Antioxidant activity in the extracts of two edible aroids.

Two neglected species of Araceae, Alocasia macrorhiza (Linn.) G. Don and Alocasia fornicata (Roxb.) Schott are important as food and ethno medicine in Asia and Africa. Their bioefficacy is documented in the Ayurveda. The solvent extracts of different edible parts of these two species like rhizomes, leaves, roots and stolons were screened for in vitro antioxidant properties using standard procedures. The successive extracts in hexane, benzene, toluene, chloroform, diethyl ether, ethyl acetate and water fraction exhibited IC(50) values in the following order, roots>rhizome>leaves for Alocasia macrorhiza and leaves>stolon for Alocasia fornicate, respectively in 2,2-diphenyl-1-picryl hydrazyl antioxidant inhibition assay. Maximum antioxidant activity was observed in diethyl ether extracts for both species. The IC(50) values were comparable with those of quercetine and ascorbic acid as standards. These results suggest that the two aroid species have antioxidant activity in their edible parts and should be extracted using diethyl ether solvent.

Congenital anomaly surveillance in England and Wales.

The National Congenital Anomaly System (NCAS) was set up in 1964, following the thalidomide epidemic, as a monitoring system designed to detect changes in the frequency of reporting of malformations. Its original aim was to detect anomalies reported within 7 days of birth. The NCAS is voluntary at all stages and covers all live- and stillbirths. It has two tiers; a 'passive system' receiving congenital anomaly notifications through a standard paper notification form, known as the SD56, and the congenital anomaly registers that send notifications electronically. Congenital anomalies are classified using the International Classification of Diseases codes and 10 monitoring groups. The Office for National Statistics performs a statistical analysis on a monthly, quarterly and annual basis, using the cumulative sum technique, which is the basis upon which surveillance alerts are raised within the system. The NCAS is now an open database where congenital anomalies can be notified whenever they are detected. The aim of this paper is to describe the current operation and uses of the NCAS based on guidelines for the evaluation of public health surveillance systems published by the Centers for Disease Control and Prevention.

Evaluation of the National Congenital Anomaly System in England and Wales.

OBJECTIVE: To evaluate the National Congenital Anomaly System (NCAS). METHODS: The NCAS in England and Wales based at the Office for National Statistics and the various regional registers that exchange data with it were examined, based on guidelines for evaluating public health surveillance systems, published by the Centres for Disease Control (CDC). Data relating to congenital anomaly notifications received from 1991 to 2002 were analysed. MAIN OUTCOME MEASURES: The main outcome measures were based on CDC standards and included the level of usefulness of the system, simplicity, flexibility, data quality, acceptability, sensitivity, representativeness, timeliness, and stability of the system. RESULTS: The NCAS has two main tiers: the "passive" system of voluntary notifications and the anomaly registers, but many reporting sources within these. It receives about 7000 notifications a year. It is inflexible and has variable data quality. The voluntary nature of reporting affects the system's acceptability. The sensitivity as compared with two regional registers (Trent and Wales) is about 33%. The congenital anomaly registers reporting to the NCAS achieve high levels of coverage and completeness. From 2003, they cover 42% of all births and account for the major proportion of the notifications. CONCLUSIONS: The NCAS serves the important function of monitoring birth defects in England and Wales, but is not currently operating in a timely or effective way. It should be adapted to meet its main objectives more effectively. More regional anomaly registers should be instituted and existing registers supported through central funds.

Primary electron-transfer dynamics in 2-phenylindole-9-cyanoanthracene system. A comparative study with 2-methylindole.

Electrochemical measurements by cyclic voltammetry predict the possibility of occurrence of photoinduced electron-transfer (PET) reactions between the ground state of 2-phenylindole (2PI) (electron donor) and the excited singlet of 9-cyanoanthracene (9CNA) molecule acting as an electron acceptor. However, 2PI should be expected to behave as a relatively weaker electron donating agent than the structurally related donor 2-methylindole (2MI) as it possesses higher oxidation potential value. Both steady-state and time-resolved spectroscopic measurements in the polar acetonitrile (ACN) and ethanol (EtOH) solvents show that the fluorescence quenching phenomenon of 9CNA in presence of 2PI is primarily due to the involvement of dynamic process which in high probability should be PET. Nevertheless, in less polar tetrahydrofuran (THF) medium, the quenching of 9CNA results from the combined effect of dynamic and static modes. The transient absorption spectra, measured by using nanosecond laser flash photolysis, of 9CNA in presence of 2PI exhibit the signature of the bands of the anionic species of 9CNA, cation of the donor 2PI and the contact neutral radical. Observations of the transient absorption at the different delays infer that ion-recombination mechanism is responsible for production of the monomeric triplets of both 9CNA and 2PI. From the transient absorption decays in ACN medium, it has been demonstrated that the diffusional separation of ions from geminate ion-pair is facilitated in the case of 2MI-9CNA pair whereas for 2PI-9CNA system the energy wasting charge recombination dominates over the process of charge dissociation. From the above observations, the possibility of developing much potential photosynthetic model compounds with the donor 2MI, rather than with the other donor 2PI molecule has been hinted.

