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Biomedical subjects

R Kumar

Publications and source records attributed to R Kumar.

At least 109 records · Page 6Linked to original sources
PubMed1990

N-nitroso compounds and their precursors in Brassica oleracea.

In the present study, Brassica a dried green vegetable from Kashmir, which is a major constituent of the local diet, was analysed for nitrosatable aliphatic amines, N-nitrosamines prior to and after nitrosation) and alkylating activity due to N-nitrosamides following nitrosation. The cooked vegetable contained 11 micrograms/kg nitrosodimethylamine and 21 micrograms/kg nitrosopyrrolidine. Nitrosation under chemical conditions yielded 1200 micrograms/kg N-methylnitrosourea.

Brassica↗
PubMed1990

Activation of ras oncogenes preceding the onset of neoplasia.

The identification of ras oncogenes in human and animal cancers including precancerous lesions indicates that these genes participate in the early stages of neoplastic development. Yet, these observations do not define the timing of ras oncogene activation in the multistep process of carcinogenesis. To ascertain the timing of ras oncogene activation, an animal model system was devised that involves the induction of mammary carcinomas in rats exposed at birth to the carcinogen nitrosomethylurea. High-resolution restriction fragment length polymorphism analysis of polymerase chain reaction-amplified ras sequences revealed the presence of both H-ras and K-ras oncogenes in normal mammary glands 2 weeks after carcinogen treatment and at least 2 months before the onset of neoplasia. These ras oncogenes can remain latent within the mammary gland until exposure to estrogens, demonstrating that activation of ras oncogenes can precede the onset of neoplasia and suggesting that normal physiological proliferative processes such as estrogen-induced mammary gland development may lead to neoplasia if the targeted cells harbor latent ras oncogenes.

Adenofibroma↗
PubMed1990

The calcium-transporting ATPase and the calcium- or magnesium-dependent nucleotide phosphatase activities of human placental trophoblast basal plasma membrane are separate enzyme activities.

The properties of calcium-stimulated ATP hydrolysis often differ from those of ATP-dependent calcium transport. We have characterized two components of calcium-stimulated ATP hydrolysis in human placental basal plasma membrane. In the absence of magnesium, component 1 apparently has saturable sites for free calcium in both the nanomolar and low micromolar range. It was stimulated by either calcium or magnesium, was unselective for nucleotide substrate, and its activity was very much greater than that of ATP-dependent calcium transport. Component 1 was inhibited by GTP, permitting measurement of component 2 with activity and magnesium stimulation comparable to ATP-dependent calcium transport. Component 2 was inhibited partially by an antibody against purified erythrocyte calcium transporter and completely by sulfhydryl reagents, whereas component 1 was unaffected. A phosphorylated intermediate of the calcium transporter co-migrated with the erythrocyte transporter on acidic sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Immunostaining after transfer to nitrocellulose revealed a doublet. The band of lower molecular weight co-migrated with that of the human erythrocyte membrane transporter. The addition of GTP permits separate measurement of ATP hydrolysis by the calcium transporter of the placental basal plasma membrane and may be useful in defining its properties in other cell membranes under a variety of conditions.

Adenosine Triphosphatases↗
PubMed1990

Growth regulation of A431 cells. Modulation of expression of transforming growth factor-alpha mRNA and 2',5'-oligoadenylate synthetase activity.

To explore bidirectional regulatory interactions between interferons and autocrine polypeptide factors, we examined the modulation of expression of transforming growth factor-alpha and 2',5'-oligoadenylate synthetase activity in A431 epidermoid carcinoma cells after treatment with interferon-gamma and transforming growth factor-alpha. Treatment of A431 cells with interferon-gamma increased steady state levels of transforming growth factor-alpha mRNA by 4-fold and increased the levels of transforming growth factor-alpha in the culture medium. There were additive growth inhibitory effects upon coaddition of exogenous transforming growth factor-alpha and interferon-gamma to the cultures. Addition of transforming growth factor-alpha to A431 cell cultures in the absence of interferon could stimulate the induction of 2',5'-oligoadenylate synthetase activity by more than 2-fold. These findings demonstrate that the induction of transforming growth factor-alpha in interferon-gamma-treated A431 cells could act to regulate interferon-induced gene(s), e.g. 2',5'-oligoadenylate synthetase, suggesting interactions between a potential autocrine growth factor and the interferon system in the growth regulation of A431 cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1990

The synthesis and biological activity of four novel fluorescent vasopressin analogs.

