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Biomedical subjects

R N Singh

Publications and source records attributed to R N Singh.

At least 19 recordsLinked to original sources
PubMed1999

A reverse transcriptase/maturase promotes splicing by binding at its own coding segment in a group II intron RNA.

Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3' exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.

Bacterial Proteins↗
PubMed1999

Transglutaminase activity in equine strongyles and its potential role in growth and development.

Transglutaminases (E.C. 2.3.3.13) are a family of Ca(2+)-dependent enzymes that stabilize protein structure by catalyzing the formation of isopeptide bonds. A novel form of transglutaminase has been identified and characterized that seem to play an important role in growth, development, and molting in adult and larval stages of filarial nematodes. The aim of this study was to identify the ubiquitous nature of this enzyme in other nematodes and to measure its significance to larval growth, molting, and development. For this purpose, equine Strongylus spp. were used. Activity of this enzyme was identified in extracts of larvae and adults of Strongylus vulgaris, S. edentatus, Parascaris equorum and Cylicocyclus insigne. The significance of transglutaminase in the early growth and development of Strongylus vulgaris, S. edentatus and S. equinus was tested by adding specific inhibitors, monodansylcadaverine (MDC) or cystamine (CS), to in vitro cultures of third (L3) and fourth stage larvae (L4). The viability, molting and growth of these nematode species were affected by both inhibitors. Cystamine promoted abnormal development of Strongylus edentatus L3, resulting in an aberrant expansion of the anterior end. Addition of these inhibitors to cultures of L4 also reduced growth of the three species. The results indicated that transglutaminase is present in a wide array of nematode parasites and may be important in growth and development of their larval stages.

Animals↗
PubMed1998

Pharmacological expression in rat hepatocytes of a gene transferred by an adenovirus vector enabled by a chimeric promoter containing multiple cyclic adenosine monophosphate response elements.

Using the adenovirus vector AdCF126(CRE8).Luc to deliver an expression cassette containing multiple cyclic adenosine monophosphate (cAMP) response elements driving the luciferase reporter gene, this study is directed toward evaluating the possibility of controlling genes transferred to the liver using pharmacological agents that raise hepatocyte cAMP levels. Infection of primary rat hepatocytes with AdCF126(CRE8).Luc yielded a low level of luciferase activity that was enhanced 16-fold by the addition of forskolin. Direct intrahepatic administration of the Ad vector in C57B1/6 mice resulted in low-level luciferase activity that was increased 76-fold by the administration of theophylline and 8-bromo-cAMP to increase cAMP levels. In contrast, animals receiving intrahepatic administration of a control vector containing a constitutively active Rous sarcoma virus (RSV) viral promoter driving the luciferase gene had no response to elevated cAMP Strikingly, delivery of the vector to the liver by the intravenous route permitted a 258-fold enhancement of liver luciferase activity following administration of the same cAMP-elevating agents. In comparison, a control Ad vector with the RSV promoter was not activated by the elevation in cAMP The maximum luciferase levels achieved by the combination of AdCF126(CRE8).Luc and pharmacological cAMP elevation was 45-fold greater than that with the RSV promoter. These results show the feasibility of using a chimeric promoter to permit pharmacological induction of high-level expression from an expression cassette transferred to the liver with an adenovirus vector, an approach that may be useful in a variety of liver-related gene-transfer strategies.

Adenoviridae↗
PubMed1998

Specific site selection in RNA resulting from a combination of nonspecific secondary structure and -CCR- boxes: initiation of minus strand synthesis by turnip yellow mosaic virus RNA-dependent RNA polymerase.