Concentration-dependent surface-enhanced resonance Raman scattering of a porphyrin derivative adsorbed on colloidal silver particles.

Surface-enhanced Raman spectra (SERS) of 5,10,15,20-tetrakis(1-decylpyridium-4-yl)-21H,23H-porphintetrabromide or Por 10 (H(2)Tdpyp) adsorbed on silver hydrosols are compared with the FTIR and resonance Raman spectrum (RRS) in the bulk and in solution. Comparative analysis of the RR and the FTIR spectra indicate that the molecule, in its free state, has D(2h) symmetry rather than C(2v). The SERS spectra, obtained on adsorption of this molecule on borohydride-reduced silver sol, indicate the formation of silver porphyrin. With the change in the adsorbate concentration, the SERS shows that the molecule changes its orientation on the colloidal silver surface. The appearance of longer wavelength band in the electronic absorption spectra of the sol has been attributed to the coagulation of colloidal silver particles in the sol. The long wavelength band is found to be red-shifted with the decrease in adsorbate concentration. The excitation profile study indicates that the resonance of the Raman excitation radiation with the original sol band is more important than that with the new aggregation band for the SERS activity. This indicates a large contribution of electromagnetic effect to surface enhancement.

Non-radiative depletion of the excited electronic states of 9-cyanoanthracene in presence of tetrahydronaphthols.

Both steady state and time resolved spectroscopic measurements reveal that the prime process involved in quenching mechanism of the lowest excited singlet (S1) and triplet (T1) states of the well known electron acceptor 9-Cyanoanthracene (9CNA) in presence of 5,6,7,8-tetrahydro-1-naphthol (TH1N) or 5,6,7,8-tetrahydro-2-naphthol (TH2N) is H-bonding interaction. It has been confirmed that the fluorescence of 9CNA is not at all affected in presence of 5,6,7,8-tetrahydro-2-methoxy naphthalene (TH2MN) both in non-polar n-heptane (NH) and highly polar acetonitrile (ACN) media. This indicates that the H-bonding interaction is crucial for the occurrence of the quenching phenomenon observed in the present investigations with TH1N (or TH2N) donors and 9CNA acceptor. In ACN solvent both contact ion-pair (CIP) and solvent-separated (or dissociated) ions are formed due to intermolecular H-bonding interactions in the excited electronic states (both singlet and triplet). In NH environment due to stronger H-bonding interactions, the large proton shift within excited charge transfer (CT) or ion-pair complex, 1 or 3(D+-H...A-), causes the formation of the neutral radical, 3(D+H-A)*, due to the complete detachment of the H-atom. It is hinted that both TH1N and TH2N due to their excellent H-bonding ability could be used as antioxidants.

Nature of non-radiative processes involved in the excited state of 9-cyanoanthracene in presence of 2-methylindole/2-methylindoline quenchers. A laser flash photolysis study to reveal the medium effects.

By using steady state and time-resolved (laser flash photolysis and single photon counting) spectroscopic techniques the quenching of the lowest excited singlet (S1) state of 9-cyanoanthracene (9CNA) by the donors (quenchers) 2-methylindole (2MI) and 2-methylindoline (2MIN) in solvents of different polarity has been studied. Both the transient absorption, by laser flash photolysis technique, and photobleaching measurements were made at the ambient temperature both in non-polar n-heptane (NH) and highly polar acetonitrile (ACN) solvents. The photobleaching efficiency (alpha) was found to depend significantly on the polarity of surrounding solvents and also on the molecular structures of the quenchers. In NH the values of alpha are found to be larger than the corresponding values observed in ACN for both 2MI and 2MIN which possess highly reactive H atom bound to the heterocyclic N atom. Following the results obtained from the transient absorption spectra of the present donor-acceptor molecules in the different polarity solvents, a scheme describing the overall reaction mechanisms of the different photoreactions involved has been proposed. The probable causes for the changes observed in the mechanisms of the photoreactions involved in the cases of 2MI and 2MIN donors have been discussed in the light of their canonical structures.