We synthesized and tested the biological properties of four fluorescent vasopressin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9, and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 microM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.

Adenylyl Cyclases↗
PubMed1990

Transfection of a human alpha-(1,3)fucosyltransferase gene into Chinese hamster ovary cells. Complications arise from activation of endogenous alpha-(1,3)fucosyltransferases.

In order to isolate a human gene encoding an alpha-(1,3)fucosyltransferase (alpha-(1,3)Fuc-T), genomic DNA from HL-60 cells was transfected by several methods into Chinese hamster ovary (CHO) cells. Colonies expressing alpha-(1,3)Fuc-T activity were identified by their ability to bind a monoclonal antibody (anti-SSEA-1) that recognizes the carbohydrate product of alpha-(1,3)Fuc-T action. CHO cells do not express alpha-(1,3)Fuc-T activity but contain at least two, silent alpha-(1,3)Fuc-T genes previously identified by their activation in the rare, dominant mutants LEC11 and LEC12. These CHO enzymes were shown to be distinguishable from the alpha-(1,3)Fuc-T activity of HL-60 cells by the latter's comparative inability to transfer fucose to paragloboside and fetuin. Based on these criteria, only 11 isolates from more than 70 putative transfectants examined were found to stably express an alpha-(1,3)Fuc-T activity typical of HL-60 cells. Genomic DNA from two of these isolates was used to generate five independent secondary transfectants with HL-60-like alpha-(1,3)Fuc-T activity. Southern analysis revealed a common DNA fragment that hybridized to an Alu probe in each secondary, providing evidence that a human alpha-(1,3)Fuc-T gene had been transfected. However, in all transfection experiments, isolates that expressed alpha-(1,3)Fuc-T activities similar to CHO-encoded enzymes were also obtained. Several lines of evidence indicated that these cells arose from activation of endogenous CHO alpha-(1,3)Fuc-T genes as a consequence of DNA transfection. These false positives complicated the identification of transfectants expressing a human alpha-(1,3)Fuc-T gene and represent an important consideration in experiments to transfect other glycosyltransferase genes.

Animals↗
PubMed1990

Studies on the determination and degradation of pyrimethamine in mammals.

Treatment of pyrimethamine with blood plasma in vitro yields a metabolite which is also produced when the drug is administered through intravenous injection in the rat. A thin layer liquid chromatographic method for quantitative and qualitative determination of pyrimethamine and its metabolite in plasma and biological tissues is described.

Animals↗
PubMed1990

Has modern management improved the outcome of infants with intracranial tumors?

Records of 92 children were reviewed who, in the first 18 months of life had an intracranial tumour, and who were registered with the Manchester Children's Tumour Registry during the 34-year period from 1953 to 1987. The incidence of intracranial tumour was 1 in 25,000 live births, and 85% of the tumours were malignant. The introduction of computed tomography (CT) in the region in 1976 improved the diagnostic accuracy and led to early diagnosis. This also coincided with refined radiotherapy and improved chemotherapy. The surgical attitude became more aggressive, and with early treatment, mortality was reduced, though the 1-month operative mortality still remains high at 15%. All survivors were available for follow-up. All children with axial tumours died within 9 years. Long-term follow-up did not suggest significant improvement in the overall survival or in the quality of life, though there was a significant improvement in the 5-year survival rate with modern management.

Actuarial Analysis↗
PubMed1990

Intracranial tumours in the first 18 months of life.

