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Biomedical subjects

G N Rao

Publications and source records attributed to G N Rao.

At least 19 recordsLinked to original sources
PubMed1992

Na+/H+ antiporter gene expression increases during retinoic acid-induced granulocytic differentiation of HL60 cells.

During differentiation of human leukemic HL60 cells into granulocytes, sustained increases in intracellular pH and Na+/H+ antiporter activity have been observed. In the present study we report that retinoic acid (RA)-induced granulocytic differentiation of HL60 cells causes an approximately 18-fold increase in the steady-state mRNA levels for the Na+/H+ antiporter. This was due to an increase in the rate of Na+/H+ antiporter gene transcription as measured by nuclear run-on analysis. Antiporter protein synthesis increased by seven-fold during RA-induced granulocytic differentiation of HL60 cells as measured by immunoprecipitation of 35S-methionine-labeled proteins with the RP1-c28 Na+/H+ antiporter antibody. No increase in antiporter mRNA was observed in response to etretinate, an analogue of retinoic acid, which did not induce differentiation. Thus, Na+/H+ antiporter gene expression is associated with RA-induced granulocytic differentiation of HL60 cells. The present findings and our previous data (Rao et al., 1991) demonstrate that Na+/H+ antiporter gene expression is a generalized feature of HL60 cell differentiation.

Carrier Proteins↗
PubMed1992

Active oxygen species stimulate vascular smooth muscle cell growth and proto-oncogene expression.

Vascular smooth muscle cells (VSMCs) proliferate in response to arterial injury. Recent findings suggest that, in addition to platelet-derived growth factors, growth factors from inflammatory cells and endothelial cells at the site of injury may contribute to VSMC proliferation. We hypothesized that a common mechanism by which endothelial cells and inflammatory cells stimulate VSMC growth could be the active oxygen species (i.e., O2-, H2O2, and .OH) generated during arterial injury. Using xanthine/xanthine oxidase to generate active oxygen species, we studied the effects of these agents on VSMC growth. Xanthine/xanthine oxidase (100 microM xanthine and 5 microunits/ml xanthine oxidase) stimulated DNA synthesis in growth-arrested VSMCs by 180% over untreated cells. Administration of the scavenging enzymes superoxide dismutase and catalase demonstrated that H2O2 was primarily responsible for xanthine/xanthine oxidase-induced VSMC DNA synthesis. H2O2 directly increased VSMC DNA synthesis and cell number (maximal at 200 microM) but decreased DNA synthesis of endothelial cells and fibroblasts. This effect was protein kinase C independent: sphingosine, a potent protein kinase C inhibitor, failed to block H2O2-induced VSMC DNA synthesis. H2O2 (200 microM) stimulated c-myc and c-fos mRNA levels by fourfold and 20-fold, respectively, as compared with quiescent levels. In contrast to DNA synthesis, H2O2 induction of c-myc and c-fos mRNA was primarily protein kinase C dependent. These findings show that H2O2 specifically increases VSMC DNA synthesis and suggest a role for this oxidant in intimal proliferation, especially after arterial injury.

Animals↗
PubMed1992

Effects of corn oil, time-related changes, and inter-laboratory variability on tumor occurrence in control Fischer 344 (F344/N) rats.

Survival, body weight, and site-specific tumor rates in untreated, corn oil gavage, and water gavage control Fischer 344 (F344/N) rats from 88 National Toxicology Program (NTP) long term carcinogenicity studies were evaluated to determine which factors were primarily responsible for inter-study variability. For male rats, previously-reported decreases in leukemia and increases in body weight, survival, and pancreatic acinar cell tumors attributable to corn oil gavage were confirmed. Corn oil did not appear to affect tumor rates in female rats. The gavage technique per se did not appear to influence tumor rates in rats of either sex. Previously reported time-related increases in certain site-specific neoplasia in control rats appeared to have stabilized in recent years, but control tumor rates are still much greater than those seen a decade ago. More recent studies continue to show increasing rates of leukemia and mammary gland tumors and decreasing survival. Female rats also continue to show time-related increases in maximum mean body weight. Inter-laboratory variability in body weight and in the rates of a number of site-specific neoplasms were also significant. High mean body weights in control groups were found to be associated with increased rates of mammary and pituitary tumors. Our evaluation supports the view that if historical control data are to be utilized in the interpretation of experimental results, primary emphasis should be given to lab and route of administration-specific tumor rates for studies that are contemporary to the study under evaluation. It also suggests that certain experimental design changes (e.g., dietary modifications) may be needed to reduce tumor rates and to increase survival.

