Functionalized conductive activated carbon-polyaniline composite anode for augmented energy recovery in microbial fuel cells.

Internal resistance is one of the limiting factors for power production in microbial fuel cells (MFC). To overcome this, current study designed polyaniline functionalized activated carbon (PANi-FAC) composite as capacitive anode with strategic electrocatalytic capability, and was comparatively assessed with SSM-PANi and bare SSM as anodes in three double chambered MFCs respectively. Power output and COD removal efficiency of PANi-FAC coated on stainless steel mesh (SSM-PANi/FAC) is superior (322 mW/m2; 87.6%) in comparison to SSM-PANi (273 mW/m2; 62.4%) and bare SSM (169 mW/m2; 54%). In addition, maximum specific capacitance of hybrid electrodes is relatively high with SSM-PANi/FAC (360.84 F/g) than SSM-PANi anode (128.26 F/g). Nyquist impedance plots showed less charge-transfer resistance (Rct) with SSM-PANi/FAC (29.9 Ω) than SSM-PANi (206.8 Ω) and SSM anodes (678 Ω). Study infers that, development of electrochemical double layer capacitance makes SSM-PANi/FAC, a potential capacitive anode for augmenting bio-electrocatalytic activity and reducing Ohmic losses.

Measuring liquidity in Indian stock market: A dimensional perspective.

Market liquidity ensures the marketability of security and is an indispensable feature of stock markets. Previous studies have emphasized the role of stock market liquidity in empirical finance. However, they have inadequately explored its multidimensional nature. This study eliminates the ambiguities related to market liquidity by precisely measuring it by using popular and proven liquidity measures. As such, the present study aims to evaluate market liquidity in terms of depth, breadth, tightness, and immediacy in the Indian equity market and also identifies crucial interdependencies between liquidity dimensions. The study selects 500 stocks constituting the NIFTY 500 index of the National Stock Exchange, India, as of 26th May 2019. The data on trading volume, bid price, ask price, the number of shares outstanding, closing share prices were retrieved for the period from 1st April 2009 to 31st March 2019. The study employs Share Turnover, Amihud Illiquidity Ratio, Relative Quoted Spreads, and Coefficient of Elasticity of Trading for liquidity measurement. The Vector Auto-Regressive (VAR) model is used to establish the simultaneous relationships between liquidity dimensions. The analysis is conducted at the aggregate market level as well as across turnover based stock groups divided based on their rankings in terms of stock specific share turnover. The empirical results evidenced the presence of consistent depth, strong breadth, and immediacy but lower tightness in the Indian equity market. The market depth and tightness appear to be relevant in determining dimensional interdependencies. Also, less frequently traded stocks exhibit higher illiquidity in the wake of lower tightness. The findings of this study will assist the investors to wisely understand the multifaceted nature of market liquidity and base their trading decisions accordingly. Moreover, the regulators of the stock exchange can devise liquidity enhancing policies based on the directional movements among liquidity dimensions.

Modified conductive polyaniline-carbon nanotube composite electrodes for bioelectricity generation and waste remediation.

Anode with good electrocatalytic capabilities is more specifically required to reduce the ohimic losses during microbial fuel cell (MFC) operation. Highly conductive polymers viz., Polyaniline (PANi) and Polyaniline/Carbon nanotube (PANi/CNT) composite were prepared by in situ oxidative chemical polymerization method. Anodes were fabricated independently by coating PANi and CNT/PANi composites on the surface of SSM. The fabricated electrodes were evaluated as anode against stainless steel mess (SSM) as cathode during MFC operation. Maximum bioelectricity generation was observed in SSM-PANi/CNT-anode with power density of 48 mW/m2 and COD removal efficiency of 80% compared with SSM-PANi-anode (38 mW/m2; 65%) and SSM-anode (28 mW/m2; 58%). Bioelectrochemical characterization of the electrode materials using cyclic voltammetry and electrochemical impedance spectroscopy showed high electrocatalytic activity of PANi/CNT composite electrode. The study concluded the efficiency of PANi/CNT composite electrodes as bioanode in operation of MFCs towards achieving increased bioelectricity production along with wastewater treatment.

Immediate impact of successful percutaneous balloon mitral valvuloplasty on right and left ventricular functions: An echocardiographic study using load independent tissue velocity imaging indices.

