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Biomedical subjects

S Narayan

Publications and source records attributed to S Narayan.

At least 73 records · Page 4Linked to original sources
PubMed1997

Apoptosis in experimental myocardial infarction in situ and in the perfused heart in vitro.

We report the appearance of apoptotic cells in experimental myocardial infarction (rabbit heart) in in situ and in vitro preparations. Apoptosis was recognized by intravital staining with Hoechst 33342 (Ho342), by nick-end labeling (TUNEL) and by DNA laddering. A steady rise in the relative number of apoptotic cardiomyocytes (apoptotic index) was noted in in situ preparations. Apoptosis was first noted 6 h after the onset of ischemia with its highest value occurring after 72 h. Apoptotic nuclei were absent in remote areas of the left and right ventricles. Apoptotic nuclei within the infarcted area showed diminished intensity of Ho342 fluorescence. Three days after ischemia, a border zone adjacent to the infarcted area consisting of apoptotic macrophages was recognized. A novel finding was the appearance of apoptotic cardiomyocytes in the isolated perfused ischemic heart. Occurring as early as 50 min after the onset of ischemia, a high apoptotic index was present adjacent to the ligature placed around the coronary artery. This observation provides the opportunity to selectively examine factors leading to apoptosis in the ischemic heart under controlled experimental conditions.

Animals↗
PubMed1997

Phosphorylation of a high molecular weight (approximately 600 kDa) protein regulates catch in invertebrate smooth muscle.

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of approximately 600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the approximately 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of approximately 600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the approximately 600 kDa protein regulates catch.

Animals↗
PubMed1997

Disruption of transcription in vitro and gene expression in vivo by DNA adducts derived from a benzo[a]pyrene diol epoxide located in heterologous sequences.

Previous studies indicated a high affinity of the transcription factor Sp1 for DNA adducts derived from benzo[a]pyrene diol epoxide (BPDE) in sequences that are not normal binding sites for Sp1. We tested for functional effects of this phenomenon in three systems in which transcription is Sp1-dependent. In an in vitro, Sp1-dependent transcription system addition of heterologous plasmid DNA containing BPDE adducts abolished production of a specific run-off transcript. This inhibition was not seen with unmodified plasmid DNA, and could be overcome by addition of purified Sp1 protein. In SL2 insect cells, high-level expression of an Sp1-dependent reporter gene, which was dependent on co-transfection of an Sp1 expression vector, was inhibited >95% by co-transfection of heterologous DNA containing BPDE adducts. This inhibition could be partially overcome by increasing the amount of the Sp1 expression vector in the transfections. In human C33A cells, expression of a transfected reporter gene driven by a GC box containing fragment of the human E2F1 promoter was enhanced by co-transfection of an Sp1 expression plasmid. Expression was inhibited 3-6-fold by co-transfection of heterologous DNA containing BPDE-DNA adducts. A similar inhibition was seen in human SAOS-2 cells, which lack functional p53 protein. These data are consistent with functionally significant sequestration of the Sp1 transcription factor by BPDE-DNA adducts in all three systems. Altered availability of transcription factors such as Sp1 in carcinogen-treated cells may disrupt patterns of gene expression.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1997

Decision making by elderly patients with cancer and their caregivers.

This study explored the scope of decisions encountered by elderly cancer patients and/or their family caregivers, and the types of decision-making assistance requested and required within one practice setting. Semistructured interviews were conducted with five cancer center nurse coordinators (CCNCs). The CCNCs were interviewed weekly for 16 weeks to identify decision-making topics addressed, assistance requested, and perceptions of assistance required during telephone conversations. The CCNCs' reports of 41 telephone conversations revealed 44 specific decision-making topics. Content analysis uncovered 11 categories: symptom management, use of chemotherapy, ancillary choices selection of a medical provider, planning for end-of-life care, alternative therapy, vacation planning, weekend-pass planning, discharge planning, family survivor issues, and involvement of adult children in the elder's care. Elderly patients and/or their family caregivers requested information and assistance with making decisions. CCNCs perceived that callers also needed information clarification, reassurance about their decisions, a listener, permission to change the treatment regimen, and help with communication among health professionals, the elderly patient, and the family.