There are few population-based studies of intracranial tumours in children. This study examines 93 brain tumours in children up to 18 months of age from over 800 childhood central nervous system tumours reported to the Manchester Children's Tumour Registry during the period 1953-1987. The incidence was 1 per 25,000 live births. Of these tumours, 85% were malignant. The three most common tumours were medulloblastoma, ependymoma and juvenile astrocytoma. Eighteen percent of the tumours were in the axial region. Twenty-five percent were connected to the ventricles. The tumour was supratentorial in two-thirds of the children under 6 months of age. A high incidence of tumours in the families of these children was found; tumours were noted in two sets of siblings.

Astrocytoma↗
PubMed1990

Cellular and segmental distribution of Ca2(+)-pump epitopes in rat intestine.

We used a monoclonal antibody (5F10) specific for the human erythrocyte plasma membrane Ca(++)-pump to demonstrate the presence and distribution of Ca(++)-pump epitopes in rat intestine. In paraffin embedded tissue sections, antibody 5F10 binds to epitopes in the basolateral membranes of absorptive cells in rat duodenum and portions of jejunum but not ileum. Western blot analysis of intestinal mucosal proteins with antibody 5F10 shows binding of antibody to major bands of Mr approximately 135,000 and Mr approximately 72,000, and to lesser bands of Mr approximately 125,000 and Mr approximately 27,000. This pattern was seen in mucosal homogenates of rat duodenal and jejunal cells and to a lesser extent in ileal cells. The Mr approximately 135,000 band corresponds to the molecular weight of Ca(++)-pumps in other tissues. The other bands correspond in size to known proteolytic fragments of the Ca(++)-pump. Slot-blot analysis of nitrocellulose immobilized mucosal homogenates shows binding of 5F10 to be greatest in duodenum and least in ileum. Ca(++)-transport studies by the everted gut sac technique show a correlation between vitamin D induction of active Ca(++)-transport and the segmental distribution of Ca(++)-pump epitopes.

Animals↗
PubMed1990

Synthesis and biological activity of novel vitamin D analogues: 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 and 24,24-difluoro-1 alpha,25-dihydroxy-26,27-dimethylvitamin D3.

We synthesized 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 (16), and 24,24-difluoro-1 alpha, 25-dihydroxy-26,27-dimethylvitamin D3 (21), from 3 beta-hydroxy-22,23-dinorcholenic acid 3-acetate. Compound 16 was found to be a highly potent vitamin D analogue with bioactivity similar to that of 25-hydroxyvitamin D3 in vivo. Compound 16 was bound by vitamin D binding protein with an affinity slightly less than that of 25-hydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with approximately the same affinity as that of 25-hydroxyvitamin D3. In the organ-culture duodenum, 16 induced the synthesis of calcium binding protein with a potency approximately 1/20 that of 1,25-dihydroxyvitamin D3. Compound 21 was also noted to be a highly potent vitamin D analogue with bioactivity in vivo similar to that of 1,25-dihydroxyvitamin D3. It was bound to vitamin D binding protein with an affinity considerably less than that of 1,25-dihydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with an affinity slightly less than that of the native hormone. In the organ-culture duodenum, 21 was noted to be about 3 times more active than 1,25-dihydroxyvitamin D3 in the induction of calcium binding protein. The introduction of fluorines at C-24 and extension of the sterol side chain at C-26 and C-27 by methylene groups results in vitamin D analogues that have biological activity in vivo similar to those of the respective nonfluorinated natural sterols.

Animals↗
PubMed1990

Synthesis and antiviral and cytotoxic activity of iodohydrin and iodomethoxy derivatives of 5-vinyl-2'-deoxyuridines, 2'-fluoro-2'-deoxyuridine, and uridine.