BACKGROUND: The impact of successful percutaneous balloon mitral valvuloplasty (PBMV) on left ventricular (LV) function has been a controversial subject. This study aimed to determine the immediate impact of PBMV on biventricular function using recent Tissue Velocity Imaging (TVI) derived load-independent indices. METHODS AND RESULTS: A total of 30 patients with severe mitral stenosis (MS) who underwent PBMV at a tertiary center of India from August 2012 to December 2013 were included in the study. Thirty age-matched and gender-matched healthy controls were also enrolled. Out of 30 patients, 27(90%) were female. Mean mitral valve area (MVA) of patients before and after PBMV was 0.78 and 1.82cm2 (p<0.001), respectively. All TVI-derived LV and RV basal systolic (IVCV, Sm and the relatively load independent IVA) and diastolic velocities (Em, Em/Am) were significantly decreased in patients with MS compared to controls (p<0.001 for all) which improved significantly after PBMV (6.4±0.7 vs 11±1.6; 5.8±0.7 vs 9.9±1.6; 1.5±0.3 vs 4.2±0.6; 6.4±0.6 vs 13.1±2.1; 0.7±0.1 vs 1.7±0.2 for mitral annulus respectively, p<0.001 for all). Increment in MVA positively correlated with Tricuspid annular plane systolic excursion (TAPSE) and tricuspid annular Sm and isovolumic contraction velocity (IVCV) and inversely with left atrium (LA) size and Pulmonary arterial systolic pressure (PASP) (p=0.01 for LA size; p<0.001 for others) while no such correlation was found with mitral annulus isovolumic acceleration (IVA) (r=-0.078; p=0.679). CONCLUSION: The improved right ventricular (RV) function appears to be predominantly due to afterload reduction, while that of LV appears to be more due to the acute relief of mechanical restraint.

Isolation and evolutionary analysis of Australasian topotype of bluetongue virus serotype 4 from India.

Bluetongue (BT) is a Culicoides-borne disease caused by several serotypes of bluetongue virus (BTV). Similar to other insect-borne viral diseases, distribution of BT is limited to distribution of Culicoides species competent to transmit BTV. In the tropics, vector activity is almost year long, and hence, the disease is endemic, with the circulation of several serotypes of BTV, whereas in temperate areas, seasonal incursions of a limited number of serotypes of BTV from neighbouring tropical areas are observed. Although BTV is endemic in all the three major tropical regions (parts of Africa, America and Asia) of the world, the distribution of serotypes is not alike. Apart from serological diversity, geography-based diversity of BTV genome has been observed, and this is the basis for proposal of topotypes. However, evolution of these topotypes is not well understood. In this study, we report the isolation and characterization of several BTV-4 isolates from India. These isolates are distinct from BTV-4 isolates from other geographical regions. Analysis of available BTV seg-2 sequences indicated that the Australasian BTV-4 diverged from African viruses around 3,500 years ago, whereas the American viruses diverged relatively recently (1,684 CE). Unlike Australasia and America, BTV-4 strains of the Mediterranean area evolved through several independent incursions. We speculate that independent evolution of BTV in different geographical areas over long periods of time might have led to the diversity observed in the current virus population.

Analysis of 12 Months Clinical Outcomes Associated with Implantation of Ultrathin (60 µm) Bare Metal Stent in an Unselected Real-world Population with Coronary Artery Disease.

