Nickel-catalyzed insertions of vinylidenes into Si-H bonds.

A nickel-catalyzed reductive cyclization of 1,1-dichloroalkenyl silanes is reported. The products of this reaction are unsaturated five- or six-membered silacycles. Intermolecular variants are also described, providing access to trisubstituted vinyl silanes that are not accessible by alkyne hydrosilylation or sila-Heck-type processes. A variety of silanes can be utilized, including those that serve as nucleophilic partners in Hiyama cross-coupling reactions. Mechanistic studies using deuterium-labelled silanes are described.

Information Retrieval and Awareness about Evidence-Based Dentistry among Dental Undergraduate Students-A Comparative Study between Students from Malaysia and Finland.

BACKGROUND: A fundamental skill in education includes the ability to search for, evaluate, and synthesize information, and this cannot be underestimated in dental education. The aim of this study was to assess how dental students from Malaysia and Finland acquire scientific information and to compare their information retrieval skills. METHODS: Fourth and fifth-year dental students from Malaysia and Finland were invited to participate. A self-administered structured questionnaire including items about the use of information sources, subjective assessment of literature retrieval skills and knowledge was used. RESULTS: A total of 226 dental students participated in the survey: 131 from Malaysia and 95 from Finland. In both countries, the highest interest for data retrieval among students was found in the oral surgery specialty. The three most used sources of information among Malaysian students were personal lecture notes, dental textbooks, and colleagues; while Finnish students used colleagues, lecture notes, and current clinical guidelines. Students' knowledge of evidence-based practice was inadequate in both student groups. Though the majority of participants reported that they had good or passable skills in literature retrieval, more students from Finland judged themselves to have at least good skills compared to those from Malaysia. CONCLUSION: Dental education in both countries includes information retrieval studies and mandatory research projects. However, students did not often use those sources that are considered essential in evidence-based dentistry. Universities should further develop educational and training interventions that guide students to use knowledge resources more effectively for critically appraising scientific evidence.

Metabolic adaptability in liver and gastrocnemius muscle of mice following subacute lead toxicity.

Lead (Pb) is one of several environmental pollutants that adversely affect human health by producing toxicity at the tissue level. The aim of the study was to understand the effects of Pb on the metabolic profiles of liver and gastrocnemius muscle of mice in relation to carbohydrate and fat metabolisms. Swiss albino mice were chosen and divided into two groups, control and Pb-treated. The Pb-treated animals were exposed to Pb at a dose of 5 mg/kg body weight for 30 days orally, which resulted in hypoglycemia, glycosuria, and increased glycogenolysis in the liver and gastrocnemius muscle of treated mice. Pyruvic acid, the end product of glycolysis decreased in muscular tissue and increased in the liver. Additionally, the activity of G-6Pase was depressed in the liver, whereas lactate dehydrogenase activity was increased in skeletal muscle only. An adaptive mechanism was initiated by stimulating glycogenolytic and retarding glycolytic activity in the liver and also by alteration of liver and muscle pyruvate dehydrogenase activity along with increased activity of malate dehydrogenase in skeletal muscle. There was enhancement of succinate dehydrogenase and nicotinamide adenine dinucleotide phosphate oxidase activities in the studied tissues. Interestingly, cholesterol, high-density lipoprotein, and low-density lipoprotein levels were elevated, whereas those of triglycerides were decreased in Pb-treated mice serum. The activities of fatty acid synthase and glyceraldehyde 3 phosphate dehydrogenase were depressed in Pb-treated mice livers. Pb also significantly altered the morphological features of the liver, skeletal muscle, and pancreas. These data suggested that subacute Pb exposure was responsible for metabolic modulation in an adaptive fashion in the liver and skeletal muscle of mice.

Transcutaneous electrical nerve stimulation (TENS) for pain management in sickle cell disease.

