumor promoters, and implicated in causation of cancer. Since carcinogen exposure can induce cell proliferation, the relationship between induction of cell proliferation and levels of DNA base oxidation was examined. Cell proliferation was induced in livers of male F344 rats by stimuli of either regeneration or hyperplasia. Levels of DNA base oxidation were evaluated by measuring 8-OH-deoxyguanosine/deoxyguanosine (8-OHdG/dG) ratios by HPLC in enzymatic digests of DNA isolates. Despite induction of cell proliferation, hepatic levels of 8-OHdG/dG were not increased at 1, 2, 3 or 5 days after any of these treatments. Results of the present work suggest that the mechanism of elevated levels of DNA base oxidation is not directly related to induction of cell proliferation.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Immunocytochemical assay for estrogen receptor with monoclonal antibody D753P gamma in routinely processed formaldehyde-fixed breast tissue. Comparison with frozen section assay and with monoclonal antibody H222.

BACKGROUND: Estrogen receptor (ER) immunocytochemical assay (ICA) can be a useful procedure in patients with breast cancer. The authors sought to study the utility of monoclonal antibody D75P3 gamma (D75) in paraffin-embedded sections of breast cancers. METHODS: Sixty-seven cases of breast cancer were studied by ICA for ER using monoclonal antibody D75 in routinely processed formaldehyde-fixed paraffin-embedded tissue, employing ficin predigestion of sections before application of D75. The results were tabulated using a modified histochemical score (HS). In 59 cases, frozen sections of tumors were also studied with D75. In 40 cases, paraffin-embedded sections were stained with both D75 and monoclonal antibody H222 (which is commercially available). Quantitative biochemical ER assays were performed by the standard dextran-coated charcoal method. RESULTS: Statistical analysis of the data showed a highly significant correlation (P < 0.0001) between biochemical ER and paraffin-embedded section HS using D75. The results of frozen section assay and paraffin-embedded section assay using D75 were also highly correlated (P < 0.0001). In addition, paraffin-embedded section HS using D75 correlated well with paraffin-embedded section HS using H222 (P < 0.0001). However, D75 HS generally were higher than H222 HS because of the more intense staining with D75, and this difference was statistically significant (P < 0.0001). Assuming that the biochemical ER data were "true," paraffin-embedded section ICA with D75 had a diagnostic sensitivity of 100%, a diagnostic specificity of 70%, a positive predictive value of 89%, and a negative predictive value of 100%. The results of biochemical ER status and paraffin-embedded section D75 ICA ER status agreed in 61 of 67 cases (91%). In all six cases where the ER status differed, the biochemical ER status was negative, and the ER status by ICA with D75 was positive, leading the authors to suspect that some of these cases may have been biochemical false-negative findings. CONCLUSIONS: It was concluded that ICA for ER with D75 using a 2-minute ficin predigestion of formaldehyde-fixed paraffin-embedded tissue is an acceptable alternative to frozen section ICA. In addition, paraffin-embedded section ICA with D75 is easier to interpret than frozen section ICA, and the results are superior to ICA with H222 on paraffin-embedded sections.

Antibodies, Monoclonal↗
PubMed1993

Enhanced robotic dissolution system with concurrent off-line analysis.

A Zymate II robotic dissolution system was modified by addition of a liquid chromatography (LC) system equipped with an Isco ISIS autosampler. Using the Concurrent EasyLab programming language, operation of this LC system was integrated into the main robotic dissolution procedure. This enabled simultaneous dissolution sampling and LC analysis of standard and pre-pulled sample solutions. The resulting dual-tasking system effects considerable efficiency of operation and results in significant time savings. Comparison of the robotic and non-robotic data indicate good correlation and statistically insignificant differences at the 95% confidence level. Validation studies confirm linearity and precision of the quantitative LC method, accuracy and precision of volume and temperature measurements and negligible vessel-to-vessel carryover.

Capsules↗
PubMed1993

Renin expression in renal proximal tubule.