INTRODUCTION: In the era of drug-eluting stents, Bare Metal Stent (BMS) has worked its way up to be recognized in several indications. Moreover, literature suggests that strut thickness has been directly related to the restenosis rate. AIM: We intended to evaluate the clinical performance of the ultrathin (60 µm) Flexinnium stent (Sahajanand Medical Technologies Pvt. Ltd. Surat, India) for treatment of a wide range of patients with coronary artery disease in routine clinical practice. MATERIALS AND METHODS: This was an observational, non-randomized, retrospective, single-arm study carried out in real-world patients at three clinical centres of India. A total of 419 consecutive patients' data was collected for the study, who underwent treatment for coronary lesions by implantation of Flexinnium stent, between April 2013 and December 2014. The primary endpoint of the study was Major Adverse Cardiac Events (MACE), a conglomerate of cardiac death, Myocardial Infarction (MI) (Q-wave and non-Q-wave), Target Lesion Revascularization (TLR) and Target Vessel Revascularization (TVR). Any incidence of Stent Thrombosis (ST) was also observed as safety endpoint. These endpoints were observed during in-hospital stay, at 30 days, six months and at 12 months follow up. All data were analysed using the Statistical Package for Social Sciences (SPSS; Chicago, IL, USA) program, version 15. RESULTS: A total of 491 lesions were treated in 419 patients having mean age of 54.1 years. A total of 525 Flexinnium stents were implanted. There were 243 (58.0%) patients with hypertension. At 12 months the total incidences of MACE were 14 (3.5%). These included 9 (2.3%) cardiac deaths, 1 (0.3%) MI, 3 (0.8%) TLRs and 1 (0.3%) TVR. There was one incidence of definite ST at 12 months follow up. CONCLUSION: Our results demonstrate that the Flexinnium stent is associated with a low 12 months incidence of MACE in a wide range of real-world population. Long-term follow up would further confirm its clinical performance profile.

Dual Infection with Bluetongue Virus Serotypes and First-Time Isolation of Serotype 5 in India.

Bluetongue is endemic in India and has been reported from most Indian states. Of late, the clinical disease is most frequently seen in the states of Andhra Pradesh, Telangana (erstwhile Andhra Pradesh state), Tamil Nadu and Karnataka. Our analysis of diagnostic samples from bluetongue outbreaks during 2010-2011 from the state of Karnataka identified bluetongue virus (BTV) serotype 5 (BTV-5) for the first time in India. One of the diagnostic samples (CH1) and subsequent virus isolate (IND2010/02) contained both BTV-2 and BTV-5. Segment 2 (seg-2) sequence data (400 bp: nucleotides 2538-2921) for IND2010/02-BTV5, showed 94.3% nucleotide identity to BTV-5 from South Africa (Accession no. AJ585126), confirming the virus serotype and also indicating that Seg-2 was derived from a Western topotype, which is in contrast to serotype 2, that belongs to an Eastern topotype. BTV-5 has been recently reported from Africa, China, French islands and the Americas. Although the exact source of the Indian BTV-5 isolate is still to be confirmed, recent identification of additional exotic serotypes in India is of real concern and might add to the severity of the disease seen in these outbreaks.

Sequential oxonium-olefin-alkyne cyclization for the stereoselective synthesis of (octahydro-1H-pyrano[3,4-c]pyridin-5-yl)methanone derivatives.

A broad range of aldehydes undergo a smooth cascade cyclization with (E)-5-(3-phenylprop-2-ynylamino)pent-3-en-1-ol in the presence of BF3·OEt2 at room temperature to furnish a novel series of (octahydro-1H-pyrano[3,4-c]pyridin-5-yl)methanone derivatives in good yields and diastereoselectivities. This cascade process provides a simple and proficient alternative for the stereoselective construction of fused pyranopiperidine derivatives.

Evaluation of moxifloxacin-hydroxyapatite composite graft in the regeneration of intrabony defects: A clinical, radiographic, and microbiological study.

BACKGROUND: The formation of new connective periodontal attachment is contingent upon the elimination or marked reduction of pathogens at the treated periodontal site. An anti-microbial agent, i.e. moxifloxacin has been incorporated into the bone graft to control infection and facilitate healing during and after periodontal therapy. MATERIALS AND METHODS: By purposive sampling, 15 patients with at least two contralateral vertical defect sites were selected. The selected sites in each individual were divided randomly into test and control sites according to split-mouth design. Test site received moxifloxacin-hydroxyapatite composite graft and control site received hydroxyapatite-placebo gel composite graft. Probing depth (PD) and Clinical attachment level (CAL) were assessed at baseline, 3, 6, 9, and 12 months. Bone probing depth (BPD) and hard tissue parameters such as amount of defect fill, percentage of defect fill, and changes in alveolar crest were assessed at baseline, 6, and 12 months. Changes in subgingival microflora were also assessed by culturing the subgingival plaque samples at baseline and at 3-month follow-up. The clinical, radiographic, and microbiological data obtained were subjected to statistical analysis using descriptive statistics, paired sample t-test, independent t-test, and contingency test. RESULTS: On intragroup comparison at test and control sites, there was a significant improvement in all clinical and radiographic parameters. However, on intergroup comparison of the same, there was no statistically significant difference between test and control sites at any interval. Although test sites showed slightly higher amount of bone fill, it was not statistically significant. There was a significant reduction in the counts of Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis at both sites from baseline to 3 months. In addition, there was a significant reduction at test sites as compared to control sites at 3-month follow-up (P = 0.003 and P = 0.013). CONCLUSION: The reduction in microbial counts found in test sites at 3-month follow-up could not bring similar significant improvements in the clinical and radiographic parameters though the test sites showed slightly higher bone fill.