Animals↗
PubMed1992

Histopathologic observations in weanling B6C3F1 mice and F344/N rats and their adult parental strains.

Weanling Fischer 344/N (F344) rats and the first filial hybrid of C57BL/6 x C3H (B6C3F1) mice and retired breeders from the parental stocks of these strains were monitored over a 5-yr-period by examining the histopathology of selected organs and comparing those results to viral and mycoplasmal serology and the intestinal tract bacterial flora of each animal on an individual basis. Serology gave no evidence of viral infection, but Mycoplasma arthriditis antibodies were detected. Reactivity of serum of adult C57BL/6 female mice with control cells or media (tissue culture, TC) was seen in a significant number of mice. TC reactivity correlated positively with lymphoid perivascular infiltrates, predominantly of the lungs, suggesting an allergic response in development of the lesions. Other lesions of note consisted of Harderian gland inflammation of rats, focal necrotizing lesions of the liver of both species, and thickening of the pleura and adjacent pulmonary interstitium of weanling rats. Embolization of bacteria from the gastrointestinal tract to the liver was considered a possible cause of the liver necrosis in both species. Although lesions of the lung and Harderian gland of the rats are similar to those caused by known viral agents, the cause of the latter could not be determined as these animals were negative for viral antibodies and the former was considered to be related to incomplete pulmonary development in the young rat. Features differentiating the lesions observed in animals of this survey from those caused by viral infection are discussed.

Aging↗
PubMed1991

Na+/H+ antiporter gene expression during monocytic differentiation of HL60 cells.

During differentiation of human promyelocytic HL60 cells into monocytes there are sustained increases in intracellular pH and Na+/H+ antiporter activity. Here we show that increased transcription and expression of the gene for the Na+/H+ antiporter precedes phorbol 12-myristate 13-acetate (PMA)-induced HL60 cell differentiation. PMA increased steady-state Na+/H+ antiporter mRNA levels approximately 50-fold within 8 h (at which time less than 15% of cells had differentiated). This increase was due to an increased transcriptional rate as determined by nuclear run on. Immunoprecipitation of [35S]methionine-labeled Na+/H+ antiporter using an antiporter fusion protein antibody (RP1-c28) showed an equivalent increase in Na+/H+ antiporter protein synthesis. The synthetic diacylglycerol, 1-oleolyl-2-acetylglycerol, an activator of protein kinase C, which unlike PMA did not cause differentiation, failed to induce Na+/H+ antiporter mRNA. Furthermore, inhibition of PMA-induced differentiation by either sphingosine or cycloheximide prevented accumulation of Na+/H+ antiporter mRNA. Together, these findings strongly suggest a close association of Na+/H+ antiporter induction with HL60 cell differentiation. The HL60 cell system is a promising model to study the mechanisms of Na+/H+ antiporter gene regulation and its function in differentiation.

Carrier Proteins↗
PubMed1991

Uric acid stimulates vascular smooth muscle cell proliferation by increasing platelet-derived growth factor A-chain expression.

Recent data suggest that uric acid is generated locally in the vessel wall by the action of xanthine oxidase. This enzyme, activated during ischemia/reperfusion by proteolytic conversion of xanthine dehydrogenase, catalyzes the oxidation of xanthine, thereby generating free radicals and uric acid. Because of the potential role of ischemia/reperfusion in vascular disease, we studied the effects of uric acid on rat aortic vascular smooth muscle cell (VSMC) growth. Uric acid stimulated VSMC DNA synthesis, as measured by [3H]thymidine incorporation, in a concentration-dependent manner with half-maximal activity at 150 microM. Maximal induction of DNA synthesis by uric acid (250 microM) was approximately 70% of 10% calf serum and equal to 10 ng/ml platelet-derived growth factor (PDGF) AB or 20 ng/ml fibroblast growth factor. Neither uric acid precursors (xanthine and hypoxanthine) nor antioxidants (ascorbic acid, glutathione, and alpha-tocopherol) were mitogenic for VSMC. Uric acid was mitogenic for VSMC but not for fibroblasts or renal epithelial cells. The time course for uric acid stimulation of VSMC growth was slower than serum, suggesting induction of an autocrine growth mechanism. Exposure of quiescent VSMC to uric acid stimulated accumulation of PDGF A-chain mRNA (greater than 5-fold at 8 h) and secretion of PDGF-like material in conditioned medium (greater than 10-fold at 24 h). Uric acid-induced [3H]thymidine incorporation was markedly inhibited by incubation with anti-PDGF A-chain polyclonal antibodies. Thus uric acid stimulates VSMC growth via an autocrine mechanism involving PDGF A-chain. These findings suggest that generation of uric acid during ischemia/reperfusion contributes to atherogenesis and intimal proliferation following arterial injury.

