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Biomedical subjects

M A Siddiqui

Publications and source records attributed to M A Siddiqui.

At least 19 recordsLinked to original sources
PubMed1992

Chemistry and anti-HIV properties of 2'-fluoro-2',3'-dideoxyarabinofuranosylpyrimidines.

The synthesis, chemistry, biochemistry, and anti-HIV activity of a series of 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)pyrimidines have been studied in an attempt to find useful anti-AIDS drugs. Synthesis is carried out via a 2,3-dideoxyribose intermediate which facilitates the preparation of analogues by removing the sugar 3'-hydroxyl group prior to, rather than after, condensation with a uracil or cytosine aglycon. The 2'-F-dd-uridine analogues 7a-d (with H, F, Cl, and CH3 substitution in the 5-position) as well as the 4-deoxy compound (12b) are nonprotective to ATH8 or CEM cells infected with HIV-1. In the corresponding cytidine series, the 5-chloro analogue (11) is inactive. However, 2'-fluoro-2',3'-dideoxyarabinosylcytosine, 10a, and its 5-fluoro analogue, 10b, are both active. While neither compounds is a potent as ddC or 5-F-ddC (2b), 10b gives complete protection against the cytopathic effects of HIV in both host cell lines. 2'-Fluoro substitution confers increased chemical and enzymatic stability on dideoxynucleosides. Even though dideoxy pyrimidine nucleosides are inherently more stable than the corresponding purine analogues toward acid-catalyzed cleavage of the glycosidic bond, 2'-fluoro substitution (10a) still increases stabilization relative to ddC (2b). No detectable deamination by partially purified cytidine deaminase is observed with the 2'-fluoro compounds 10a, 10b, or 11 under conditions which rapidly deaminate cytidine. A small amount of 2'-F-dd-ara-U (7a) is formed from 10a in monkey plasma after greater than 24 h of exposure. The octanol-water partition coefficients for the dideoxynucleosides in this study indicate their hydrophilic character, with log P values varying from -0.28 to -1.18.

Animals↗
PubMed1992

Localization of the gene coding for ventricular myosin regulatory light chain (MYL2) to human chromosome 12q23-q24.3.

Human myosin light chain-2 (MYL2) is an important protein involved in the regulation of myosin ATPase activity in smooth muscle. In cardiac muscle, the precise role of MYL2 is not well understood; however, an increase in ventricular MYL2 is observed during myocardial hypertrophy in cardiac patients with valve stenosis. The chromosomal location of the gene coding for MYL2 was identified using a cloned cDNA for human MYL2. Southern blot analysis of DNA from a human/rodent somatic cell hybrid mapping panel showed that the BamHI fragment that hybridized with this cDNA probe was concordant with chromosome 12. The 768-bp cDNA was hybridized to human metaphase chromosomes. The results revealed a significant clustering of silver grains over chromosome 12 bands q23-q24.3, indicating that the gene coding for MYL2 is located in this region.

Cardiomegaly↗
PubMed1992

A single transcription factor binds to two divergent sequence elements with a common function in cardiac myosin light chain-2 promoter.

The cardiac myosin light chain-2 (MLC-2) gene promoter contains several positive and negative cis-acting sequences that are involved in the regulation of its expression. We describe here the properties of two activator sequences, elements A and P, and their DNA-binding factors (ABFs). Element A (CCAAAAGTGG), located at -61, has homology with the evolutionarily conserved sequence CC(A/T)6GG, present in the genes of many contractile proteins. Element P (TAACCTTGAAAGC), located 114 bp upstream of element A, is conserved in both chicken and rat cardiac MLC-2 gene promoters. Deletion mutagenesis demonstrated that these two elements are involved in the positive regulation of MLC-2 gene transcription. At least two sequence-specific element A-binding proteins, ABF-1 and ABF-2, were identified by gel shift analysis of the fractionated cardiac nuclear proteins. ABF-1 binds to element A with strict dependence on the internal element A sequence AAAAGT. In contrast, ABF-2 exhibits a relaxed sequence requirement, as it recognizes the consensus CArG and CCAAT box sequences as well. ABF-2 also recognizes the distal element P despite the fact that the sequences of elements A and P are divergent. DNase I footprinting, methylation interference, and gel shift analyses demonstrated unequivocally that the element A-DNA affinity-purified protein ABF-2 binds to element P with sequence specificity. Since both elements A and P play a positive regulatory role in MLC-2 gene transcription and bind to a single protein (ABF-2), it would appear that ABF-2 is a key transcription factor with the ability to recognize divergent sequence elements involved in a common regulatory pathway during myogenesis.

Animals↗
PubMed1992

Changes in sarcolemmal proteins in subacute myocardial infarction in the dog.