Isolation of Bluetongue Virus 24 from India - An Exotic Serotype to Australasia.

Bluetongue (BT) is a viral disease of ruminants and is caused by different serotypes of bluetongue virus (BTV), which is transmitted by several species of Culicoides midges. The disease is endemic in tropical areas, and incursions have been observed in some of the temperate areas. Twenty-seven recognized serotypes of BTV have been reported so far. Some serotype viruses have been shown to circulate in certain geographical areas. BTV-24 has been reported from Africa, the Mediterranean and the Americas, whereas it is exotic to Australasia. Here, we report isolation of BTV-24 from India and show that it has high sequence homology in genome segment 2 with other Western isolates of BTV-24. Entry of this serotype into Australasian region is a cause of concern.

A tandem Prins spirocyclization for the stereoselective synthesis of tetrahydrospiro[chroman-2,4'-pyran] derivatives.

A novel cascade strategy has been developed for the synthesis of tetrahydrospiro[chroman-2,4'-pyran] derivatives by condensation of aldehydes with 2-(5-hydroxy-3-methylenepentyl)phenol. The reaction proceeds smoothly in the presence of BF3·OEt2 under mild conditions in a highly stereoselective manner leading to a single diastereomer. This approach is simple and convenient for the synthesis of pharmacologically relevant spirochroman scaffolds.

Molecular Typing of Bluetongue Viruses Isolated Over a Decade in South India.

Bluetongue (BT) is an arthropod-borne viral disease mostly of sheep. Bluetongue virus (BTV) is a segmented double-stranded RNA virus belonging to the genus Orbivirus of family Reoviridae and is transmitted by midges belonging to Culicoides spp. The disease is endemic in the tropics and subtropics, and the incidence is high in southern India. Twenty-six serotypes of BTV have been reported worldwide. Although most of the serotypes have been reported in India, information regarding currently circulating serotypes is essential to develop control programs. Both serological assays and nucleic acid-based assays have been used for typing BTV. Segment 2, which codes for the outer capsid protein VP2, is the target for PCR-based typing; however, the VP2 sequence diversity among viruses belonging to the same serotype but isolated from different geographical areas makes it essential to develop geographical based reagents. In this study, reverse transcription PCR was developed based on sequences of Indian isolates of BTV (serotypes 1, 2, 9, 10, 12, 16, 21 and 23), and this was applied to type 52 isolates obtained during the last decade. It was found that multiple serotypes circulate, with involvement of more than one serotype infecting individual animals and herds over a period in a given area. Detection of circulating serotypes and estimation of herd immunity against different serotypes of BTV may provide important information for predicting the distribution of these serotypes and inclusion of serotypes in vaccines.

Epidemiology of Bluetongue in India.

Bluetongue (BT) is an insectborne endemic disease in India. Although infections are observed in domestic and wild ruminants, the clinical disease and mortality are observed only in sheep, especially in the southern states of the country. The difference in disease patterns in different parts of the country could be due to varied climatic conditions, sheep population density and susceptibility of the sheep breeds to BT. Over the five decades after the first report of BT in 1964, most of the known serotypes of bluetongue virus (BTV) have been reported from India either by virus isolation or by detection of serotype-specific antibodies. There have been no structured longitudinal studies to identify the circulating serotypes throughout the country. At least ten serotypes were isolated between 1967 and 2000 (BTV-1-4, 6, 9, 16-18, 23). Since 2001, the All-India Network Programme on Bluetongue and other laboratories have isolated eight different serotypes (BTV-1-3, 9, 10, 12, 16, 21). Genetic analysis of these viruses has revealed that some of them vary substantially from reference viruses, and some show high sequence identity with modified live virus vaccines used in different parts of the world. These observations have highlighted the need to develop diagnostic capabilities, especially as BT outbreaks are still declared based on clinical signs. Although virus isolation and serotyping are the gold standards, rapid methods based on the detection of viral nucleic acid may be more suitable for India. The epidemiological investigations also have implications for vaccine design. Although only a handful serotypes may be involved in causing outbreaks every year, the combination of serotypes may change from year to year. For effective control of BT in India, it may be pertinent to introduce sentinel and vector traps systems for identification of the circulating serotypes and to evaluate herd immunity against different serotypes, so that relevant strains can be included in vaccine formulations.