Adult↗
PubMed1997

Hypertrophy of colonic smooth muscle: structural remodeling, chemical composition, and force output.

The cellular basis of adaptations occurring during the development of megacolon was studied with the lethal spotted mouse model. Age-dependent changes in the length-force characteristics of the colon reach a steady state by 3-4 mo and include an increased relative force development at very short muscle lengths. In megacolon the following occur: 1) structural remodeling expressed as a greater increase in the fraction of maximum force production at short lengths, a shift of optimum length (Lo) to longer lengths, and no change in force per square centimeter; 2) hypertrophy and hyperplasia of both circular and longitudinal muscle; 3) high resting compliance consistent with no disproportionate change in collagen or elastin composition; 4) marked distension so that in situ circumference approximately 1.8 Lo, where active force production is low, and 5) slack length approximately 0.65 Lo, as in normal colon. Biochemical remodeling in megacolon includes disproportionate increases in ATP and phosphocreatine concentration, with 3.5-fold more preformed phosphagen than in normal colon. The myosin concentration is the same in both muscles, but the actin concentration is 1.5-fold greater in megacolon. Most of the cellular changes in megacolon would facilitate high active force output from the muscle at much larger intestinal diameters.

Actins↗
PubMed1997

Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation.

Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.

Actins↗
PubMed1996

Activation of the human DNA polymerase beta promoter by a DNA-alkylating agent through induced phosphorylation of cAMP response element-binding protein-1.

Treatment of cells with the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase beta (beta-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG-induced up-regulation. Recently, we described a kinetic mechanism for transcriptional regulation of the beta-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up-regulates the promoter by stimulating formation of closed preinitiation complex (RPc). Here, using a CRE-dependent chimeric beta-pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RPc samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the beta-pol promoter after treatment of cells with the DNA-alkylating agent MNNG.

Alkylating Agents↗
PubMed1996

Purification and characterization of a DNA polymerase beta promoter initiator element-binding transcription factor from bovine testis.

A low-abundance DNA-binding protein for the DNA polymerase beta (beta-pol) promoter initiator element was purified from bovine testis. The transcriptional initiator element (Inr) of the mammalian beta-pol promoters characterized is highly conserved, and the bovine beta-pol promoter Inr has the sequence -11CAGAGGCGGCCATTGTT+6. The purified initiator element-binding protein (Inr-BP) binds with high affinity to an oligonucleotide corresponding to the beta-pol promoter Inr (Kd = 5 pM), and increasing ionic strength decreases stability of the protein-DNA complex. Mutational analysis of the Inr shows that the purified Inr-BP binds with sequence specificity to the sequence CCAT at -2 to +2 of the Inr, but that seven residues on the 5' side and three residues on the 3' side of the CCAT sequence are required also. Using an in vitro transcription assay with HeLa cell nuclear extract, we find that the endogenous Inr-BP is required for transcriptional activity of the beta-pol promoter; addition of purified Inr-BP restores activity to the nuclear extract depleted in Inr-BP by affinity chromatography. These results, based upon the sequence specificity for DNA binding, indicate that Inr-BP is a YY1-like protein and suggest that it is a required transcription factor in beta-pol gene expression.

Animals↗
PubMed1996

Ocular malaria. A clinical and histopathologic study.

PURPOSE: Clinical and histopathologic findings of ocular lesions in malaria rarely have been described. This study reports lesions in three patients with malaria, with a histopathologic study of eyes obtained at autopsy of one of these patients. METHODS: Various ocular lesions were documented in three patients with malarial infection, with histopathologic study of one patient. In all three patients, the diagnosis of malaria was confirmed by the demonstration of malarial parasites in peripheral smears and by fluorescent microscopy of acridine orange-stained buffy coat preparations of venous blood. RESULTS: Multiple superficial blotchy retinal hemorrhages over the posterior pole were seen in the first patient, whereas acute bilateral panuveitis and secondary glaucoma were seen in the second, which on resolution showed multiple blotchy superficial retinal hemorrhages with perivasculitis. The third patient had cerebral malaria and was found to have subconjunctival and retinal hemorrhages. This patient died of pulmonary thromboembolism, and the eyes were obtained at autopsy. On gross examination, there were multiple retinal hemorrhages in the posterior pole. Histopathologic study showed cytoadherence of parasitized erythrocytes as well as schizonts and gametocytes of Plasmodium vivax within the retinal and choroidal blood vessels. CONCLUSION: The authors' findings suggest that patients with unexplained blotchy retinal hemorrhages should be investigated for malarial infection, especially if they reside or have travelled in endemic areas.