Angiotensinogen, angiotensin-converting enzyme, and renin constitute the components of the renin-angiotensin system. The mammalian renal proximal tubule contains angiotensinogen, angiotensin-converting enzyme, and angiotensin receptors. Previous immunohistochemical studies describing the presence of renin in the proximal tubule could not distinguish synthesized renin from renin trapped from the glomerular filtrate. In the present study, we examined the presence of renin activity and mRNA in rabbit proximal tubule cells in primary culture and renin mRNA in microdissected proximal tubules. Renin activity was present in lysates of proximal tubule cells in primary culture. Cellular renin content in cultured proximal tubule cells was increased by incubation with 10(-5) M isoproterenol and 10(-5) M forskolin by 150 and 110%, respectively. In addition, renin transcripts were detected in poly(A)+ RNA from cultured proximal tubule cells by RNA blots under high stringency conditions. In microdissected tubules from normal rats, renin mRNA was not detectable with reverse transcription and polymerase chain reaction. However, in tubules from rats administered the angiotensinogen-converting-enzyme inhibitor, enalapril, renin was easily detected in the S2 segment of the proximal tubule. We postulate the existence of a local renin-angiotensin system that enables the proximal tubule to generate angiotensin II, thereby providing an autocrine system that could locally modulate NaHCO3 and NaCl absorption.

Animals↗
PubMed1993

Chronic regulation of the Na/H antiporter.

This review focuses on studies from our laboratory investigating the mechanisms of chronic regulation of the Na/H antiporter in renal and nonrenal cells. Tissue culture provides an ideal tool for investigating this problem because it avoids many complicating effects that would occur in an intact animal during a chronic study. Chronic decreases in extracellular fluid pH cause an increase in Na/H antiporter activity that is dependent on protein synthesis and associated with an increase in NHE-1 (isoform of the sodium-hydrogen antiporter) mRNA abundance. This effect is associated with acid-induced increases in a number of immediate early genes, including c-fos, c-jun, junB, and egr-1. In primary cultures of rabbit proximal tubule cells, activation of protein kinase C for 2 hours causes an increase in Na/H antiporter activity that persists 24 hours later, is dependent on transcription and translation, and is associated with an increase in NHE-1 mRNA abundance. Chronic activation of protein kinase A in opossum kidney (OKP) cells causes an increase in Na/H antiporter activity that persists 16 to 20 hours later and is dependent on protein synthesis. This latter effect is of particular interest because it is opposite in direction to the acute inhibitory effect of protein kinase A on the Na/H antiporter in these cells.

Animals↗
PubMed1992

Role of protein kinase C and transcription factor AP-1 in the acid-induced increase in Na/H antiporter activity.

Chronic incubation of cultured renal tubular epithelial cells in acid medium causes an increase in Na/H antiporter activity that persists after removal from acid, is dependent on protein synthesis, and is associated with an increase in Na/H antiporter mRNA. Chronic activation of protein kinase C has similar effects in these cells. The present studies examined the role of protein kinase C in the effect of acid incubation. Incubation of MCT cells in acid for 24 h caused a 50% increase in Na/H antiporter activity. This was prevented by inhibition of protein kinase C, either with sphingosine or by protein kinase C downregulation. Pertussis toxin pretreatment did not prevent the increase in antiporter activity. Acid incubation caused an increase in transcription factor AP-1 activity, as shown by an increase in expression from a reporter gene containing six tandem AP-1 binding sites. This was associated with transient increases in c-fos and c-jun mRNAs. This response is typical of that for gene activation by protein kinase C. These studies demonstrate that acid activation of the Na/H antiporter requires protein kinase C and is associated with c-fos and c-jun expression and increased AP-1 activity.

Animals↗
PubMed1992

Cobalamin deficiency associated with methylmalonic acidemia in a cat.

A 9-month-old sexually intact male longhair cat was examined because of lethargy, anorexia, cold intolerance, and failure to thrive since acquisition at an early age. Clinical signs of disease were less pronounced when the cat was fed a low-protein diet. Anemia, hypoglycemia, low total CO2 content, and hyperammonemia were detected. The cat was euthanatized. Urine obtained immediately before euthanasia contained a large amount of methylmalonic acid. Total serum cobalamin concentration was low. Hepatic methylmalonic-CoA mutase activity, with and without the addition of coenzyme adenosylcobalamin, was consistent with a cobalamin deficiency. Methylmalonic acidemia secondary to a putative defect in cobalamin absorption was diagnosed.