Isolation and Complete Genome Sequencing of Bluetongue Virus Serotype 12 from India.

Bluetongue virus (BTV) causes disease mainly in sheep, but can be transmitted via other domestic and wild ruminants, resulting in pecuniary burden and trade restrictions. Segmented genome with the possibility of reassortment, existence of 26 serotypes, geographical restriction in the distribution of many of the serotypes, use of live attenuated vaccines and the lack of complete sequences of viruses isolated from several parts of the globe have complicated our understanding of the origin, movement and distribution of BTV. Recent efforts in genome sequencing of several strains have helped in better comprehending BTV epidemiology. In an effort to contribute to the genetic epidemiology of BTV in India, we report the isolation and complete genome sequencing of a BTV serotype 12 virus (designated NMO1). This is the first BTV-12 isolated from India and the second BTV-12 to be sequenced worldwide. The analysis of sequences of this virus suggests that NMO1 derived its segments from viruses belonging to western topotype viruses, as well as those from South-East Asia and India. The results have implications for understanding the origin, emergence/re-emergence and movement of BTV as well as for the development of vaccines and diagnostics based on robust epidemiological data.

WIN OVER study: Efficacy and safety of olmesartan in Indian hypertensive patients: results of an open label, non-comparative, multi-centric, post marketing observational study.

BACKGROUND: Hypertension is a global health problem. Multiple classes of drugs including angiotensin receptor blockers (ARBs) are available for the treatment of hypertension. Olmesartan is a relatively newer ARB used in hypertension management. OBJECTIVE: To assess the efficacy and safety of WIN-BP (Olmesartan 20 mg/40 mg) tablet in Indian patients with hypertension. MATERIAL AND METHODS: An open label, non-comparative, multi-centric, real world post marketing observational study included Indian adult hypertensive patients who were treated with olmesartan 20 mg/40 mg tablet once daily for six months. The primary outcome was reduction of systolic blood pressure (SBP) to <140 mmHg and diastolic BP (DBP) to <90 mmHg at 3 and 6 months after initiation of treatment with olmesartan. All reported adverse events were recorded. RESULTS: A total of 8940 patients were enrolled in this study. Baseline SBP of 164 mmHg was reduced to 153, 145, 134 and 130 mmHg at the end of 15 days, 1, 3 and 6 months respectively. Similarly, baseline DBP of 100 mmHg was reduced to 93, 89, 84 and 82 mmHg at the end of 15 days, 1, 3 and 6 months respectively. The reduction in both systolic and diastolic blood pressure from day 15 to month 6 was statistically significant (p < 0.0001) with olmesartan treatment. The percentage of responders for both systolic and diastolic blood pressure increased consistently from day 15 to month 6. Only 0.08% patients reported the adverse events. No serious adverse event was reported in the study. CONCLUSION: Olmesartan 20 mg/40 mg is effective and well tolerated without any serious adverse events in patients with hypertension.

Prevalence, Incidence, and Severity of Sorghum Diseases in Western Kenya.

