Sammer Siddiqui

Association of angiotensin converting enzyme gene polymorphisms with left ventricular hypertrophy.

The corresponding author has indicated to the journal that this paper should be retracted as it is a duplicate publication. The author apologizes to the journal and its readers for any inconvenience caused.

Head and Neck Cancer Susceptibility: A Genetic Marker in the Methylenetetrahydrofolate Reductase Gene

Progress in the elucidation of molecular genetic changes that lead to the development of tumors should soon bring novel diagnostic and therapeutic procedures into clinical practice. In this respect, methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism that affects DNA methylation and synthesis. DNA methylation is an epigenetic feature that influences cellular development and function. Germ line mutation C>T at nucleotide 677 of the MTHFR gene, which results in increased thermolability and diminished enzyme activity, is oncogenic, i.e. should be a contributor to molecular changes leading to cancerous phenotypes (it has also been shown independently to be implicated in cardiovascular disease phenotypes). Interestingly, it has been shown that MTHFR T677 allele homozygosity confers a sixfold increased risk for esophageal squamous cell carcinoma in Northern China. The purpose of this study was twofold: (1) to evaluate the putative association of MTHFR C677T and epithelial squamous cell carcinoma (ESCC) in Pakistan, and (2) to investigate whether de novo MTHFR C677T mutations are involved in the determination of ESCC phenotypes. We recruited 50 ESCC patients referred to the Otolaryngology Clinic of the Aga Khan University Hospital, and 54 age- and gender-matched control (disease-free) subjects. Our results show that T allele frequencies were 0.18 ± 0.05 in cases vs. 0.24 ± 0.05 in controls (as compared with 0.63 vs. 0.41 in the report from China). Although the association is not statistically significant, T alleles are actually more common amongst controls in the Pakistani population, which is the opposite of what would be expected and what has been reported amongst Chinese. Yet the frequency of deleterious T alleles is lower in Pakistan (range 0.18–0.24) than in other parts of the world. Our results indicate that MTHFR C677T cannot form the basis for a genetic test aimed at evaluating an individual’s genetic susceptibility to ESCC in Pakistan. As the conversion of precancerous submucous fibrosis into overt cancer is a frequent occurrence in Pakistan, we proceeded to extract DNA samples in all ESCC patients, from whole blood, cancerous tissues and neighboring normal tissues. We sought to determine whether C677T genotypes were different in the three tissue samples from each ESCC patient. In all patients, identical genotypes (and therefore allele frequencies) were systematically observed in all three samples. This indicates that de novo MTHFR 677TC>T mutations are not part of the molecular etiology of ESCC. In conclusion, we can rule out a major involvement of the MTHFR gene in the determination of ESCC in Pakistan.

Fluoroquinolones inhibit HCV by targeting its helicase.

BACKGROUND HCV has infected >170 million individuals worldwide. Effective therapy against HCV is still lacking and there is a need to develop potent drugs against the virus. In the present study, we have employed two culture models to test the activity of fluoroquinolone drugs against HCV: a subgenomic replicon that is able to replicate independently in the cell line Huh-8 and the Huh-7 cell culture model that employs cells transfected with synthetic HCV RNA to produce the infectious HCV particles. Fluoroquinolones have also been shown to have inhibitory activity against certain viruses, possibly by targeting the viral helicase. To tease out the mechanism of the antiviral activity of fluoroquinolones, their effect on HCV NS3 helicase protein was also tested. METHODS Huh-7 cells producing the HCV virion as well as Huh-8 cells were grown in the presence or absence of 12 different fluoroquinolones. Afterwards, Huh-7 and Huh-8 cells were lysed and viral RNA was extracted. The extracted RNA was reverse transcribed and quantified by real-time quantitative PCR. Fluoroquinolones were also tested on purified NS3 protein in a molecular-beacon-based in vitro helicase assay. RESULTS To varying degrees, all of the tested fluoroquinolones effectively inhibited HCV replication in both Huh-7 and Huh-8 culture models. The inhibition of HCV NS3 helicase activity was also observed with all 12 of the fluoroquinolones. CONCLUSIONS Fluoroquinolones inhibit HCV replication possibly by targeting the HCV NS3 helicase. These drugs hold promise for the treatment of HCV infection.

Immunohistochemical localization of G protein βγ subunits in the lateral wall of the rat cochlea

The role of G protein‐mediated signal transduction in the production of endolymph, an extracellular fluid of unusual ionic composition, is beginning to be understood. The identity of Gα subunits in the stria vascularis and the spiral ligament of the lateral wall of the cochlear duct is well established. However, little is known about the presence of βγ subunits. This study used immunohistochemistry to investigate the distribution of G protein βγ subunits in the lateral wall of the cochlea. Temporal bones of 6‐ to 8‐week‐old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit‐specific polyclonal antibodies. The results show that the pattern of immunoreactivity varies for the G protein β1–4 and γ1–3, 5 and 7 subunits in the stria vascularis and spiral ligament. In the stria vascularis, immunoreactivity was detected for β2, β3, β4, γ1, γ2 and γ7 subunits. All five types of fibrocytes in the spiral ligament exhibited positive staining for γ2 and γ7. However, immunoreactivity for β1–4 subunits was variable. Immunoreactivity for γ3 and γ5 subunits was not detected in the lateral cochlear wall. The expression pattern of G protein βγ subunits in lateral wall provides a basis for interpreting the functions of G protein‐coupled receptors in cochlear fluid homeostasis.

