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M Sharma

Publications and source records attributed to M Sharma.

At least 145 records · Page 8Linked to original sources
PubMed1999

The promoter region of human prepro-nociceptin gene and its regulation by cyclic AMP and steroid hormones.

The promoter and upstream regulatory region of the human prepro-nociceptin gene has been cloned from adaptor-ligated genomic DNA libraries by polymerase chain reaction. This 1.7 kb region contains several potential binding sites for transcription factors, among which are binding sites for TF-IID, cyclic AMP response element binding protein, glucocorticoid receptor and estrogen receptor. Multiple start points for the transcription of prepro-nociceptin are identified by an 'oligoribonucleotide-capping' method, but the major one is located at -558(G). Promoter activity assays using luciferase reporter gene constructions with the 1.7 kb fragment and a series of deletion mutations demonstrate that the core promoter is located in the region from -639 to -521 (a region surrounding the major transcription start point -558). A TATA-box motif displays weak promoter activity. An increase of cellular cyclic AMP levels by forskolin treatment up-regulates prepro-nociceptin transcription. Estrogen also up-regulates gene transcription whereas glucocorticoid down-regulates transcription, each through their corresponding receptor response elements. These regulatory effects can be blocked either by mutations of the potential cyclic AMP- or estrogen receptor response elements or by the application of antagonists for glucocorticoid and estrogen receptors. These findings provide a molecular basis for the regulatory mechanisms of human prepro-nociceptin gene expression.

Base Sequence↗
PubMed1999

Regulation of cyclin-dependent kinase 5 catalytic activity by phosphorylation.

Cyclin-dependent kinase 5 (cdk5) is found in an active form only in neuronal cells. Activation by virtue of association with the cyclin-like neuronal proteins p35 (or its truncated form p25) and p39 is the only mechanism currently shown to regulate cdk5 catalytic activity. In addition to cyclin binding, other members of the cdk family require for maximal activation phosphorylation of a Ser/Thr residue (Thr(160) in the case of cdk-2) that is conserved in all cdks except cdk8. This site is phosphorylated by cdk-activating kinases, which, however, do not phosphorylate cdk5. To examine the possible existence of a phosphorylation-dependent regulatory mechanism in the case of cdk5, we have metabolically labeled PC12 cells with (32)P(i) and shown that the endogenous cdk5 is phosphorylated. Bacterially expressed cdk5 also can be phosphorylated by PC12 cell lysates. Phosphorylation of cdk5 by a PC12 cell lysate results in a significant increase in cdk5/p25 catalytic activity. Ser(159) in cdk5 is homologous to the regulatory Thr(160) in cdk2. A Ser(159)-to-Ala (S159A) cdk5 mutant did not show similar activation, which suggests that cdk5 is also regulated by phosphorylation at this site. Like other members of the cdk family, cdk5 catalytic activity is influenced by both p25 binding and phosphorylation. We show that the cdk5-activating kinase (cdk5AK) is distinct from the cdk-activating kinase (cyclin H/cdk7) that was reported previously to neither phosphorylate cdk5 nor affect its activity. We also show that casein kinase I, but not casein kinase II, can phosphorylate and activate cdk5 in vitro.

Amino Acid Sequence↗
PubMed1999

Prevention of quinone-mediated DNA arylation by antioxidants.

High performance liquid chromatographic (HPLC) analysis showed that the prototype antioxidant ascorbate (vitamin C) inhibits the DNA adducts induced by synthetic estrogen diethylstilbestrol (DES) and the antiestrogen metabolite 4-hydroxytamoxifen (4-OHTam). Treatment of salmon testes DNA with 4-OHTam quinone or 4-OHTam in the presence of horseradish peroxidase and hydrogen peroxide (H(2)O(2)) generated the same DNA adduct profile. Vitamin C and N-acetylcysteine (NAC) inhibited the formation of 4-OHTam-dG adducts in a dose-dependent manner. To determine whether the same antioxidants also protect cellular DNA, HL-60 cells were used as cell culture model. Cells treated with 10 microM 4-OHTam in the presence of 1 microM H(2)O(2 )for 24 h gave 4-OHTam-dG adducts approximately 4 x 10(-7), n = 3. Treatment of the cells with 100 microM 4-OHTam, without H(2)O(2), produced the same level of adducts. Supplementation of the incubation media with vitamin C (2.5 mM) or NAC (5 mM) inhibited the formation of DNA adducts. Thus, antioxidants may protect susceptible cells from genotoxicity associated with 4-OHTam activation.