Acridine Orange↗
PubMed1996

Volumetric measurement of multifocal brain lesions. Implications for treatment trials of vascular dementia and multiple sclerosis.

This pilot study examined the reproducibility of serial magnetic resonance (MR) measurements of brain, ventricular, sulcal, and lesion volumes in patients with ischemic brain disease using an image analysis protocol designed at the University of Cincinnati. Five patients with a clinical history of brain ischemia had two separate MR brain imaging studies using the standard clinical MR imaging protocol at the University of Cincinnati Medical Center. The MR images on both film and tape were digitized and then analyzed according to the standardized image analysis protocol. Based on tape data, variability in volume measurements between the two MR studies, as measured by the coefficient of variation, ranged from 1% for intracranial volume to 8% for ventricular volume. Variability based on film data was slightly greater, ranging from 2% for intracranial volume to 12% for lesion volume. As part of a multicenter treatment trial of vascular dementia, this method was then used to analyze MR films in 13 patients with vascular dementia who all had an MR study at baseline and at 1 year. The mean annual change in lesion volume was 4 +/- 5 cm3 (a 24% increase from the baseline lesion volume); in ventricular volume, 7 +/- 8 cm3 (a 10% increase from baseline); and in sulcal volume, 13 +/- 25 cm3 (a 5% increase from baseline). This method of image analysis, using MR film or tape-generated data, can provide reproducible serial measurements of brain, ventricular, sulcal, and ischemic lesion volumes. This method, if applied in randomized treatment trials of vascular dementia or multiple sclerosis, can be used to monitor disease progression and to evaluate the effectiveness of a given therapy.

Aspirin↗
PubMed1995

The bovine DNA polymerase beta promoter: cloning, characterization and comparison with the human core promoter.

The core promoter of the human DNA polymerase beta (beta Pol)-encoding gene (POL beta) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755-12763]. To study the homologous promoter (ppol beta) in a bovine system, we cloned and characterized the 5'-flanking region of the bovine gene (pol beta). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5'-flanking region was isolated from a testis library in bacteriophage lambda EMBL3. S1 nuclease mapping and primer extension analysis of the 5'-end of the pol beta mRNA identified the major transcription start point (tsp), which is located 142-bp 5' of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5'-deletion mutants demonstrated that a fragment of only 91-bp 5' of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOL beta). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Sp1-binding sites, and the spacing separating them.

Animals↗
PubMed1995

DNA damage-induced transcriptional activation of a human DNA polymerase beta chimeric promoter: recruitment of preinitiation complex in vitro by ATF/CREB.

Treatment of hamster cells in culture with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces DNA polymerase beta (beta-pol) gene expression and cellular levels of the enzyme. Transcriptional activity of a cloned beta-pol promoter in transient expression assays is also stimulated. Among the requirements for these responses are methylation damage to genomic DNA, cellular cAMP-dependent protein kinase, and the ATF/CREB site of the cloned beta-pol promoter. In the present study, HeLa cell nuclear extract from MNNG-treated cells was much more active in an in vitro transcription assay than nuclear extract from normal cells. By using an oligonucleotide affinity column to deplete the nuclear extract of ATF/CREB, we showed that the difference was due to ATF/CREB activator. Purified ATF/CREB activator from MNNG-treated cells was approximately 10-fold more active than ATF/CREB purified from normal cells as a transcriptional activator for the depleted nuclear extract. ATF/CREB in the extract from normal cells is known to activate in vitro transcription by increasing the rate of promoter clearance [Narayan, S., Widen, S. G., Beard, W. A., & Wilson, S. H. (1994) J. Biol. Chem. 269, 12755-12763]. With ATF/CREB from MNNG-treated cells, the amount of preinitiation complex formed was much greater than with ATF/CREB from normal cells, and the kinetics of both the closed to open preinitiation complex isomerization and promoter clearance were altered. These results indicate that the mechanism of transcriptional activation secondary to DNA alkylation damage is recruitment of more preinitiation complex and alteration of the kinetic scheme of transcription initiation.