Previous studies have shown degradation of cardiac structural proteins and disruption of the sarcolemma as a result of acute myocardial infarction. However, there is no evidence to date on changes in sarcolemmal membrane proteins induced by experimental subacute myocardial infarction. We studied subepicardial layers overlying myocardial infarct 4 days following ligation of the left anterior descending coronary artery in 12 dog hearts. We first demonstrated that this layer provides the anatomic-electrophysiologic substrate for reentrant arrhythmias using activation mapping techniques and histologic correlations. The makeup of membrane proteins was studied using SDS polyacrylamide gel electrophoresis, peptide mapping, and laser densitometry. Sarcolemmal membrane proteins were isolated by ultracentrifugation through a sucrose gradient. We found that a sarcolemmal polypeptide (MW 126,000; n = 12) in the normal tissues has a different mobility than the corresponding protein (MW 124,000; n = 12) of the ischemic tissues although their peptide analysis appeared similar, suggesting that the protein undergoes a post-translational modification. In addition, two proteins (MW 75,000; n = 12 and MW 88,000; n = 12) were present in greater amount in the ischemic than in the control tissues suggesting either acceleration in protein synthesis or slow down of degradation turnover. These results demonstrate that specific changes occur in membrane proteins subjected to ischemic insults which might be responsible for membrane alterations following ischemia and may contribute to the abnormal electrophysiologic properties and arrhythmia seen in vivo at this stage.

Animals↗
PubMed1992

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.

3T3 Cells↗
PubMed1992

Effect of cytosol on the regulation of expression of myosin heavy chain genes during cardiac hypertrophy.

In vitro translation of RNA transported from the rat heart nuclei has suggested that the transport of translatable messages from hypertrophic heart nuclei is greater than from sham-operated heart nuclei. An increased translation activity was observed in cell-free system with RNA transported from sham-operated heart nuclei in presence of hypertrophic heart cytosol, than from sham-operated heart cytosol. Similar results were obtained when myosin heavy chain (MHC) mRNA in the transported RNA was analyzed by slot-blot hybridization using beta cDNA probe. Immunoprecipitation analysis of the translated products using beta MHC isozyme specific antibody indicated that the increased levels of beta MHC specific mRNA in the RNA transported from sham-operated heart nuclei in presence of hypertrophic heart cytosol than sham-operated heart cytosol. Direct quantitation of alpha and beta MHC messengers by slot-blot hybridization analysis of transported RNA using oligomeric probes corresponding to the 3' untranslated regions of alpha and beta MHC mRNAs revealed that an increased transport of both alpha and beta MHC specific mRNAs from sham-operated heart nuclei in the presence of hypertrophic heart cytosol occurs, of which beta MHC mRNA is more than that of alpha MHC. In contrast, slot-blot hybridization analysis of the radioactive RNA synthesized during transcription in vitro in nuclei obtained from sham-operated as well as hypertrophic hearts has shown an increased synthesis of alpha in sham nuclei and that of beta in hypertrophic heart nuclei. These results suggest that both transcriptional and post-transcriptional regulation may be operative in the expression of alpha and beta MHC genes during the development of cardiac hypertrophy.

Animals↗
PubMed1992

fos-mediated repression of cardiac myosin light chain-2 gene transcription.

The transcription of the chicken cardiac myosin light chain-2 (MLC-2) promoter containing a 1.3 Kb 5'-flanking DNA segment is repressed upon co-transfection with an expression vector (pMMV) containing the proto-oncogene fos in embryonic chicken cardiac muscle cells in culture. Similar concentrations of co-transfectants containing other genes e.g. luciferase were ineffective. To identify the DNA element(s) in MLC-2 gene that responds to fos-mediated inhibition, 5'-sequential deletion mutants of MLC-2 promoter were tested in a transient transfection assay. A mutant, in which the 5' distal sequence was deleted upto -1200 bp upstream of the mRNA start site was sensitive to fos inhibition, but the mutant containing -1130 bp was not, suggesting that a fos responsive element (FRE) is located between -1130 to -1200 bp upstream of the transcription initiation site. The same FRE sequence was also responsive to fos-inhibition in chicken skeletal muscle cells as well. Since over-expression of fos is implicated in repression of myogenic process, the selective inhibition of MLC-2 promoter activity by fos and identification of FRE sequence potentially important in understanding the relationship between myogenesis and the oncoprotein-mediated signal pathway(s).

Animals↗
PubMed1992

Identification of cis-acting DNA elements involved in the regulation of angiotensinogen gene expression.