A turnip yellow mosaic virus RNA-dependent RNA polymerase activity was used to study the template requirements for in vitro minus strand synthesis, which is initiated specifically opposite the 3'-CCA that terminates the 3'-tRNA-like structure. A deletion survey confirmed earlier results suggesting the absence of minus strand promoter elements upstream of the pseudoknotted acceptor stem and 3'-terminus. Reiteration of this 27-nt domain provided two competing initiation sites. By varying the added downstream element, it was shown that the pseudoknotted domain could be functionally replaced by various simple stem/loops, although with some decrease in activity. The addition of varying numbers of consecutive -CCA- triplets to the 3' end of the tRNA-like structure resulted in accurate initiation from each added triplet. A similar spectrum of initiations occurred with an unstructured RNA consisting of 12 consecutive -CCA- triplets and no additional viral sequence. Substitution mutations revealed no influence on minus strand synthesis of the identity of the nucleotide immediately upstream of a -CC- initiation site, but a preference for a purine immediately downstream. The introduction of secondary structure into the linear template showed that the usage of potential -CCR- initiation sites is influenced by nonspecific secondary structure. We conclude that specificity arises from the requirement that a -CCR- sequence be sterically accessible. This mechanism is only applicable to interactions that do not involve RNA unwinding during site selection, but may be used commonly in positive strand RNA virus replication and be applicable to other RNA-protein interactions.

Base Sequence↗
PubMed1997

Neurobiology of the gustatory systems of Drosophila and some terrestrial insects.

Insects have been favorites for the study of taste perception in the last few decades. They have been used for anatomical, behavioral, developmental, genetic, and physiological studies related to gustation and feeding response. Several genes known to affect the formation of gustatory sensilla or alter the feeding behavior of insects such as Drosophila are known. Studies related to signal transduction, coding of gustatory information, and the nature and constitution of genes involved in taste perception have also been taken up with insects in recent years. The understanding of basic mechanisms of taste perception in insects is likely to lead to better management of useful as well as harmful insects.

Animals↗
PubMed1997

The regulation of Streptococcus mutans glucan-binding protein A expression.

The S. mutans GBP-A is hypothesized to be constitutively expressed and to contribute to the sucrose-dependent colonization of S. mutans. To investigate GBP-A expression, a reporter gene encoding chloramphenicol acetyltransferase (CAT) was placed downstream of the gbpA promoter and CAT activity was measured under conditions that would be associated with the sucrose-dependent colonization of S. mutans. Expression of GBP-A was optimal under anaerobiosis and neutral pH conditions, and correlated with optimal growth. The addition of sucrose to the growth medium did not elevate the expression of GBP-A.

Carrier Proteins↗
PubMed1997

Turnip yellow mosaic virus RNA-dependent RNA polymerase: initiation of minus strand synthesis in vitro.

An RNA-dependent RNA polymerase (RdRp) activity was detergent-solubilized from the chloroplast membranes of Chinese cabbage leaves infected with turnip yellow mosaic virus (TYMV). The template-dependent, micrococcal nuclease-treated activity synthesized full-length minus strands from TYMV RNA and 3'-fragments as short as a 28-nucleotide-long RNA comprising the amino acid acceptor stem of the 3'-tRNA-like structure (TLS). Minus strands were shown to arise by de novo initiation with the insertion of GTP opposite the penultimate (C) residue of the 3'-terminal -CCA. The TYMV RdRp activity was template specific in that poly(A) RNA was not copied, and alfalfa mosaic virus (AIMV) RNA, which does not contain a 3'-TLS, was a very poor template. However, other viral RNAs with a 3'-TLS and in vitro transcripts of tRNAs were copied to varying degrees. Fully modified tRNAs were either inactive or poorly active templates, and AIMV 3'-RNA, even when provided with a 3'-terminal -ACCA, was not copied detectably. A potential role of the acceptor stem pseudoknot as a promoter element was assessed with mutations that drastically altered the structure and sequence of the pseudoknot, revealing only a twofold effect in decreasing template activity. The data show that RNAs with both a tRNA-like conformation and a -CCA 3'-terminus are potential templates for TYMV RdRp and suggest that promoter elements are not limited to the acceptor stem pseudoknot.

Base Sequence↗
PubMed1997

Surfactant therapy improves pulmonary function in infants with Pneumocystis carinii pneumonia and acquired immunodeficiency syndrome.

Pneumocystis carinii pneumonia (PCP) is an important cause of acute respiratory failure in HIV-infected children. PCP may initiate acute respiratory distress syndrome (ARDS) by adversely affecting surfactant physiology. We report improved pulmonary function following administration of bovine lipid extract surfactant to two infants with AIDS-related PCP/ARDS.

AIDS-Related Opportunistic Infections↗
PubMed1997

Elevated interleukin-1 receptor antagonist levels in pediatric sepsis syndrome.