Absorption↗
PubMed1992

Unusual prostatic mass in a dog.

Marginal surgical resection and castration were successfully used to treat a dog with a large benign mass involving only the left lobe of the prostate. Pathologic findings were compatible with unusual nodular hyperplasia or adenoma, with smooth muscle proliferation. Although the definitive diagnosis remains controversial, the mass was termed nodular hyperplasia because of histologic and clinical similarities to the disease in human beings.

Adenoma↗
PubMed1992

Long-term activation of protein kinase c causes chronic Na/H antiporter stimulation in cultured proximal tubule cells.

To examine the role of protein kinase C as a chronic regulator of proximal tubule Na/H antiporter activity, the effect of phorbol 12-myristate 13-acetate (PMA) on the Na/H antiporter was studied in cultured proximal tubule cells. Short-term activation of protein kinase C by 5 min exposure to PMA caused an acute increase in Na/H antiporter activity that was not prevented by cycloheximide or actinomycin D and did not persist 24 h later. Long-term activation of protein kinase C by 2 h exposure to PMA caused a dose-dependent increase in Na/H antiporter activity 24 h later. This latter effect was due to protein kinase C activation in that it was inhibited by sphingosine and was not seen with 4 alpha-PMA, an inactive analogue. The chronic effect of PMA was inhibited by 10 nM actinomycin D or 7 microM cycloheximide. Proximal tubule cells exposed to PMA for 2 h demonstrated a two- to threefold increase in Na/H antiporter mRNA (mRNANa/H) abundance 4 h later. In conclusion, short-term activation of protein kinase C leads to a transient increase in Na/H antiporter activity that is independent of transcription and translation, whereas long-term activation of protein kinase C causes a persistent increase in antiporter activity that is dependent on transcription and translation and is associated with increased mRNANa/H abundance. This latter effect may mediate increased Na/H antiporter activity in a number of chronic conditions.

Animals↗
PubMed1992

The impact of molecular biology on understanding renal signal transduction.

The information that cells require to grow and prosper comes from diverse sources, adjacent to them and from considerable distances. Through evolution, cells have developed multiple mechanisms to acquire and process information. When a signaling mechanism has been successful, it has been used repeatedly with relatively minor modifications, and consequently is seen throughout biology. Specificity comes from using precise combinations of signaling molecules to achieve different ends in different cell types. Among the signaling systems that fit this description include the receptor and nonreceptor tyrosine kinases, G protein-coupled receptors, cell attachment receptors, guanylate cyclases, ligand-gated ion channels, the second messenger dependent and independent protein kinases and phosphatases, and transcription factors. The consequence of this conservation of molecular mechanisms is that many basic regulatory systems, which cannot be dissected free of the complexity of mammals, can be understood in relatively simple, manipulable experimental systems. With information from simple systems and the tools of genetics and molecular biology, complex problems such as human physiology and disease may be understood and eventually controlled.

Animals↗
PubMed1992

Comparison of estrogen receptor immunocytochemical assay in frozen and paraffin sections.

Frozen and paraffin sections of 11 breast carcinomas were stained for estrogen receptors (ER) using the same rat monoclonal primary antibody, D75P3, as the marker and alkaline phosphatase as the chromogen-linking enzyme. The results of this staining process were assessed visually and with the microTICAS image analysis system to determine the degree of correlation between frozen and paraffin-embedded tissue. In all specimens, some fraction of the nuclei stained positively. This included two specimens selected for their biochemically negative assay; one of them stained strongly positively with D75P3. The results of quantitative analysis support the visually apparent correlation between the two types of samples in terms of both overall staining pattern and intensity of nuclear staining. Although the conclusions of this pilot study are limited because of the small number of cases, this method of staining establishes the feasibility of representative ER determination in archival paraffin-processed material. The additional information provided by this method is potentially useful in stratifying patients in prospective studies on the basis of the efficacy of hormonal therapy in biochemically ER positive breast tumors.

Antibodies, Monoclonal↗