Angiotensinogen is the precursor molecule of one of the most potent vasoactive substances, angiotensin-II. Angiotensinogen is normally synthesized in the liver and secreted into the plasma where it is converted into angiotensin-II by the combined proteolytic action of renin and angiotensin converting enzyme. Angiotensinogen levels in the plasma are modulated by a number of pathological and physiological factors. In order to understand the regulation of angiotensinogen gene expression, we have constructed an expression vector in which 688 bp of the 5'-flanking region of the rat angiotensinogen gene were attached to the chloramphenicol acetyl transferase (CAT) coding sequence. We have also obtained 5'-sequential deletion mutants from the rat angiotensinogen promoter attached to the CAT gene, and have identified multiple cis-acting DNA sequences involved in the regulation of angiotensinogen gene expression by transient transfection of these recombinant DNA molecules in human hepatoma cell lines, Hep3B, and HepG2.

Angiotensinogen↗
PubMed1992

Mechanism of tissue-specific transcription: interplay between positive and negative regulatory factors.

At least four regulatory cis-acting DNA sequences, CCAAAAGTGG (element A), TTATTTTTA (element B), TATTTATT (element C), and TATTACCTTTAT (element S), were identified in cardiac myosin light chain-2 (MLC2) proximal promoter as target sites for sequence-specific binding of nuclear proteins. For muscle-specific transcription, the proximal promoter (-53 to +1) consisting only of elements B and C is required. Addition of element A to this promoter results in a muscle-specific up-regulation, whereas the addition of element S exerts a negative effect on transcription. The negative and positive regulatory effects of elements S and A respectively were demonstrated by site-specific mutations of the promoter following transient transfection of cardiac muscle cells in culture. Elements S and A interact separately with distinct nuclear protein factor present in both muscle and non-muscle cells, even though their regulatory activities are restricted to muscle cells. Among the multiple complexes resulting from the interaction of nuclear proteins and elements S and A DNAs, one requires both S and A sequences together for binding. Element B, which exerts a muscle-specific positive effect on transcription, binds to a nuclear protein present in cardiac muscle, but not in non-muscle cells. DNA-protein binding assays and mutational analysis of the MLC2 promoter suggest that the contribution of the functionally opposed cis-elements depends upon an interplay between the positively and negatively acting DNA-binding proteins via protein-protein interactions to mediate opposite regulatory effects on gene transcription.

Animals↗
PubMed1991

The black disease of Arabia, Sowda-onchocerciasis. New findings.

Sowda, the localized asymmetrical lesion of onchocerciasis endemic in Yemen and Southern Saudi Arabia, is characterized by hyperpigmented lichenified papular lesions on one leg with intense pruritus. There is enlargement of femoral and inguinal lymph glands. In our study, even the long standing cases do not show elephantiasis of the leg or genitalia. Microfilaria appeared to be scarce and adult worms could not be detected clinically, as well as by ultrasonography (except in one case). There was no significant lymphatic obstruction; such cases were studied by contrast lymphangiography and isotope lymphangiography.

Adolescent↗
PubMed1991

Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element.

Physiological expression of the cardiac muscle myosin light-chain 2 (MLC-2) gene in chickens is restricted to cardiac muscle tissue only, at least during the late embryonic to adult stages of development. The mechanism by which cardiac MLC-2 gene expression is repressed in differentiated noncardiac muscle tissues is unknown. Using sequential 5'-deletion mutants of the cardiac MLC-2 promoter introduced into primary skeletal muscle cells in culture, we have demonstrated that a 89-bp region, designated the cardiac-specific sequence (CSS), is essential for repression of cardiac MLC-2 expression in skeletal muscle. Removal of the CSS sequence alone allows transcription in skeletal muscle cells without affecting the transcriptional activity of the promoter in cardiac muscle cells. DNase I footprinting and gel shift assays indicate that protein binding to sequences in the CSS domain occurs readily in nuclear extracts obtained from skeletal muscle but not in extracts isolated under identical conditions from cardiac muscle. Thus, it appears that a negative regulatory mechanism accounts for the lack of expression of the cardiac MLC-2 gene in skeletal muscle and that the CSS element and its binding proteins are important functional components of the regulatory apparatus which ensures the developmental program for cardiac tissue-specific gene expression.

Animals↗
PubMed1990

Role of dietary fibre on platelet adhesiveness.

Fifty five adult patients of diabetes mellitus and fourteen normal healthy subjects matched for age and sex were studied to see the effect of dietary fibre on platelet adhesiveness. In patients of diabetes mellitus the platelet adhesiveness 2 1/2 hrs. after a load of glucose was significantly higher (p = less than 0.001) as compared to normal individuals under the same conditions. After a glucose load with fibre the platelet adhesiveness in patients with diabetes mellitus fell significantly (p = less than 0.001). Although there was a fall in platelet adhesiveness in normal subjects also, it was statistically not significant. Supplements of fibre in the diet of diabetic patients may prevent or delay the vascular complications.