BACKGROUND: Sickle cell disease (SCD), one of the most common inherited disorders, is associated with vaso-occlusive pain episodes and haemolysis leading to recurrent morbidity, hospital admissions and work or school absenteeism. The crises are conventionally treated with opioids, non-opioids and other adjuvants with the risk of developing complications, addictions and drug-seeking behaviour. Different non-pharmacological treatments, such as transcutaneous electrical nerve stimulation (TENS) have been used for managing pain in other painful conditions. Hence, the efficacy of TENS for managing pain in SCD needs to be reviewed. OBJECTIVES: To assess the benefits and harms of TENS for managing pain in people with SCD who experience pain crises or chronic pain (or both). SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Haemoglobinopathies Register, comprising of references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. We also searched online trial registries and the reference lists of relevant articles and reviews. Date of the last search: 26 Febraury 2020. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and quasi-RCTs, where TENS was evaluated for managing pain in people with SCD. DATA COLLECTION AND ANALYSIS: Two review authors independently assessed the eligibility of the trials identified by the literature searches according to the inclusion criteria. Two review authors then independently extracted data, assessed for risk of bias using the Cochrane standard tool and rated the quality of evidence using the GRADE guidelines. MAIN RESULTS: One double-blind cross-over RCT with 22 participants with SCD (aged 12 to 27 years) was eligible for inclusion. Following stratification into four pain crises severity grades, participants were then randomised to receive TENS or placebo (sham TENS). The trial was concluded after 60 treatment episodes (30 treatment episodes of each treatment group). There is a lack of clarity regarding the trial design and the analysis of the cross-over data. If a participant was allocated to TENS treatment for an episode of pain and subsequently returned with a further episode of a similar degree of pain, they would then receive the sham TENS treatment (cross-over design). For those experiencing a pain episode of a different severity, it is not clear whether they were re-randomised or given the alternate treatment. Reporting and analysis was based on the total number pain events and not on the number of participants. It is unclear how many participants were crossed over from the TENS group to the sham TENS group and vice versa. The trial had a high risk of bias regarding random sequence generation and allocation concealment; an unclear risk regarding the blinding of participants and personnel; and a low risk regarding the blinding of the outcome assessors and selective outcome reporting. The trial was small and of very low quality; furthermore, given the issue with trial design we were unable to quantitatively analyse the data. Therefore, we present only a narrative summary and caution is advised in interpreting the results. In relation to our pre-defined primary outcomes, the included trial did not report pain relief at two to four weeks post intervention. The trial authors reported that no difference was found in the changes in pain ratings (recorded at one hour and four hours post intervention) between the TENS and the placebo groups. In relation to our secondary outcomes, the analgesic usage during the trial also did not show any difference between groups. Given the quality of the evidence, we are uncertain whether TENS improves overall satisfaction as compared to sham TENS. The ability to cope with activities of daily living was not evaluated. Regarding adverse events, although one case of itching was reported in the TENS group, the site and nature of itching was not clearly stated; hence it cannot be clearly attributed to TENS. Also, two participants receiving 'sham' TENS reported a worsening of pain with the intervention. AUTHORS' CONCLUSIONS: Since we have only included one small and very low-quality trial, with a high risk of bias across several domains, we are unable to conclude whether TENS is harmful or beneficial for managing pain in people with SCD. There is a need for a well-designed, adequately-powered, RCT to evaluate the role of TENS in managing pain in people with SCD.

Rapid detection of Escherichia coli using fiber optic surface plasmon resonance immunosensor based on biofunctionalized Molybdenum disulfide (MoS2) nanosheets.

The molybdenum disulfide (MoS2) nanosheets functionalized fiber optic surface plasmon resonance (SPR) immunosensor has been reported for the sensitive detection of Escherichia coli (E. coli). The MoS2 nanosheets were prepared by chemical exfoliation method. The synthesised nanostructures were characterized for their structural, morphological and optical properties. The E. coli monoclonal antibodies were successfully immobilized on the MoS2 functionalized sensing platform via hydrophobic interactions. An alternative method simplifying the antibodies immobilization process by functionalization of 2D nanomaterial (MoS2 nanosheets) for rapid (~15 mins) bacterial quantification is presented in this study. The immunosensor uses wavelength interrogation method and a strong linear relationship (R2 = 0.994) was observed between spectral response of immunosensor and different concentration of E. coli. The nonspecificity and cross-reactivity studies of the developed immunosensor were investigated with detection of Salmonella Typhimurium and Staphylococcus aureus. To demonstrate the practical application, spiked samples of water and orange juice were analysed with acceptable recovery results. The label-free immunosensor exhibits better performance, detection limit (94 CFU/mL), high sensitivity (2.9 nm/1000 CFU mL-1; 3135 nm/RIU) and profound specificity as compared to conventional fiber optic SPR sensor (detection limit: 391 CFU/mL, sensitivity: 0.6 nm/1000 CFU mL-1; 1646 nm/RIU). This sensing platform shows promising applications in regular water and food quality monitoring for various pathogenic microorganisms.