OBJECTIVE: To measure plasma levels of interleukin-1 beta, interleukin-1 receptor antagonist (IL-Ira), and tumor necrosis factor alpha in children with sepsis syndrome. STUDY DESIGN: A prospective, observational study of 14 patients aged 5 months to 13 years with sepsis syndrome admitted to a pediatric intensive care unit. Cytokine levels were measured by enzyme-linked immunosorbent assay at baseline and at a 12, 24, and 48 hours and compared with the levels of 21 age-matched control subjects. RESULTS: The mean pediatric risk of mortality score was 16.1. Bacterial and viral sepsis was confirmed in five and three patients, respectively. Compared with the levels in the control subjects (mean level of IL-Ira: 654 pg/ml), the IL-Ira levels were elevated in the septic patients, with mean values of 17855 (p < 0.001), 12771 (p < 0.001), 9182 (p < 0.01), and 2296 pg/ml (p = not significant) at baseline and at 12, 24, and 48 hours, respectively. The IL-Ira level was greater than 1000-fold higher than the IL-1 beta level at all time points in 13 of 14 septic patients. CONCLUSIONS: At the time of hospital admission, circulating IL-Ira levels in a cohort of children with sepsis syndrome were at concentrations known to block IL-1 receptors. Thus additional benefit from exogenous IL-Ira therapy would be questionable. Further studies are indicated to determine whether there is a population of patients with sepsis who could benefit from administration of exogenous IL-Ira.

Adenoviridae↗
PubMed1997

Ability of a chimeric cAMP-responsive promoter to confer pharmacologic control of CFTR cDNA expression and cAMP-mediated Cl- secretion.

Based on the theoretical concern that chronic over-expression of the exogenous CFTR protein could be associated with adverse effects following gene transfer, we have constructed a replication-deficient adenovirus (Ad) vector containing the normal human CFTR cDNA controlled by a chimeric, cAMP-regulatable promoter responsive to agents that elevate intracellular cAMP levels. Studies with the IB3 human CF-derived respiratory epithelial line as a model target for CF gene therapy and forskolin to elevate cAMP levels demonstrated that following infection with the AdCF126(CRE8) CFTR vector, there was a marked increase in CFTR mRNA levels after forskolin addition. There was an associated correction of cAMP-mediated Cl- secretion that could be further increased with additional forskolin. cAMP-mediated Cl- secretion was corrected with vector doses as low as 0.2 MOI, a dose that can be achieved in vivo in humans. These observations suggest the feasibility of using a regulatable promoter for gene therapy for CF, with the promoter and gene product stimulated by the same class of pharmacologic agents.

Adenoviridae↗
PubMed1997

Diethylcarbamazine: effect on lysosomal enzymes and acetylcholine in Wuchereria bancrofti infection.

We measured the levels of lysosomal enzymes and acetylcholine in Wuchereria bancrofti-infected asymptomatic microfilaraemic human serum, and found a significant decrease in the activity of beta-glucuronidase and acid phosphatase compared to normal serum. Acetylcholine levels were also decreased during infection. However, after giving diethylcarbamazine (6 mg/kg body wt/day) the level of lysosomal enzymes and acetylcholine increased and reached a normal value after two weeks of therapy. It is proposed that parasites secrete acetylcholinesterase in the circulation which degrades acetylcholine. Since acetylcholine stimulates the release of lysosomal enzymes and phagocytosis, the immune response of the host is suppressed during infection. During diethylcarbamazine (DEC) therapy the parasitic enzyme is inhibited by the drug and the normal level of acetylcholine is resumed, which again stimulates the release of lysosomal enzyme and the process of phagocytosis.

Acetylcholine↗
PubMed1996

Regulatable promoters for use in gene therapy applications: modification of the 5'-flanking region of the CFTR gene with multiple cAMP response elements to support basal, low-level gene expression that can be upregulated by exogenous agents that raise intracellular levels of cAMP.