Expression of G protein α subunits in the lateral wall of the rat cochlea

Expression of five G protein α subunits was investigated in the rat cochlea by reverse transcription‐polymerase chain reaction (RT‐PCR) in order to understand their role in the cochlear signal transduction mechanisms. Immunohistochemical techniques were employed to study their distribution in the lateral wall of the cochlea. Total RNA was extracted with guanidine thiocyanate from cochleas and brains of 14–21‐day‐old rats. The extract was treated with DNase to degrade genomic DNA. After RT, the resulting cDNA was amplified by PCR using primers specific for the nucleotide sequences representing α subunits of heterotrimeric G proteins. The results indicated that mRNA for all five α subunits was expressed in the brain and cochlear samples. For immunohistochemical localization, temporal bones of 6‐week‐old rats were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde and processed for embedding in paraffin wax. The dewaxed, midmodiolar sections of the cochlea were incubated with subunit‐specific polyclonal antibodies. The pattern of immunoreactivity varied for the five G protein α subunits studied in the stria vascularis and spiral ligament. The significance of these findings and the role of G protein α subunits in cochlear fluid homeostasis are discussed.

Use of the SYBR Green dye for measuring helicase activity.

Here we describe a non-radioactive assay that exploits the fluorescent dye SYBR Green to measure the helicase enzyme activity. SYBR Green I emits fluorescence upon intercalation with double-stranded DNA or RNA. The fluorescence is lost proportionally as the nucleic acid is converted to single strands by a helicase, and this decrease in fluorescence intensity can be used to measure the activity of the helicase enzyme. The reaction was prepared by mixing a double-stranded substrate with the helicase enzyme, buffer, ATP and SYBR Green I. After completion, the reaction was terminated by EDTA and fluorescence was measured. Using this technique, a linear increase in substrate release was observed with increasing time and helicase concentrations. The assay described here is speedy, efficient and economical; it holds promise for use in large-scale screening of drugs that target helicases.

Temporal expression of calcium/calmodulin-dependent adenylyl cyclase isoforms in rat articular chondrocytes: RT-PCR and immunohistochemical localization

A multitude of signalling cascades are implicated in the homeostasis of articular chondrocytes. However, the identity of these signalling pathways is not fully established. The 3, 5′‐cyclic AMP‐mediated signalling system is considered to be a prototype. Adenylyl cyclase (AC) is an effector enzyme responsible for the synthesis of cAMP. There are 10 mammalian AC isoforms and some of these are differentially regulated by calcium/calmodulin (Ca2+/CaM). Ca2+ is known to play an important role in the development and maintenance of skeletal tissues. Ca2+/CaM‐dependent AC isoforms and their temporal expression in articular chondrocytes in rats were identified using RT‐PCR and immunohistochemistry techniques. All Ca2+/CaM‐dependent AC isoforms were expressed in chondrocytes from all age groups examined. Each isoform was differentially expressed in developing and adult articular chondrocytes. Generally, expression of AC isoforms was observed to increase with age, but the increase was not uniform for all Ca2+/CaM‐dependent AC isoforms. Expression of Ca2+/CaM‐dependent AC isoforms along with other signalling molecules known to be present in articular chondrocytes indicate complicated and multifactorial signalling cascades involved in the development and homeostasis of articular cartilage. The significance of these findings in terms of articular chondrocyte physiology is discussed.

Simian Virus 40 Large T Antigen as a Model to Test the Efficacy of Flouroquinolones against Viral Helicases

Simian virus 40 large T-antigen (SV40 LT-Ag) is a 708 amino acid nuclear phosphoprotein. Among many functions of LT-Ag is its ability to perform as an ATPase-helicase, catalyzing the unwinding of viral genome during replication. The LT-Ag has been employed in the studies of helicase structure and function, and has served as a model helicase for the screening of antiviral drugs that target viral helicase. In this study, using in vitro enzyme assays and in silico computer modeling, we screened a batch of 18 fluoroquinolones to assess their potential as antivirals by virtue of their inhibition of the LT-Ag helicase. We found all fluoroquinolones to be inhibitory to the helicase activity of LT-Ag. In our docking analysis, most of these tested drugs showed similarity in their interactions with LT-Ag. Our study shows the potential of fluoroquinolones as antiviral drugs and of SV40 LT-Ag as a model protein for screening drugs against viral helicases.