Animals↗
PubMed1991

A PC based computer program to aid the preparation of solvent systems for the HPLC.

An interactive computer program, SOLCOMP, is developed which calculates the volumes of the components that are to be added to obtain either a fresh solvent mixture or a new solvent system from the old one of required composition. The code is implemented in MicroSoft FORTRAN and GWBASIC which runs on any IBM compatible PC under MSDOS environment.

Algorithms↗
PubMed1991

A microcomputer program to analyze the CD spectrum of proteins and nucleic acids--use of LOTUS 1-2-3 spread sheet.

A user friendly interactive computer program, CIRDIC, is developed which calculates the molar ellipticity and molar circular dichroic absorption coefficients from the CD spectrum. This, in combination with LOTUS 1-2-3 spread sheet, will give the spectra of above parameters vs wavelength. The code is implemented in MicroSoft FORTRAN 77 which runs on any IBM compatible PC under MSDOS environment.

Algorithms↗
PubMed1991

Light intensity-associated eye lesions of Fischer 344 rats in long-term studies.

Albino rats and mice are sensitive to light and the recommended illumination of animal rooms (75-125 ft-candles) is known to cause retinal damage. When a room is illuminated by ceiling lights, animals in the cages of the top row and, to some extent, in the side columns of cage racks will be exposed to higher light intensity than those in the other cages of the rack. In 2-yr chemical carcinogenicity studies of the National Toxicology Program (previously the Carcinogenicity Bioassay Program of the National Cancer Institute), Fischer 344 rats were group-housed in hanging drawer-type clear polycarbonate cages. During the course of the chronic studies, a number of rats developed opacity of the eye. Ocular examination indicated chronic uveitis, deep interstitial keratitis, cataract formation leading to panophthalmitis, and in severe cases, phthisis bulbi. Histologic examination showed cataract and retinal degeneration. Incidences of these lesions were highest (greater than 55%) in the rats of the top rows and lowest in those of the bottom rows (less than 10%) of cages with no relation to chemical treatment, indicating an association with light intensity. The incidence of these eye lesions was markedly decreased (less than 15%) by decreasing the light intensity of the animal room to less than 50 ft-candles at 5 ft from the floor and rotating the cages in each column of a rack from top to bottom when cages or racks were changed.

Animals↗
PubMed1990

Differential regulation of Na+/H+ antiporter gene expression in vascular smooth muscle cells by hypertrophic and hyperplastic stimuli.

The Na+/H+ antiporter is a ubiquitous transmembrane protein that plays a vital role in cell growth via regulation of intracellular Na+ and H+. In vascular smooth muscle cells (VSMC), vasoconstrictors and mitogens rapidly activate the antiporter, suggesting that both should have growth promoting effects. Indeed, angiotensin II increases VSMC protein and volume (hypertrophy), but does not increase cell number (hyperplasia). In the present work we investigated whether alterations in the steady state levels of Na+/H+ antiporter mRNA might differentiate these VSMC growth responses. Differences in function of the Na+/H+ antiporter appeared likely because exposure of growth-arrested VSMC for 24 h to 100 nM angiotensin II decreased intracellular pH from 7.08 to 6.99, while exposure to 10% calf serum caused an increase to 7.18. Simultaneous measurement of Na+/H+ antiporter mRNA levels, using the human c28 cDNA, revealed a 25-fold increase in response to serum (as well as to platelet-derived and fibroblast growth factors), but no change in response to angiotensin II. All agonists increased mRNA levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase approximately 3-fold. The increase in Na+/H+ antiporter mRNA induced by serum was first apparent within 2 h and peaked 24 h after treatment. These results demonstrate that expression of Na+/H+ antiporter mRNA in VSMC is dependent on growth state: hyperplastic agonists (serum, platelet-derived and fibroblast growth factor) increase the steady state levels of Na+/H+ antiporter mRNA while a hypertrophic agonist (angiotensin II) does not.