Metabolic adaptability in hexavalent chromium-treated renal tissue: an in vivo study.

BACKGROUND: Hexavalent chromium [Cr(VI)], an environmental pollutant that originates mostly from anthropogenic sources, is a serious threat to human health. After entering into cells, Cr(VI) is capable of producing excessive free radicals and causing tissue damage. The present study aims to reveal the toxic manifestation of Cr(VI) on the metabolic activity of renal tissue. METHODS: Male Swiss albino mice were treated orally with potassium dichromate (K2Cr2O7) at a dose of 10 mg/kg body weight for a period of 30 days. Important tricarboxylic acid (TCA) cycle enzyme activities like isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, as well as the activities of enzymes involved in oxidative phosphorylation such as Nicotinamide adenine dinucleotide (NADH) dehydrogenase, were measured. Additionally, transaminase and protease (pronase, cathepsin and trypsin) activities, tissue protein and free amino nitrogen were estimated in renal tissue. Glucose-6-phosphatase, glucose-6-phosphate dehydrogenase and alkaline phosphatase activities, as well as lactic acid, pyruvic acid and chromium contents, of kidneys were determined following standard protocols. Kidney histology was performed by hematoxylin and eosin staining. RESULTS: Cr(VI) suppresses the rate-limiting enzymes of the TCA cycle and oxidative phosphorylation indicating an inhibition of renal ATP production. It decreases protease activity by eliminating the protein substrates and alters the gluconeogenic pathway. Cr(VI) worsens the normophysiological attributes of renal tissue by enhancing the activity of alkaline phosphatase, pointing towards kidney disease. Histopathological observations confirmed these biochemical results through the presence of chronic tubular nephritis and altered glomerular structure. Cr(VI) retention occurs to a greater extent in renal tissue, which intensifies the toxic manifestation of this pollutant in the kidney. CONCLUSIONS: Cr(VI) disrupts the metabolic interaction between carbohydrates and proteins in mammalian renal tissue.

Catalytic Reductive Vinylidene Transfer Reactions.

Methylenecyclopropanes are important synthetic intermediates that possess strain energies exceeding those of saturated cyclopropanes by >10 kcal/mol. This report describes a catalytic reductive methylenecyclopropanation reaction of simple olefins, utilizing 1,1-dichloroalkenes as vinylidene precursors. The reaction is promoted by a dinuclear Ni catalyst, which is proposed to access Ni2(vinylidenoid) intermediates via C-Cl oxidative addition.

A clinicopathologic and epidemiologic study of chronic white lesions in the oral mucosa.

Invasive oral squamous cell carcinoma is often preceded by the presence of clinically identifiable premalignant changes of the oral mucosa, including white lesions. We conducted a cross-sectional, observational study to assess the clinicopathologic and epidemiologic aspects of chronic oral mucosal white lesions to determine the necessity of early biopsy in these cases. Our study population was made up of 77 patients-50 males and 27 females, aged 15 to 70 years (mean: 42.9)-who presented with white lesions persisting for at least 4 weeks. All but 3 patients underwent a biopsy; the 3 exceptions were diagnosed with smear-proven candidiasis. Patients with moderate or severe dysplasia underwent an excision biopsy. The buccal mucosa was the single most common site of white lesions, occurring in 15 patients (19.5%), although 21 patients (27.3%) exhibited a diffuse involvement of the oral mucosa. Of the 77 patients, 59 (76.6%) had concerning findings: premalignant lesions in 45 patients (58.4%) and malignant lesions in 14 (18.2%). Also, dysplasia was seen in 8 patients (10.4%), all of whom had premalignant lesions. Tobacco chewing (p = 0.008) and betel quid chewing (p = 0.029) were significantly associated with the development of premalignant and malignant lesions; a longer duration of tobacco chewing (≥10 yr) was significantly associated with a higher risk of malignant but not premalignant lesions (p = 0.031). Finally, illiteracy was a significant risk factor for premalignant and malignant lesions (p = 0.03). Our findings support the necessity of biopsy in every case. Early detection of oral carcinoma by biopsy of all oral white lesions would not only prevent patients from undergoing disfiguring surgery and chemoradiation, but it also would increase the 5-year survival rate.

Dinuclear oxidative addition reactions using an isostructural series of Ni2, Co2, and Fe2 complexes.