Acetylcysteine↗
PubMed1999

Binding of the bacteriophage T4 transcriptional activator, MotA, to T4 middle promoter DNA: evidence for both major and minor groove contacts.

During infection, the bacteriophage T4 transcriptional activator MotA, the co-activator AsiA, and host RNA polymerase are needed to transcribe from T4 middle promoters. Middle promoters contain a -10 region recognized by the sigma(70)subunit of RNA polymerase and a MotA box centered at -30 that is bound by MotA. We have investigated how the loss or modification of base determinants within the MotA box sequence 5'TTTGCTTTA3' (positions -34 to -26 of a middle promoter) affects MotA function. Gel retardation assays with mutant MotA boxes are consistent with the idea that MotA uses minor groove contacts upstream and major groove contacts downstream of the center GC, and does not require any specific base feature at the C.G base-pair at position -30. In particular, the 5-methyl residue on the thymine residue at position -29, a major groove contact, contributes to MotA binding, while converting the T.A at -32 to a C. I base-pair, a change that affects the major but nor the minor groove, yields a MotA box that is similar to wild-type. However, methylation interference analyses indicate that neither the binding of MotA nor the binding of polymerase/MotA/AsiA to the middle promoter PuvsXis inhibited by premethylation of guanine and adenine residues, suggesting that binding does not require minor groove contact with any specific T.A base-pair. Using gel retardation analyses, we calculate an apparent dissociation constant of 130 nM for MotA binding to the wild-type MotA box. Previous work has shown that the N-terminal region of MotA is needed for an interaction between MotA and sigma(70). We suggest that this MotA-sigma(70)interaction helps to stabilize the relatively weak interaction of MotA with the -30 region of middle promoter DNA.

Bacteriophage T4↗
PubMed1999

Multiple splice patterns of cyclic AMP response element-binding protein mRNA in the central nervous system of the rat.

The alternative splicing pattern of cyclic AMP response element-binding protein (CREB) in the central nervous system (CNS) of the rat has been investigated by an exon-flanking polymerase chain reaction (PCR) strategy. A series of RT-PCR studies with primer pairs flanking all possible alternative splicing sites (corresponding to a genomic region with at least one full exon and two flanking introns) has revealed multiple splice patterns in nine regions of the rat CNS. These include some novel transcripts that lack the phosphorylation site and a segment of the leucine zipper region which is crucial for dimerization and DNA binding. Some isoforms previously reported as testis-specific were also detected in the rat CNS. The findings from this study, which include differential splicing patterns among CNS regions, suggest a complex expression and functional regulation of CREB in the CNS.

Alternative Splicing↗
PubMed1999

Identification of substrate binding site of cyclin-dependent kinase 5.

Cyclin-dependent kinase 5 (CDK5), unlike other CDKs, is active only in neuronal cells where its neuron-specific activator p35 is present. However, it phosphorylates serines/threonines in S/TPXK/R-type motifs like other CDKs. The tail portion of neurofilament-H contains more than 50 KSP repeats, and CDK5 has been shown to phosphorylate S/T specifically only in KS/TPXK motifs, indicating highly specific interactions in substrate recognition. CDKs have been shown to have a high preference for a basic residue (lysine or arginine) as the n+3 residue, n being the location in the primary sequence of a phosphoacceptor serine or threonine. Because of the lack of a crystal structure of a CDK-substrate complex, the structural basis for this specific interaction is unknown. We have used site-directed mutagenesis ("charged to alanine") and molecular modeling techniques to probe the recognition interactions for substrate peptide (PKTPKKAKKL) derived from histone H1 docked in the active site of CDK5. The experimental data and computer simulations suggest that Asp86 and Asp91 are key residues that interact with the lysines at positions n+2 and/or n+3 of the substrates.