A series of new 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine and uridine compounds (11, 16) was synthesized by the regiospecific addition of HOI to the vinyl substituent of 5-vinyl-2'-deoxyuridine (10a), 5-vinyl-2'-fluoro-2'-deoxyuridine (10b), 5-vinyluridine (10c), and (E)-5-(2-iodovinyl)-2'-deoxyuridine (4b). Treatment of the iodohydrins 11a-c with methanolic sulfuric acid afforded the corresponding 5-(1-methoxy-2-iodoethyl) derivatives (12a-c). In contrast, reaction of 5-(1-hydroxy-2-iodoethyl)-2'-deoxyuridine (11a) with sodium carbonate in methanol afforded a mixture of 5-(1-hydroxy-2-methoxyethyl)-2'-deoxyuridine (13) and 2,3-dihydro-3-hydroxy-5-(2'-deoxy-beta-D-ribofuranosyl)- furano[2,3-d]pyrimidin-6(5H)-one (14). The most active compound, 5-(1-methoxy-2-iodoethyl)-2'-deoxyuridine (12a, ID50 = 0.1 micrograms/mL), which exhibited antiviral activity (HSV-1) 100-fold higher than that of the 5-(1-hydroxy-2-iodoethyl) analogue (11a), was less active than IVDU or acyclovir (ID50 = 0.01-0.1 micrograms/mL range). The C-5 substituent in the 2'-deoxyuridine series was a determinant of cytotoxic activity, as determined in the in vitro L1210 screen, where the relative activity order was CH(OH)CHI2 (16) greater than CH(OMe)CH2I (12a) greater than CH(OH)CH2I (11a) congruent to CH(OH)CH2OMe (13). The 2'-substituent was also a determinant of cytotoxic activity in the 5-(1-hydroxy-2-iodoethyl) (11a-c) and 5-(1-methoxy-2-iodoethyl) series of compounds, where the relative activity profile was 2'-deoxyuridine greater than 2'-fluoro-2'-deoxyuridine greater than uridine (11a greater than 11b greater than or equal to 11c; 12a greater than 12b greater than 12c). The most active cytotoxic agent (16), possessing a 5-(1-hydroxy-2,2-diiodoethyl) substituent (ED50 = 0.77 micrograms/mL), exhibited an activity approaching that of melphalan (ED50 = 0.15 micrograms/mL). All compounds tested, except for 13 and 14, exhibited high affinity (Ki = 0.035-0.22 mM range relative to deoxyuridine, Ki = 0.125) for the murine NBMPR-sensitive erythrocyte nucleoside transport system, suggesting that these iodohydrins are good permeants of cell membranes.

Animals↗
PubMed1990

Internalization of vasopressin analogs in kidney and smooth muscle cells: evidence for receptor-mediated endocytosis in cells with V2 or V1 receptors.

To determine whether receptor-mediated endocytosis occurs in vasopressin-responsive cells, we developed a model system using synthetic fluorescent-labeled vasopressin analogs and A10 (smooth muscle) and LLC-PK1 (kidney epithelial) cells in culture; these cell lines express V1 and V2 vasopressin cell surface receptor types, respectively. We used epifluorescence microscopy to examine the binding, internalization, and intracellular destination of [1-(2-mercapto)propionic acid,8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) and [1-(2-mercapto)propionic acid,8-lysine-N6-carboxyfluorescein]vasopressin (F-MLVP) in these cells. The rhodamine-labeled fluorescent vasopressin analog, R-MLVP, initially bound in a diffuse manner at the cell surface of both A10 and LLC-PK1 cells and could be displaced by excess unlabeled [8-arginine]vasopressin. After incubation at 37 degrees C, bound ligand rapidly aggregated into small clusters or patches, which were internalized in a manner consistent with receptor-mediated endocytosis. Subsequent processing of internalized ligand-receptor complexes appeared to differ between A10 and LLC-PK1 cells. In the case of LLC-PK1 cells, ligand was delivered to a tightly focused lysosome compartment in the perinuclear region of the cell, and receptor molecules were replenished at the cell surface. The lysosomal location of ligand was supported by the quenching of fluorescence in the internalized vesicles when F-MLVP was used as a fluorescent tracer. In the case of A10 cells, ligand became localized to a vesicular compartment and reappearance of receptor at the cell surface was limited. Our data are consistent with the occurrence of receptor-mediated endocytosis of vasopressin in cells with V1 and V2 receptors.