A family of low-valent Ni2, Co2, and Fe2 naphthyridine-diimine (NDI) complexes is presented. Ligand-based π* orbitals are sufficiently low-lying to fall within the metal 3d manifold, resulting in electronic structures that are highly delocalized across the conjugated [NDI]M2 system. This feature confers stability to metal-metal interactions during two-electron redox reactions, as demonstrated in a prototypical oxidative addition of allyl chloride.

Comparison of a microsliced modified chondroperichondrium shield graft and a temporalis fascia graft in primary type I tympanoplasty: A prospective randomized controlled trial.

We conducted a prospective, randomized, controlled trial to compare outcomes in type I tympanoplasty patients who received an autologous microsliced modified cartilage perichondrium shield graft (cartilage group) and those who received an autologous temporalis muscle fascia graft (fascia group). Our three outcomes measures were (1) anatomic success rates at 3 months, (2) hearing results at 6 months, and (3) rates of morphologic success (i.e., the absence of reperforation, retraction, and graft displacement) at 2 years among those in each group who had an intact graft at 3 months. Of 56 patients who were initially enrolled and who underwent one of these type I tympanoplasty procedures, 51 completed the study-28 in the cartilage group and 23 in the fascia group. The former was made up of 11 males and 17 females, aged 15 to 48 years (mean: 27.4), and the latter included 9 males and 14 females, aged 15 to 52 years (mean: 31.7). The overall graft take rate at 3 months with respect to perforation closure (anatomic success) was 93.3% in the cartilage group and 91.7% in the fascia group, which was not a statistically significant difference. The mean hearing gain at 6 months was 11.7 ± 7.6 dB in the cartilage group and 12.6 ± 6.0 dB in the fascia group-again, not statistically significant. At 2 years, morphologic success rates were 92.3 and 81.0%, respectively-again, not statistically significant. We conclude that autologous microsliced modified cartilage perichondrium shield graft tympanoplasty is as effective as conventional temporalis fascia tympanoplasty in terms of graft take rates and functional results. Indeed, medium-term outcomes (2-yr follow-up) revealed that sustainable morphologic success was actually better with the cartilage technique than with the fascia technique because it was associated with fewer revision surgeries.

Epistaxis Due to Leech Infestation in Nose: A Report of Six Cases and Review of Literature.

The aim of this study is to report unusual cause of epistaxis due to leech infestation in nose in hilly area and its management. The study was carried out for a period of 4 years (2008-2012) in a secondary level hospital in hilly area of Darjeeling, West Bengal, India with data collected from the OPD and Emergency register of the patients. This retrospective case series consisted of six cases. All the cases presented with unilateral recurrent epistaxis and foreign body nose. Anterior rhinoscopy revealed fleshy greenish brown mobile mass inside the nasal cavity which was removed by forceps. The animate foreign body was identified as leech in all the cases. To conclude, in hilly areas leech infestation can present as animate foreign body in nose and it should be considered as important cause of epistaxis.

Evaluating the Effect of Catalyst Nuclearity in Ni-Catalyzed Alkyne Cyclotrimerizations.

An evaluation of catalyst nuclearity effects in Ni-catalyzed alkyne oligomerization reactions is presented. A dinuclear complex, featuring a Ni-Ni bond supported by a naphthyridine-diimine (NDI) ligand, promotes rapid and selective cyclotrimerization to form 1,2,4-substituted arene products. Mononickel congeners bearing related N-donor chelates (2-iminopyridines, 2,2'-bipyridines, or 1,4,-diazadienes) are significantly less active and yield complex product mixtures. Stoichiometric reactions of the dinickel catalyst with hindered silyl acetylenes enable characterization of the alkyne complex and the metallacycle that are implicated as catalytic intermediates. Based on these experiments and supporting DFT calculations, the role of the dinuclear active site in promoting regioselective alkyne coupling is discussed. Together, these results demonstrate the utility of exploring nuclearity as a parameter for catalyst optimization.

Protective effect of resveratrol on fluoride induced alteration in protein and nucleic acid metabolism, DNA damage and biogenic amines in rat brain.