This study focuses on the design, construction, and evaluation of a chimeric promoter for gene therapy applications where it is desirable to have low-level basal expression of the newly transferred gene, which can be induced to higher levels of expression by the administration of pharmacologic agents that can be safely used locally and/or systemically in humans. To achieve this, a chimeric promoter was constructed using fragments of the 5'-flanking region of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, and multiple tandem repeats of the consensus sequence and flanking elements of the cAMP response element (CRE), promoter sequences that support increased transcription in response to elevations in intracellular cAMP levels. Preliminary studies using plasmid vectors demonstrated that: (i) the 5'-flanking sequences from the CFTR gene have low promoter activity in the human airway epithelial cell lines; (ii) chimeras using -718 bp fragment from the 5'-flanking sequence of CFTR gene as the base, with the addition of 4-10 units of a 25-bp sequence containing the CRE consensus sequence, were all inducible by a rise in intracellular cAMP, with the chimera having eight CRE repeats the most responsive; and (iii) a CF126(CRE8) chjimera, consisting of the -126 bp fragment from the 5'-flanking region of CFTR gene together with eight CRE repeats, yielded low-level basal activity but maximal upregulation by cAMP, resulting in expression of the reporter gene that was 51-58% of an RSV-LTR control. On the basis of these observations, replication-deficient adenoviral vectors containing the CF126(CRE8) chimera and the luciferase reporter gene [AdCF126(CRE8).Luc] or the Escherichia coli lacZ (beta-Gal) reporter gene [AdCF126(CRE8). beta gal] were constructed. In several human airway epithelial cell lines, the AdCF126 (CRE). Luc vector provided low basal activity, but was significantly upregulated by agents that increase cAMP levels. Intranasal administration of the beta-Gal-expressing AdCF126(CRE8) beta gal vector into C57B1/6 mice demonstrated cAMP-induced upregulation of the reporter gene in airway epithelial cells. Quantification of the inducibility of the basal promoter activity in the airway epithelium using the AdCF126(CRE8). Luc vector demonstrated an 11-fold upregulation of the basal promoter activity in the lung with the administration of a phosphodiesterase inhibitor and a cAMP analog. These observations demonstrate the feasibility of using a chimeric promoter comprised of a minimal fragment of the CFTR 5'-flanking region, together with added multiple CRE, to control genes delivered in vivo. Importantly, because there are many drugs used in humans that raise cAMP, the concept of using a cAMP-regulatable promoter may also be a useful approach to enhance the safety, efficacy, and feasibility of a variety of human gene therapy strategies.

Animals↗
PubMed1996

Interaction of Escherichia coli RecA protein with LexA repressor. I. LexA repressor cleavage is competitive with binding of a secondary DNA molecule.

Essential to the two distinct cellular events of genetic recombination and SOS induction in Escherichia coli, RecA protein promotes the homologous pairing and exchange of DNA strands and the proteolytic cleavage of the LexA repressor, respectively. Since both of these activities require single-stranded DNA (ssDNA) and ATP, the inter-relationship between these reactions was investigated and found to display many parallels. The extent of active complex formed between RecA protein and M13 ssDNA, as measured by both ATP hydrolysis and LexA proteolysis, is stimulated in a similar manner by either a reduction in magnesium ion concentration or the presence of single-stranded DNA binding (SSB) protein. However, unexpectedly, SSB protein inhibits both LexA proteolysis and ATP hydrolysis (in assays containing repressor) at concentrations of RecA protein that are substoichiometric to the ssDNA, arguing that LexA repressor affects the competition between RecA and SSB proteins for limited ssDNA binding sites. Additionally, attenuation of LexA repressor cleavage in the presence of double-stranded DNA or by an excess of ssDNA suggests that interaction of the RecA nucleoprotein filament with either LexA repressor or a secondary DNA molecule is mutually exclusive. The significance of these results is discussed in the context of both the regulation of inducible responses to DNA damage, and the competitive relationship between the processes of SOS induction and genetic recombination.

Adenosine Triphosphate↗
PubMed1996

Human growth hormone deletion mutant (hGH44-191) binds with high affinity to lactogenic receptors but not to somatogenic receptors.