Base Sequence↗
PubMed1995

Structural organization of the mouse DNA repair gene, N-methylpurine-DNA glycosylase.

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, removes N-alkylpurines and other purine lesions induced in DNA by simple alkylating carcinogens. A mouse MPG cDNA clone was isolated from a lambda recombinant phage library of BALB/c mouse lung cell and characterized. Using the mouse MPG cDNA as a probe, the complete mouse MPG gene was isolated in two overlapping lambda recombinant genomic clones. The 6-kb gene has four exons containing 1,002 bp of coding sequence. The transcription start site was identified in the genomic sequence by primer extension of MPG mRNA from a mouse lung fibroblast cell line. The location of this transcription start site was confirmed by in vitro transcription with the promoter-containing plasmid template. Promoter function of the sequence 5' upstream of the transcription initiation site was shown by transient expression of the firefly luciferase reporter gene under the control of this sequence in transfected human and mouse cells. The mouse MPG promoter contains no TATA box, but has a CAAT element and is G.C-rich with putative AP2 elements and SP1-complementary sequences.

3T3 Cells↗
PubMed1995

Pregnancy induced hypertension: changes in coagulation profile of newborns.

Coagulation parameters and platelet count were studied in 30 neonates of mothers with pregnancy induced hypertension (PIH). 30 neonates born to normotensive mothers were taken as controls. The test group was further subdivided as neonates born to mothers with gestational hypertension, pre-eclamptic toxemia and eclampsia. The values of Prothrombin Time, Partial Thromboplastin Time with Kaolin, Thrombin Time, Fibrinogen Degradation Products were significantly raised and Fibrinogen and Platelet count were reduced significantly in both term and preterm test groups as compared to controls. The derangement in coagulation parameters was more marked with increasing severity of PIH.

Blood Coagulation Disorders↗
PubMed1994

RNA polymerase II transcription. Rate of promoter clearance is enhanced by a purified activating transcription factor/cAMP response element-binding protein.

The core promoter of the human DNA beta-polymerase (beta-pol) gene is regulated by proteins binding at 3 GC boxes and the single activating transcription factor/cAMP response element (ATF/CRE) centered at -45; the central 8 residues of this ATF/CRE match the ATF/CRE consensus sequence, TGACGTCA. Previously, we purified a beta-pol promoter ATF/CRE-binding protein (named palindrome-binding protein or PBP) from bovine testes and found that this protein is a beta-pol promoter transcriptional activator in vitro using a HeLa nuclear extract transcription system (Widen, S. G., and Wilson, S. H. (1991) Biochemistry 30, 6296-6305). In this study, we determined the mechanism of in vitro transcriptional activation by this purified PBP. We used a PBP-depleted HeLa nuclear extract transcription system with an artificial promoter containing a solitary activator element corresponding to the entire 22-nucleotide beta-pol promoter ATF/CRE-binding site. Kinetic analyses of the 180-nucleotide run-off product formation indicated that stimulation of transcriptional activity by PBP was due entirely to an increase in the rate constant for promoter clearance. Thus, under our conditions, the purified PBP had no effect on the rate of closed preinitiation complex formation or for the closed complex to open complex transition. Instead, the rate of productive initiation leading to the 180-nucleotide transcript was stimulated by PBP. We found that the rate of closed preinitiation complex formation was not in rapid equilibrium with promoter and RNA polymerase II, in contrast to the model with prokaryotic RNA polymerase transcription. The results also indicated that PBP binding to the ATF/CRE is required for the stimulation of promoter clearance. These studies define the kinetic mechanism of a purified ATF/CRE-binding protein in stimulation of the in vitro transcription of a designed mammalian promoter.