Fluoride, a well-established environmental carcinogen, has been found to cause various neurodegenerative diseases in human. Sub-acute exposure to fluoride at a dose of 20mg/kgb.w./day for 30 days caused significant alteration in pro-oxidant/anti-oxidant status of brain tissue as reflected by perturbation of reduced glutathione content, increased lipid peroxidation, protein carbonylation, nitric oxide and free hydroxyl radical production and decreased activities of antioxidant enzymes. Decreased proteolytic and transaminase enzymes' activities, protein and nucleic acid contents and associated DNA damage were observed in the brain of fluoride intoxicated rats. The neurotransmitters dopamine (DA), norepinephrine (NE) and serotonin level was also significantly altered after fluoride exposure. Protective effect of resveratrol on fluoride-induced metabolic and oxidative dysfunctions was evaluated. Resveratrol was found to inhibit changes in metabolic activities restoring antioxidant status, biogenic amine level and structural organization of the brain. Our findings indicated that resveratrol imparted antioxidative role in ameliorating fluoride-induced metabolic and oxidative stress in different regions of the brain.

Ameliorative effect of resveratrol against fluoride-induced alteration of thyroid function in male wistar rats.

Resveratrol (3,4,5-trihydroxystilbene), a polyphenol and well-known natural antioxidant has been evaluated for its protective effect against fluoride-induced metabolic dysfunctions in rat thyroid gland. Fluoride, the most abundant anions present in groundwater throughout the world, creates a major problem in safe drinking water and causes metabolic, structural, and functional injuries in different organ systems. Sub-acute exposure to sodium fluoride at a dose of 20 mg/kg b.w./day orally to rat for 30 days induces thyroidal dysfunction including suppressed synthetic machinery of the thyroid gland to produce nucleic acids and thyroid hormones, mainly T3 and T4. Other functional changes are alteration of certain metabolic enzyme activities like Na(+)-K(+)-ATPase, thyroid peroxidase, and 5,5'-deiodinase. Structural abnormality of thyroid follicles by fluoride intoxication clearly indicates its thyrotoxic manifestation. Resveratrol supplementation in fluoride-exposed animals appreciably prevented metabolic toxicity caused by fluoride and restored both functional status and ultra-structural organization of the thyroid gland towards normalcy. This study first establishes the therapeutic efficacy of resveratrol as a natural antioxidant in thyroprotection against toxic insult caused by fluoride.

Myoepithelial neoplasm of nasal cavity: an uncommon tumor presenting with an unusual clinical presentation.

Myoepithelial tumors are rare malignant tumors of salivary glands with uncertain biological behaviors and clinical course. Parotid gland is the common involvement site. In this article, we present a rare case of myoepithelial neoplasm of nasal cavity in an 11-year-old boy in the light of literature data.

Identification of facial nerve during parotidectomy: a combined anatomical & surgical study.

To find out the most easily identifiable and anatomically consistent landmark for identification of facial nerve during parotid surgery. Ten cadaveric dissections and ten live parotid surgeries for different types of parotid tumours were done. Cadaveric dissection was performed in the Department of Anatomy and the surgeries were done in the Department of ENT and Head and Neck surgery of R. G. Kar Medical College of Kolkata. The distance of the facial nerve trunk from three most commonly used landmarks (viz., tympanomastoid suture, tragal pointer and posterior belly of digastric muscle) was measured in both cadaver and live patients. The ease of identification of the nerve trunk using each of the landmarks, particularly during live surgery was also assessed. The mean distance of the tympanomastoid suture from the facial nerve trunk was 3.5 mm (cadaver) and 3.87 mm (live surgery), the tragal pointer was found to be at a mean distance of 16.61 mm (cadaver) and 16.36 mm (live surgery) and in case of the posterior belly of digastric muscle it was 7.41 mm (cadaver) and 8.03 mm (live surgery). During live surgery the posterior belly of digastric was found to be the most easily identifiable landmark with a consistent anatomical relationship with the nerve trunk. The posterior belly of digastric muscle is the most easily identifiable and a very consistent landmark for facial nerve dissection during parotidectomy. When supplemented with the tragal pointer, accuracy in identifying the facial nerve trunk is very high, thereby avoiding inadvertent injury to the nerve trunk.

Ameliorative effects of oleanolic acid on fluoride induced metabolic and oxidative dysfunctions in rat brain: Experimental and biochemical studies.