To assess the prevalence and severity of sorghum diseases in western Kenya, a 2-year survey was conducted (July 1995 and 1996), in 91 and 109 farmers' fields, respectively. Fields were generally <0.5 ha and production environment ranged from warm-humid to warm-semi-arid. Fourteen foliar and six panicle diseases were observed, with limited variation in disease prevalence and severity between the 2 years. The most common foliar diseases observed were (in decreasing order of prevalence) oval leaf spot (Ramulispora sorghicola), rust (Puccinia purpurea), ladder leaf spot (Cercospora fusimaculans), zonate leaf spot (Gloeocercospora sorghi), gray leaf spot (Cercospora sorghi), leaf blight (Exserohilum turcicum), and anthracnose (Colletotrichum sublineolum); with prevalence ranging from 95 to 97% of fields for oval leaf spot, and 44 to 65% of fields for anthracnose. Head smut (Sporisorium reilianum), was observed in 73 to 75% of fields, covered kernel smut (S. sorghi) 42 to 43% of fields, and loose smut (S. cruenta) 14 to 24% of fields. Head smut incidence was >25% in 3% of fields surveyed. Grain yield reduction from smut diseases alone was estimated to be 5%. Out of eight probability distribution functions compared, the double Gaussian model best described the frequency of disease severity levels for most diseases. Based on the best-fitting model, the proportion of fields with disease severity level thought to cause yield loss (severity rating >5 on a 1 to 9 scale, where 1 = no disease) was calculated as 26.6% for oval leaf spot, 15.3% for rust, 14.8% for anthracnose, 4.8% for ladder leaf spot, and 1.5% for leaf blight. The production environment influenced the prevalence of disease severity. Severe anthracnose, leaf blight, and ladder leaf spot were confined to fields in the humid LM1 and LM2 agro-ecological zones, rust was ubiquitous, and severe gray leaf spot was more prevalent in the dryer LM4 zone.

Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence.

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases.

Prognosis of epilepsy associated with single CT enhancing lesion: a long term follow up study.

This is a retrospective analysis to study the long term prognosis of epilepsy associated with single CT enhancing lesion (SCTEL). Follow up CT scan showed resolution of the lesion in all of the 102 patients. Seizures did not recur in 64 (63%) patients after starting antiepileptic drugs. Twenty eight (27.5%) patients had recurrence of seizures for a median period of 2 months before remission was achieved. In the remaining ten (10%) patients seizures remitted only after albendazole therapy and the median period of seizure recurrence was 8 months. Sixteen (42%) of the 38 patients who had recurrence of seizures had type B CT lesion (ring lesion with central enhancing area, probably scolex) (P<0.02 (95% CI 3.2-40.3)). Patients with type B CT lesion had more numbers of seizures and also longer intervals between first and last seizure. Antiepileptic drugs were withdrawn in all the 102 patients. The mean period of follow up was 45 months (range 19-101). Only one patient had a relapse and his follow up CT showed gliotic scar at the site of the previous lesion. We conclude that epilepsy associated with SCTEL is a benign form of epilepsy and seizures recur as long as the lesion persists. Antiepileptic drugs can safely be withdrawn once the follow up CT shows resolution of the lesion.

Probing the role of cysteine residues in the EcoP15I DNA methyltransferase.

Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase. Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide. 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein. Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577. All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays. The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis. Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity. In contrast, the C344A enzyme showed a marked loss of enzymatic activity. More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA. Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive. Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding.

Functional analysis of conserved motifs in type III restriction-modification enzymes.

EcoP1I and EcoP15I are members of type III restriction-modification enzymes. EcoPI and EcoP15I DNA methyltransferases transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the second adenine residues in their recognition sequences, 5'-AGACC-3' and 5'-CAGCAG-3' respectively. We have altered various residues in two highly conserved sequences, FxGxG (motif I) and DPPY (motif IV) in these proteins by site-directed mutagenesis. Using a mixture of in vivo and in vitro assays, our results on the mutational analysis of these methyltransferases demonstrate the universal role of motif I in AdoMet binding and a role for motif IV in catalysis. All six cysteine residues in EcoP15I DNA methyltransferase have been substituted with serine and the role of cysteine residues in EcoP15I DNA methyltransferase catalysed reaction assessed. The Res subunits of type III restriction enzymes share a distant sequence similarity with and contain the motifs characteristic of the DEAD box proteins. We have carried out site-directed mutagenesis of the conserved residues in two of the helicase motifs of the EcoP1I restriction enzyme in order to investigate the role of motifs in DNA cleavage by this enzyme. Our findings indicate that certain conserved residues in these motifs are involved in ATP hydrolysis while the other residues are involved in coupling restriction of DNA to ATP hydrolysis. Taken collectively, these results form the basis for a detailed structure-function analysis of EcoP1I and EcoP15I restriction enzymes.