Angiotensin II↗
PubMed1990

Refinement of long-term toxicity and carcinogenesis studies.

The chance that alternatives will completely replace animals for toxicology research in the foreseeable future is nil. Continual refinement of animal toxicity and carcinogenesis studies, however, can be an effective means of reducing the numbers of animals used and conserving time and resources without compromising scientific quality. We must continue to strive to find species and strains that can metabolize chemicals similar to humans, are small enough to be housed in large numbers, and have low prevalence of spontaneous lesions with sufficient life span to express the toxic and carcinogenic potential of chemicals. Adequate care of animals with control of variables such as light, temperature, diet, bedding, diseases, and genetic characters of laboratory animals will decrease the variability. Humane considerations and euthanasia of animals with large masses and other conditions interfering with eating and drinking, major injuries and ulcers related to husbandry and treatment, and diseases indicating pain and suffering will help not only to alleviate further pain and distress but also to facilitate collection of tissues without secondary complications for detection of chemical treatment-related lesions. Limiting the duration of studies to decrease the variability due to age-associated changes will also refine long-term studies. Other considerations for refinement of carcinogenesis studies include selection of the most sensitive sex of one or more species for evaluation of selected chemicals in a class where toxic and carcinogenic potential of other representative chemicals are known. Genetically engineered animal models with known oncogenes may reduce the duration and increase the sensitivity of carcinogenesis studies with a reduction in the use of animals.

Animal Husbandry↗
PubMed1990

Organization and nucleotide sequence of the 3' end of the human CAD gene.

Aspartate transcarbamylase (ATCase) is found as a monofunctional protein in prokaryotes and as a part of a multifunctional protein in fungi and animals. In mammals, this enzyme along with carbamyl phosphate synthetase II and dihydroorotase (DHOase) is encoded by a single gene called CAD. To determine the relationship between gene structure and the enzymatic domains of human CAD, we have isolated genomic clones of the human gene and sequenced the region corresponding to the 3' end of the gene. This includes exons encoding the end of the domain for DHOase, the complete domain for ATCase, and the bridge region connecting the two enzymatic domains. Three findings emerged. First, in comparing the human coding sequence to that obtained for other species that have a CAD gene, the length of the bridge region is conserved but its sequence is not. This is in contrast to the strong degree of positional identity observed for the segments of CAD encoding the DHOase and ATCase domains. Second, sets of exons appear to correspond to specific domains and subdomains of the encoded protein. Third, while overall there is a strong conservation of protein sequence among the ATCases of all species, reflecting conservation in catalytic function, two particular regions of the enzyme are more highly conserved among species where ATCase is a domain of a multifunctional protein as opposed to species where it is a monofunctional protein. Such findings may indicate regions of the ATCase domain that provide important structural contacts or functional channels when part of a multifunctional protein.

Amino Acid Sequence↗
PubMed1990

Bovine lens membrane proteins: MP70, MP64, and MP38 are products of the same gene.

We have carried out limited microsequence analysis of bovine lens intrinsic membrane proteins having molecular weights of 70, 64, and 38 kD. These three polypeptides all have an identical amino acid terminal sequence, at least for the first 17 amino acid residues, indicating a common origin. When calf lens RNA was hybridized with a labeled antisense oligonucleotide common to the amino acid sequence of these three polypeptides, a single message with an apparent molecular size of 2.6 kb was detected. Together, these results indicate that bovine lens MP70, MP64, and MP38 are products of the same gene and that the lower molecular weight polypeptides are the result of degradation (processing) of lens MP70 at its COOH-terminal end.

Amino Acid Sequence↗
PubMed1990

Growth, body weight, survival, and tumor trends in F344/N rats during an eleven-year period.

Time trends for growth, body weight, survival and tumor prevalences in 144 diet control groups with a total of 5,184 male F344/N rats and 146 diet control groups with a total of 5,289 female rats of NCI-NTP 2-yr chemical carcinogenicity studies started during an 11-yr period (1971 to 1981) in 11 toxicology testing laboratories were evaluated. Male and female rats in more recent studies grew faster and attained a higher body weight than rats from earlier studies. Survival of males showed a significantly decreasing trend over time, which may have been related to diseases associated with increasing body weight, prevalence of leukemia and changes in criteria for euthanasia of moribund animals. The time trend for survival of females was not significant. There were highly significant (p less than 0.001) positive time trends for prevalences of leukemia, anterior pituitary tumors and thyroid C-cell tumors in both sexes, adrenal pheochromocytomas in males and mammary tumors and endometrial stromal polyps in females. The prevalence of mammary tumors in females and pituitary tumors in males had a highly significant (p less than 0.01) positive association with body weight. Histological reevaluation of tumor prevalences in approximately 250 rats of each sex at each of 4 different time periods indicated that changes in diagnostic criteria may have contributed to but could not totally explain the increased prevalence of leukemia. Changes in diagnostic criteria and the amount of tissue examined may have contributed to the increased prevalence of anterior pituitary tumors in both sexes and adrenal pheochromocytomas in males. Interlaboratory variability and changes in diet may also have contributed to the time-related trends.