Amino Acid Sequence↗
PubMed1999

Evaluation of extent of shortening versus velocity of shortening at the endocardium and midwall in hypertensive heart disease.

To assess the incremental value of velocity of shortening velocity parameters compared with simpler, more widely used, extent of shortening parameters in compensated left ventricular hypertrophy, we studied 52 patients with left ventricular hypertrophy and 63 age-matched controls. Velocity parameters did not provide incremental information beyond that obtained by extent of shortening parameters.

Aged↗
PubMed1999

CT and PET lung image registration and fusion in radiotherapy treatment planning using the chamfer-matching method.

PURPOSE: We present a validation study of CT and PET lung image registration and fusion based on the chamfer-matching method. METHODS AND MATERIALS: The contours of the lung surfaces from CT and PET transmission images were automatically segmented by the thresholding technique. The chamfer-matching technique was then used to register the extracted lung surfaces. Arithmetic means of distance between the two data sets of the pleural surfaces were used as the cost function. Matching was then achieved by iteratively minimizing the cost function through three-dimensional (3D) translation and rotation with an optimization method. RESULTS: Both anatomic thoracic phantom images and clinical patient images were used to evaluate the performance of our registration system. Quantitative analysis from five patients indicates that the registration error in translation was 2-3 mm in the transverse plane, 3-4 mm in the longitudinal direction, and about 1.5 degree in rotation. Typical computing time for chamfer matching is about 1 min. The total time required to register a set of CT and PET lung images, including contour extraction, was generally less than 30 min. CONCLUSION: We have implemented and validated the chamfer-matching method for CT and PET lung image registration and fusion. Our preliminary results show that the chamfer-matching method for CT and PET images in the lung area is feasible. The described registration system has been used to facilitate target definition and treatment planning in radiotherapy.

Aged↗
PubMed1999

The bacteriophage T4 transcriptional activator MotA accepts various base-pair changes within its binding sequence.

During infection, bacteriophage T4 regulates three sets of genes: early, middle, and late. The host RNA polymerase is capable of transcribing early genes, but middle transcription requires the T4-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein, both of which bind to the sigma 70 (sigma70) subunit of RNA polymerase. MotA also binds a DNA sequence (a MotA box), centered at position -30. The identification of more than 20 middle promoters suggested that a strong match to the MotA box consensus sequence (t/a)(t/a)TGCTT(t/c)A was critical for MotA activation. We have investigated how specific base changes within the MotA box sequence affect MotA binding and activation in vitro, and we have identified seven new middle promoters in vivo. We find that an excellent match to the sigma70 -10 consensus sequence, rather than an excellent match to the MotA box consensus sequence, is an invariant feature of MotA-dependent promoters. Many single base changes in the MotA box are tolerated in binding and activation assays, indicating that there is more flexibility in the sequence requirements for MotA than was previously appreciated. We also find that using the natural T4 DNA, which contains glucosylated, 5-hydoxymethylated cytosine residues, affects the ability of particular MotA box sequences to activate transcription. We suggest that MotA and AsiA may function like certain eukaryotic TAFs (TATA binding protein (TBP) associated factors) whose binding to TBP results in transcription from new core promoter sequences.

Bacteriophage T4↗
PubMed1999

Myostatin, a transforming growth factor-beta superfamily member, is expressed in heart muscle and is upregulated in cardiomyocytes after infarct.