Animals↗
PubMed1990

Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiates complex N-linked carbohydrate formation.

This laboratory has previously identified a human gene encoding N-acetylglucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101) by complementation of the glycosylation defect in the Lec1 Chinese hamster ovary (CHO) cell mutant. A phage lambda library prepared from genomic DNA of a tertiary Lec1 transfectant (3 degrees T) has now been used to obtain clones encoding an active GlcNAc-TI enzyme. A small genomic DNA fragment [approximately 4.6 kilobases (kb)], isolated from an Alupositive lambda clone, conferred human GlcNAc-TI activity upon transfection into Lec1 cells. An approximately 1.3-kb probe generated from this DNA fragment detected unique but distinct DNA fragments in human and CHO genomic DNA. The probe also hybridized to a poly(A)+ RNA of approximately 2.7 kb in human and CHO cells and allowed the isolation of a full-length cDNA encoding human GlcNAc-TI activity. The overall features of the cDNA and deduced protein sequence (445 amino acids) are typical of other Golgi transferases that are type II transmembrane proteins. Northern blot analysis with the same probe showed that Lec1 mutant cells also possessed an approximately 2.7-kb poly(A)+ RNA, indicating that the lec1 mutation is a point mutation.

Amino Acid Sequence↗
PubMed1990

Sandwich enzyme immunoassays for detection of Salmonella typhi.

Enzyme immunoassays were developed using monoclonal antibodies raised against somatic (O), flagellar (H) and capsular (Vi) antigens of Salmonella typhi. The assay based on anti-O monoclonal antibodies could specifically detect S. typhi and soluble lipopolysaccharide (LPS) isolated from S. typhi. Anti-H MoAbs detected motile S. typhi and soluble flagellar antigen. Monoclonal antibodies against capsular polysaccharide could detect Vi-containing S. typhi as well as soluble Vi antigen. The three assays reported here detected S. typhi with 100% sensitivity in blood culture broths obtained from bacteriologically confirmed typhoid patients and were negative with blood specimens containing Salmonella senftenberg, E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis or Streptococcus (alpha-hemolytic) derived from patients with pyrexia. The assays, however, did not demonstrate the presence of soluble antigens in sera and urine samples obtained from typhoid patients.

Animals↗
PubMed1990

Rotavirus-specific antibody response in saliva of infants with rotavirus diarrhea.

The reliability of saliva as an indicator of rotavirus infection was assessed among 15 infants (3-12 months) with rotaviral and 15 with nonrotaviral diarrhea. Paired salivary samples collected during acute and convalescent phases were tested for rotavirus-specific IgA and IgM by an ELISA. The sensitivity of IgA or IgM alone to predict infection was 53.3% and 46.6%, respectively; used in conjunction, the sensitivity rose to 80%. It seems that infants with rotaviral diarrhea mount mucosal antibody responses as reflected in their saliva; possibly salivary antibodies could be used to evaluate vaccine "take" in rotavirus vaccine trials.

Antibodies, Viral↗
PubMed1990

Ring or disc-like enhancing lesions in partial epilepsy in India.

One-hundred unselected children presenting with partial motor seizures were subjected to clinical work-up and cranial computed tomography (CT scan). Seventy-three children had an abnormal scan. On these, 56 patients had either a ring (35 patients) or a disc-like (21 patients) enhancing lesion which coincided with the probable site of origin of the seizures in all cases. The lesions were surrounded by perifocal oedema which was occasionally severe enough to produce a mass effect. The significance of these disc or ring lesions is discussed in the light of recent literature. All patients with such lesions were put on three-drug anti-tubercular therapy along with anticonvulsants. A repeat scan could be obtained in 20 patients, 10 weeks to 11/2 years after the initial scan. This showed a decrease in size or disappearance of the lesion in the majority (13 patients). Four patients showed a spot of calcification in the area of the original lesion and another two showed a typical 'target sign', i.e. calcification within a ring. It is concluded that these ring or disc lesions represent tuberculomas which are, thus, the commonest cause of partial motor seizures in children in this area.

Brain↗