The carboxyl-terminal hGH fragment, hGH44-191, displays diabetogenic activity. To understand whether this biological activity is mediated through somatogenic or lactogenic receptors, we investigated the ability of hGH44-191 to bind both receptor classes. We found that hGH44-191 could not compete with [125I]hGH or [125I]bGH for bovine liver somatogenic binding sites. Additionally, hGH44-191 could not displace [125I]hGH from the somatogenic receptor sites when human liver microsomes were used as the receptor source. In contrast, hGH44-191 effectively competed with [125I]hGH for lactogenic receptor sites of lactating mammary gland microsomes and of bovine liver microsomes. In summary, hGH44-191 does not bind to somatogenic receptors but does not bind to lactogenic receptors. These data suggest that the biological actions of hGH44-191 could be mediated through lactogenic receptors.

Animals↗
PubMed1995

Fine structure and primary sensory projections of sensilla located in the sacculus of the antenna of Drosophila melanogaster.

Sensilla lining the inner walls of the sacculus on the third antennal segment of Drosophila melanogaster were studied by light and transmission electron microscopy. The sacculus consists of three chambers: I, II and III. Inside each chamber morphologically distinct groups of sensilla having inflexible sockets were observed. Chamber I contains no-pore sensilla basiconica (np-SB). The lumen of all np-SB are innervated by two neurons, both resembling hygroreceptors. However, a few np-SB contain one additional neuron, presumed to be thermoreceptive. Chamber II houses no-pore sensilla coeloconica (np-SC). All np-SC are innervated by three neurons. The outer dendritic segments of two of these neurons fit tightly to the wall of the lumen and resemble hygroreceptor neurons. A third, more electron-dense sensory neuron, terminates at the base of the sensillum and resembles a thermoreceptor cell. Chamber III of the sacculus is divided into ventral and dorsal compartments, each housing morphologically distinct grooved sensilla (GS). The ventral compartment contains thick GS1, and the dorsal compartment has slender sensilla GS2. Ultrastructurally, both GS1 and GS2 are double-walled sensilla with a longitudinal slit-channel system and are innervated by two neurons. The dendritic outer segment of one of the two neurons innervates the lumen of the GS and branches. On morphological criteria, we infer this neuron to be olfactory. The other sensory neuron is probably thermoreceptive. Thus, the sacculus in Drosophila has sensilla that are predominantly involved in hygroreception, thermoreception, and olfaction. We have traced the sensory projections of the neurons innervating the sacculus sensilla of chamber III using cobaltous lysine or ethanolic cobalt (II) chloride. The fibres project to the antennal lobes, and at least four glomeruli (VM3, DA3 and DL2-3) are projection areas of sensory neurons from these sensilla. Glomerulus DL2 is a common target for the afferent fibres of the surface sensilla coeloconica and GS, whereas the VM3, DA3 and DL3 glomeruli receive sensory fibres only from the GS.

Animals↗
PubMed1995

Transglutaminase-catalyzed reaction is important for molting of Onchocerca volvulus third-stage larvae.

Highly insoluble proteins, which are probably cross-linked, are common in the cuticle and epicuticle of filarial parasites and other nematode species. We have investigated the possible involvement of transglutaminase (TGase)-catalyzed reactions in the development of Onchocerca volvulus fourth-stage larvae (L4) by testing the effects of TGase inhibitors on the survival of third-stage larvae (L3) and the molting of L3 to L4 in vitro. The larvae were cultured in the presence of three specific TGase inhibitors: monodansylcadaverine, cystamine, and N-benzyloxycarbonyl-D,L-beta-(3-bromo-4,5-dihydroisoxazol-5-yl)-al anine benzylamide. None of the inhibitors reduced the viability of either L3 or L4. However, the inhibitors reduced, in a time- and dose-dependent manner, the number of L3 that molted to L4 in vitro. Molting was completely inhibited in the presence of 100 to 200 microM inhibitors. Ultrastructural examination of L3 that did not molt in the presence of monodansylcadaverine or cystamine indicated that the new L4 cuticle was synthesized, but there was an incomplete separation between the L3 cuticle and the L4 epicuticle. The product of the TGase-catalyzed reaction was localized in molting L3 to cuticle regions where the separation between the old and new cuticles occurs and in the amphids of L3 by a monoclonal antibody that reacts specifically with the isopeptide epsilon-(gamma-glutamyl)lysine. These studies suggest that molting and successful development of L4 also depends on TGase-catalyzed reactions.

Animals↗