Myostatin is a secreted growth and differentiating factor (GDF-8) that belongs to the transforming growth factor-beta (TGF-beta) superfamily. Targeted disruption of the myostatin gene in mice and a mutation in the third exon of the myostatin gene in double-muscled Belgian Blue cattle breed result in skeletal muscle hyperplasia. Hence, myostatin has been shown to be involved in the regulation of skeletal muscle mass in both mice and cattle. Previous published reports utilizing Northern hybridization had shown that myostatin expression was seen exclusively in skeletal muscle. A significantly lower level of myostatin mRNA was also reported in adipose tissue. Using a sensitive reverse transcription-polymerase chain reaction (RT-PCR) technique and Western blotting with anti-myostatin antibodies, we show that myostatin mRNA and protein are not restricted to skeletal muscle. We also show that myostatin expression is detected in the muscle of both fetal and adult hearts. Sequence analysis reveals that the Belgian Blue heart myostatin cDNA sequence contains an 11 nucleotide deletion in the third exon that causes a frameshift that eliminates virtually all of the mature, active region of the protein. Anti-myostatin immunostaining on heart sections also demonstrates that myostatin protein is localized in Purkinje fibers and cardiomyocytes in heart tissue. Furthermore, following myocardial infarction, myostatin expression is upregulated in the cardiomyocytes surrounding the infarct area. Given that myostatin is expressed in fetal and adult hearts and that myostatin expression is upregulated in cardiomyocytes after the infarction, myostatin could play an important role in cardiac development and physiology.

Animals↗
PubMed1999

Primary mediastinal nonseminomatous germ cell tumors: the influence of postchemotherapy pathology on long-term survival after surgery.

OBJECTIVES: The treatment of nonseminomatous germ cell tumors with cisplatin-based chemotherapy followed by aggressive surgical resection of residual disease is one of the most successful models for multimodality cancer therapy. We reviewed the case histories of 91 patients treated at our institution from 1981 to 1998 with primary mediastinal nonseminomatous germ cell tumors to evaluate variables that may influence survival after surgery. METHODS: Twelve of the 91 patients did not undergo postchemotherapy resection because of progressive disease. Seventy-nine of them underwent 82 thoracic surgical procedures and are the basis of this review. The majority (71/75) had elevated serum tumor markers, 75% (n = 50) of which returned to normal levels after first- or second-line chemotherapy. RESULTS: There were 3 operative deaths and 1 late death, attributed to pulmonary complications. Twenty-four patients died of recurrent disease and 3 of leukemia, for an overall survival of 61% after an average follow-up of 48 months. The pathologic findings of complete tumor necrosis (n = 19) and benign teratoma (n = 28) in the surgical specimen predicted excellent and good long-term survival, respectively, which was statistically better than that of patients having persistent nonseminomatous germ cell tumors (n = 24) or carcinomatous/sarcomatous degeneration (n = 8). CONCLUSIONS: Primary nonseminomatous germ cell tumors of the mediastinum can be cured with a multimodality therapy, particularly in the subset of patients with postchemotherapy pathologic findings of tumor necrosis and teratoma. Survival is poor but possible in patients with unfavorable pathologic findings after chemotherapy, currently justifying an aggressive surgical approach in patients with otherwise operable disease.

Adolescent↗
PubMed1999

Presence of opioid receptor-like (ORL1) receptor mRNA splice variants in peripheral sensory and sympathetic neuronal ganglia.

The expression of ORL1 receptor mRNA splice variants is determined in peripheral sensory and sympathetic ganglia and compared to mRNA expression for the three classic opioid receptor subtypes (mu, delta, and kappa) using the method of reverse transcription-polymerase chain reaction. ORL1, mu, delta and kappa receptor subtype mRNAs are present in human dorsal root ganglia (DRG) and trigeminal ganglia and rat DRG. ORL1, mu and delta receptor subtype mRNAs are present in rat superior cervical ganglia and only ORL1 and delta receptor mRNAs are present in rat lumbar sympathetic ganglia. Both the ORL1 mRNA splice variants are present in sensory and sympathetic ganglia, however, expression of the shorter ORL1 receptor mRNA dominates over expression of the longer splice variant in rat brain and DRG, whereas, expression of the longer splice variant is dominant in sympathetic ganglia.

Aged↗
PubMed1999

Alternative exon splicing of cyclic AMP response element-binding protein in peripheral sensory and sympathetic ganglia of the rat.