Beneficial effects of oleanolic acid on fluoride-induced oxidative stress and certain metabolic dysfunctions were studied in four regions of rat brain. Male Wistar rats were treated with sodium fluoride at a dose of 20 mg/kg b.w./day (orally) for 30 days. Results indicate marked reduction in acidic, basic and neutral protein contents due to fluoride toxicity in cerebrum, cerebellum, pons and medulla. DNA, RNA contents significantly decreased in those regions after fluoride exposure. Activities of proteolytic enzymes (such as cathepsin, trypsin and pronase) were inhibited by fluoride, whereas transaminase enzyme (GOT and GPT) activities increased significantly in brain tissue. Fluoride appreciably elevated brain malondialdehyde level, free amino acid nitrogen, NO content and free OH radical generation. Additionally, fluoride perturbed GSH content and markedly reduced SOD, GPx, GR and CAT activities in brain tissues. Oral supplementation of oleanolic acid (a plant triterpenoid), at a dose of 5mg/kgb.w./day for last 14 days of fluoride treatment appreciably ameliorated fluoride-induced alteration of brain metabolic functions. Appreciable counteractive effects of oleanolic acid against fluoride-induced changes in protein and nucleic acid contents, proteolytic enzyme activities and other oxidative stress parameters indicate that oleanolic acid has potential antioxidative effects against fluoride-induced oxidative brain damage.

Effects of type 3 thyroplasty on voice quality outcomes in puberphonia

@#p style=text-align: justify;strongOBJECTIVES:/strong To assess effects of type 3 thyroplasty on outcomes of voice quality in puberphonia.METHODS:br /Design:/strong Prospective cohortbr /strongSetting:/strong Tertiary Referral Hospitalbr /strongParticipants:/strong Six patients with puberphonia who failed voice therapy, aged 16-25 years, who consulted at the ENT Outpatient department between September 2010 and September 2012, underwent type 3 thyroplasty.Pre-operative and 6-month post-operative voice analysis by voice recordings, Voice Handicap Index (VHI), GRBAS score and real time acoustic analysis (perturbation) using Dr Speech software (University version 4.0, Voice Tech Corporation, USA [Tiger Electronics] with habitual fundamental frequency (FO), jitter % and shimmer % as parameters were performed.Results: Mean pre-operative VHI and GRBAS scores were 53 and 75.67, respectively, whereas post-operative scores were 29 and 25.00, respectively. (P-value for VHI was 0.004 and that of GRBAS was 0.00). On acoustic analysis, mean pre-operative habitual fundamental frequency (FO), jitter % and shimmer % was 245.82 Hz, 0.21 and 2.34, respectively, whereas post-operative mean was 140.78 Hz (P=0.00), 0.19 (P=0.04) and 1.52 (P=0.00), respectively.Conclusion: The mainstay of treatment of puberphonia is voice therapy. Thyroplasty provides a suitable management option in those cases who fail to respond by voice therapy./p

Evanescent wave assisted nanomaterial coating.

In this work we present a novel nanomaterial coating technique using evanescent wave (EW). The gradient force in the EW is used as an optical tweezer for tweezing and self-assembling nanoparticles on the source of EW. As a proof of the concept, we have used a laser coupled etched multimode optical fiber, which generates EW for the EW assisted coating. The section-wise etched multimode optical fiber is horizontally and superficially dipped into a silver/gold nanoparticles solution while the laser is switched on. The fiber is left until the solution recedes due to evaporation leaving the fiber in air. The coating time usually takes 40-50 min at room temperature. The scanning electron microscope image shows uniform and thin coating of self-assembled nanoparticles due to EW around the etched section. A coating thickness <200 nm is achieved. The technique could be useful for making surface-plasmon-resonance-based optical fiber probes and other plasmonic circuits.

Differential scanning calorimetric approach to study the effect of melting region upon transcription initiation by T7 RNA polymerase and role of high affinity GTP binding.

Transcription initiation by T7 RNA polymerase is a multistep process consisting of the transition from closed to open complex. The promoters of bacteriophage T7 share a consensus sequence of 23 base pairs, from -17 to +6, relative to transcription start site (+1). In the present study, we have characterized T7 RNA polymerase-promoter complexes by means of fluorescence spectroscopy and differential scanning calorimetry. We have examined the effect of high affinity GTP binding upon the equilibrium of the transition from closed to open complex. We have employed the promoter containing 23 base pair consensus sequence and two variants containing Adenine-Thymine and Guanine-Cytosine stretches in the melting region of the promoter sequence. Variation in the nucleotide sequence of melting region does not have any effect upon the affinity of promoter-T7 RNAP complex. On the other hand, alteration of the base sequence in the melting region of the promoter affects the isomerization process among the closed and open complexes. When the initiating nucleotide GTP is prebound to T7 RNA Polymerase, the isomerization process is affected only in case of the promoter with consensus sequence.