Adrenal Gland Neoplasms↗
PubMed1990

Growth, body weight, survival, and tumor trends in (C57BL/6 X C3H/HeN) F1 (B6C3F1) mice during a nine-year period.

Time trends for growth, body weight, survival and tumor prevalences in 121 diet control groups with a total of 4,636 male B6C3F1 mice and 123 diet control groups with a total of 4,758 female mice of NCI-NTP 2-yr chemical carcinogenicity studies started during a 9-yr period (1973 to 1981) in 11 laboratories were evaluated. Male and female mice did not show substantial changes in growth patterns. Both sexes had highly significant time trends with decreasing body weights in the more recent studies. This apparent trend was due to high body weights during the first 3 yr and highly significant interlaboratory variability. Time trends for survival of both sexes were not significant. Prevalences of liver tumors, lung tumors, and lymphoma in males and lung tumors in females did not show significant time trends. There were significant positive time trends for prevalences of liver tumors and lymphoma in female mice, but the trends were not significant when adjusted for interlaboratory variability. The positive time trend for anterior pituitary tumors of females was highly significant and may be due in part to an increase in the amount of pituitary tissue examined in the more recent studies. Histological reevaluation of liver and anterior pituitary tissue in 208-249 female mice at each of 4 different times periods did not substantially change the prevalences or the time trends. The major factor influencing time trends in mice appeared to be interlaboratory variability.

Animals↗
PubMed1990

Tissue reaction to an implantable identification device in mice.

Long-term toxicity and carcinogenicity studies require positive identification of animals. Due to the unreliability of traditional methods, it was necessary to investigate more dependable identification methods that can be read directly or by electronic means. A two-year study to determine the stability of and tissue reaction to a microchip glass-sealed device implanted in subcutaneous tissue of mice was conducted. Seventy B6C3F1 mice of each sex were anesthetized and implanted with the microchip. The devices were read by an electronic detector and palpated at periodic intervals. Ten mice of each sex were necropsied at 3 months and at 15 months with the remaining animals necropsied at 24 months. Of the 140 devices implanted, 3 were lost and 4 failed during the 24-month study. Devices were palpable and appeared to be fixed at one location with no obvious swelling due to inflammation or palpable masses around the implants for 24 months. At the 3, 15, and 24 month necropsies, implants were encapsulated by connective tissue. Light microscopic evaluation indicated that the capsule around the implants was thin and composed of fibrocytes and mature collagen fibers, with minimal to mild inflammation and occasional granulomatous reaction. Neoplastic changes were not observed in the tissue around the glass-sealed devices with polypropylene cap for up to 24 months.

Animal Identification Systems↗
PubMed1990

Molecular cloning and complete nucleotide sequence of the cDNA encoding a bovine lens intrinsic membrane protein (MP19).

Recently, we reported the partial characterization of bovine lens intrinsic membrane proteins having apparent SDS-PAGE derived molecular mass of 19, 21, and 23 kDa, and determined that they contained identical NH2- terminal amino acid sequences for the first 20 amino acids. From this amino acid sequence information, a mixed synthetic oligonucleotide was constructed and used to screen a calf lens lambda gt11 cDNA library in order to isolate and characterize the cDNA coding for this membrane polypeptide(s). Two separate cDNA clones were isolated and sequenced, and were found to have an identical sequence of 883 bases with an open reading frame coding for a polypeptide of 173 amino acids, having a molecular mass of 19,683 Daltons. The first 20 amino acids of the translated sequence were identical to that determined by our laboratory previously, and the last seven amino acids were identical to that recently determined by another laboratory from analysis of the extracted polypeptides, indicating that this cDNA is the authentic molecule coding for MP19.

Amino Acid Sequence↗