Adult↗
PubMed1989

Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. Identification of a 250-base pair fragment which confers cardiac-specific expression.

The present study characterized the structure, organization, and expression of the rat cardiac myosin light chain (MLC) -2 gene. The rat cardiac MLC-2 gene has seven exons which display complete conservation with the exon structure of the rat fast twitch skeletal MLC-2 gene. A 250-base pair (bp) sequence of the 5'-flanking region contains CArG motifs and additional cis elements, each greater than 10 bp in length, which were conserved in sequence and relative position with the chick cardiac MLC-2 gene. A series of MLC-2/luciferase fusion genes consisting of nested 5' deletions of the MLC-2 5'-flanking region were constructed and transfected into primary neonatal rat myocardial cells and a non-myocardial cell line (CV-1), demonstrating that this 250 bp of the MLC-2 5'-flanking region was sufficient to confer cardiac specific expression on a luciferase reporter gene. This study suggests the presence of important proximal regulatory sequences in the MLC-2 5'-flanking region which are capable of directing the cardiac specific expression of the rat cardiac myosin light chain-2 gene.

Amino Acid Sequence↗
PubMed1989

Characterization of a rat myosin alkali light chain gene expressed in ventricular and slow twitch skeletal muscles.

Mammalian cardiac muscle contains two myosin alkali light chains: 1) the atrial light chain (MLC1A), and 2) the ventricular light chain (MLC1V) predominantly expressed either in the atrium or in the ventricle. In this report we describe the isolation and characterization of the complete gene for rat MLC1V. The rat MLC1V gene is approximately 6.5 kb long and the mRNA coding sequences are organized in 7 different exons. Comparison of this gene sequence with other known MLC1 gene sequences revealed that the exon-intron organization is highly conserved within the MLC1 gene family. The derived protein sequence of rat MLC1V showed a higher sequence homology with human ventricular (96%) MLC1V than with rat fast skeletal MLC1f (74%), suggesting functional similarities between different MLC1V proteins. S1 nuclease mapping and primer extension analysis demonstrated that this gene is expressed only in ventricular and slow twitch skeletal muscle tissues and is transcribed from the same promoter and transcription initiation site.

Amino Acid Sequence↗
PubMed1989

Genital herpes simplex virus infections: laboratory confirmation in diverse patient groups.

The laboratory diagnosis of genital herpes simplex virus (HSV) infections in various patient groups with diverse clinical manifestations including asymptomatic pregnant women with history of genital herpes and in a high prevalence group consisting of male patients with recurring genital ulcerations is described. HSV was detected by conventional cell culture and also by a CPE-enhancement technique using human embryonic lung fibroblasts (MRC-5) and Vero cells with subsequent typing of isolate by HSV type-specific monoclonal antibodies. A definite improvement in HSV diagnosis was noted by the use of the CPE-enhancement technique with the reduction of turn-around time to 24 h at which 61.1% and 50% isolation rates were detected in MRC-5 cells in comparison to 38.9% and 16.6% by conventional cell culture in the high prevalence patient population and asymptomatic pregnant women, respectively. Delayed development of CPE and reduced isolation rates were detected using Vero cells. Follow-up sampling of subsequent episodes of genital ulcerations in those with history of recurrence but an initial negative cell culture result, increased the isolation rate substantially and enabled the confirmation of diagnosis of genital HSV infection.

Cells, Cultured↗
PubMed1989

Adverse events following DTP immunization in Maryland, 1987.

Between January 1, 1987 and December 31, 1987, a total of 184 adverse events were reported to have occurred within 28 days following diphtheria, tetanus, pertussis (DTP) immunization in Maryland. More than half the reports (54 percent) were of serious or major vaccine reactions. Screaming episodes were the most frequently reported serious reaction, having occurred in 64 (35 percent) of the reports.

Child↗
PubMed1988

Expression of ventricular-type myosin light chain messenger RNA in spontaneously hypertensive rat atria.

Using cloned DNA probes specific for two isoforms of cardiac myosin light chains (MLCs), nonphosphorylatable MLC1 and phosphorylatable, regulatory MLC2, we have observed that the MLC1 messenger RNA of ventricular type does not appear in detectable amounts in atrial cells of either normotensive Wistar-Kyoto rat strain (WKY) or spontaneously hypertensive rat strain (SHR). The messenger RNA of regulatory isoform of ventricular MLC2, on the other hand, is found in threefold excess in atria of SHR relative to that of age-matched WKY. The increased level of MLC2 messenger RNA is present even in 6-week-old SHR atria where there is no established overloading of the heart. Thus, it appears that the increased expression of the regulatory MLC2 gene in SHR atrial cells is a predetermined event, which, most likely, participates in functional adaptation of the myocardium in response to pressure overload and subsequent hypertrophy.

Animals↗