Activating Transcription Factor 2↗
PubMed1994

Phosphorylation of pp62 and pp54 src-like proteins in a rat intestinal cell line in response to gastrin.

Intracellular mechanisms that mediate mitogenic effects of gastrin remain largely unknown. The present studies were designed to examine if protein tyrosine kinases (PTKs) mediate growth effects of gastrin on a rat intestinal epithelial cell line (IEC-6 cells). Gastrin (< 10 nM) was mitogenic for IEC-6 cells. PTK activity of cell membranes was stimulated in response to 0.01-10.0 nM and 0.05-10.0 microM gastrin in a double biphasic manner. Cells labeled with H3(32)PO4 were stimulated with gastrin and cellular proteins immunoprecipitated with phosphotyrosine antibodies. Endogenous proteins were phosphorylated in a dose- (100% effective dose = 0.1-1.0 nM) and time-dependent manner; at > 10 nM gastrin, the second peak of response was not measured in intact cells. Thus the growth and phosphorylation response of intact cells to gastrin was similar. Both high [dissociation constant (Kd) = 1 nM]- and low (Kd = approximately 0.1 microM)-affinity gastrin binding sites are present on IEC-6 cells. The results of the present study suggest that occupancy of both high- and low-affinity gastrin-binding sites can potentially activate membrane-associated PTKs. However, in intact cells, occupancy of low-affinity sites apparently attenuates kinase activity resulting in reduced protein phosphorylation. Eight protein bands [with relative molecular weight (M(r)) of 32-145 kDa] were tyrosine phosphorylated in intact cells in response to 0.1-1.0 nM gastrin, including two pp60 src-like proteins (with M(r) of 54 and 62 kDa). Thus the growth response pattern of a target cell to gastrin may depend on the stimulation of kinases and other factors (phosphatases?) that phosphorylate and/or dephosphorylate several proteins including c-src-like proteins in a dose-dependent manner.

Animals↗
PubMed1994

Palliation of esophageal carcinoma. Laser and photodynamic therapy.

Laser therapy is a well-established, relatively safe, rapid, and highly effective method of palliation for the dysphagia that usually accompanies esophageal and esophagogastric cancer. It is the treatment of choice in many patients, although there remains some disagreement regarding technique and predictors of outcome. The major limitation of laser therapy is the need for repeated treatments, although the interval between treatments may be lengthened by concomitant external beam or endoluminal radiotherapy. When laser therapy is available, use of an esophageal stent should be reserved for special circumstances, such as esophagopulmonary fistulas or extrinsic compression. In addition, stent placement usually is effective when laser photoablation fails or must be performed too frequently. It remains to be seen whether or not technical improvements in esophageal stents will reduce the frequency of complications associated with these devices. Other promising modalities that may be less expensive and more readily available, such as the BICAP tumor probe or injection therapy, deserve further study. It appears that most of these methods are complementary and different modalities may be suited to different types of lesions. The results of phase III clinical trials with PDT, now underway, should help to define the role of this promising modality in the overall scheme of treatment for esophageal cancer. The concept of PDT is attractive, although refinements in photosensitive compounds and methods of light delivery may be needed. Current information suggests a moderately high complication rate for PDT, although this may decrease with technical improvements and increasing experience. Issues surrounding the palliation of esophageal cancer are complex. Whereas the tendency is to focus on technical aspects of therapy and the relief of dysphagia, broader aspects of a patient's quality of life cannot be ignored. Ultimately, the choice of therapy may depend as much on a patient's psychosocial circumstances as on the appearance of the lesion. For instance, the patient who lives at a great distance from the center where laser therapy is performed may be better served by placement of an esophageal stent despite the higher complication rate for this procedure. PDT would be inappropriate for the patient who wishes to spend the remaining few months of life outdoors in the sun. Guiding the patient to the best choice requires the skills of a physician as much as the technical ability of an endoscopist.

Deglutition Disorders↗