Alternative splicing patterns of cyclic AMP response element-binding protein (CREB) in dorsal root ganglia, lumbar sympathetic ganglia and several peripheral tissues of the rat have been investigated by an exon-flanking polymerase chain reaction strategy. A series of RT-PCR with primer pairs flanking all possible alternative splicing sites (corresponding to a genomic region with at least one full exon and two flanking introns) has revealed multiple tissue specific splice variants. These include some novel transcripts that lack the phosphorylation site and part of the leucine zipper region which is crucial for dimerization and DNA binding. Some isoforms previously reported as testis-specific were also detected in rat peripheral ganglia and other tissues. Notably, splicing patterns are specific for some regions. Some of the splice variants indicate inhibitory functions due to lacking phosphorylation sites or partially missing DNA-binding or leucine zipper domains. These findings suggest a complex expression and functional regulation of CREB in peripheral tissues including dorsal root and sympathetic ganglia.

Alternative Splicing↗
PubMed1999

Growth factors controlling muscle development.

The enlarged muscles of certain breeds of cattle, such as the Belgian Blue, have been shown to result from a marked increase in the number of normal sized muscle fibers. Originally insulin-like growth factors (IGFs) were implicated in this myofiber hyperplasia, as IGFs have been shown to stimulate myoblast proliferation as well as maintain fiber differentiation. Recently it has been reported that mice lacking a myostatin gene, a member of the TGFbeta superfamily, have enhanced skeletal mass resulting from increased muscle fiber number and size. Mutations in this gene have been found in double-muscled cattle, indicating that myostatin is an inhibitor of muscle growth. Myostatin is expressed early in gestation and then maintained to adulthood in certain muscles. Myostatin expression in bovine muscle is highest during gestation when muscle fibers are forming and some of the myogenic regulatory factors have elevated expression over the same period as myostatin. Molecular expression of the IGF axis does not differ between Belgian Blue and normal muscled cattle, and IGF-II mRNA is increased throughout formation of secondary fibers in both breeds. However, myostatin and MyoD expression in muscle differ between normal and hypertrophied muscle cattle breeds. This evidence strongly suggests that lack of myostatin is associated with an increase in fiber number which then results in a marked increase in potential muscle mass in double-muscled cattle.

Amino Acid Sequence↗
PubMed1999

Experimental studies of chlamydiosis in Japanese quails.

Two-week-old Japanese quails were infected intratracheally with six strains of Chlamydia psittaci isolated from calf pneumonia, swine pneumonia, goat abortion, sheep abortion, kid enteritis, and calf conjunctivitis, respectively. The Japanese quails from infected and control groups were closely observed for clinical symptoms. In order to examine the gross and microscopic lesions, quails in each group were sacrificed at 2, 5, 7, 10, 14, 21 and 30 days post infection. Alterations induced by pneumonic strains were more severe than those found in abortion isolates whereas the isolate of conjunctivitis failed to induce any lesion in the quail. Chlamydia psittaci was successfully recovered from lungs, spleen and intestinal contents of sacrificed quails. Calf pneumonia and goat abortion strains could be propagated in quails for a longer period (30 days), than in swine pneumonia, sheep abortion, kid enteritis (15 days each) and calf conjunctivitis isolates (7 days). The control quails were normal and no isolation could be made from them.

Animals↗
PubMed1999

The interaction of Rubia cordifolia with iron redox status: a mechanistic aspect in free radical reactions.

The present publication investigates the antioxidant property and mechanistic aspect of alcoholic extract of R. cordifolia. The extract of R. cordifolia has shown significant inhibitory effect on FeSO4 induced lipid peroxidation. Study with iron redox status showed that R. cordifolia extract reduced or oxidixed; Fe3+ or Fe2+ respectively, in a dose dependent manner. Results with superoxide anion (O2-.) and hydroxyl radical (OH.), showed no radical scavenging activity. The alcoholic extract significantly maintains the reduced glutathione content both in time and dose dependent manner. It also reduced the rate of depletion of reduced glutathione (GSH) level in presence of ferrous sulphate (FeSO4) and cumene hydroperoxide (CHP). On the basis of these observations, it can be concluded that the antioxidant property of R. cordifolia is due to a direct interaction with iron.

Animals↗