Surface-Enhanced Raman Scattering of Rhodamine 123 in Silver Hydrosols and in Langmuir-Blodgett Films on Silver Islands.

Surface-enhanced Raman scattering(SERS) of the Rhodamine 123 (Rh 123) molecule on ion-induced silver colloids has been studied. A time-dependent study of the SER spectra at a particular pH confirms charge transfer interaction between the probe molecule and the metal. The SER spectra of Rh 123 in Ag sol is compared with that of the molecules organized in a monolayer on silver island films by the Langmuir-Blodgett (LB) technique. The origin of high SERS activity of Rh 123 molecules in a monolayer on a silver island film is shown to be due to physisorption whereas in the ion-induced colloidal SERS both physisorption and chemisorption machanisms are involved. From these results, the contribution of charge transfer interaction to SERS in Ag sol has been estimated. In monolayer SERS, all the in-plane and out-of-plane (of xanthene ring) modes are more or less equally enhanced. This indicates that the xanthene plane of Rh 123 molecule organized in a LB film is oriented neither flat nor perpendicular to the silver island surface but is tilted. Copyright 2001 Academic Press.

Constituents of Asteracantha longifolia.

The isolation of two aliphatic esters and betulin from the aerial parts of Asteracantha longifolia is reported.

Mg2+-dependent ATPase activity in cardiac myofibrils from the insulin-resistant JCR:LA-cp rat.

There is a great deal of information presently available documenting a cardiomyopathic condition in insulin-deficient models of diabetes. Less information is available documenting a similar status in non insulin-dependent models of diabetes. We have studied the functional integrity of the myofibrils isolated from hearts of JCR:LA rats. The JCR:LA rat is hyperinsulinemic, hyperlipidemic, glucose intolerant and obese. As such, it carries many of the characteristics found in humans with non insulin-dependent diabetes mellitus. These animals also have many indications of heart disease. However, it is not clear if the hearts suffer from vascular complications or are cardiomyopathic in nature. We examined Mg2+-dependent myofibrillar ATPase in hearts of JCR:LA-cp/cp rats and their corresponding control animals (+/?) and found no significant differences (P> 0.05). This is in striking contrast to the depression in this activity exhibited by cardiac myofibrils isolated from insulin-deficient models of diabetes. Our data demonstrate that myofibrillar functional integrity is normal in JCR:LA-cp rats and suggest that these hearts are not in a cardiomyopathic state. Insulin status may be critical in generating a cardiomyopathic condition in diabetes.

Synthesis and studies on spectroscopic as well as electron donating properties of the two alkoxy benzo[b]thiophenes.

Synthesis, characterization, steady state and time resolved, using time correlated single photon counting as well as laser flash photolysis techniques, spectroscopic investigations were made for two alkoxy benzo[b]thiophene molecules: 5-methoxy benzo[b]thiophene (5MBT) and 5-methoxymethyl benzo[b]thiophene (5MMBT). In both non-polar n-heptane (NH) and polar acetonitrile (ACN) solvents and at ambient temperature the electronic absorption spectra of these thiophenes exhibit different band systems whose assignments were made from the measurements of the steady state excitation polarization spectra. Steady state fluorescence spectra of these molecules in the different polarity solvents show the presence of non-specific interactions. From the redox properties of the benzothiophenes, measured by cyclic voltammetry, their electron donating properties were observed in the presence of the well-known electron acceptor 9cyanoanthracene (9CNA). Further, detailed studies by laser flash photolysis techniques show that ion-recombination mechanism predominates after the initial excitation of the acceptor moiety using the third harmonic of Nd:YAG laser. This recombination together with the external heavy atom effect (the donor containing 'sulphur' atom) appears to be responsible for the formation of the triplet of the monomeric acceptor 9CNA. From the steady state experiments it is shown that both in non-polar NH and highly polar ACN the quenching in the fluorescence emission of 9CNA in the presence of the benzothiophene donors is brought about primarily by the external heavy atom effect and in ACN, although the presence of the photoinduced ET reaction is confirmed, this process seems, from the observed bimolecular dynamic quenching rate, kq to be significantly masked by the external heavy atom effect.

Surface enhanced Raman scattering of 2,2' biquinoline adsorbed on colloidal silver particles.

Surface enhanced Raman scattering (SERS) in silver sol and normal Raman spectra in the bulk and in solution of 2,2' biquinoline (BQ) molecule have been investigated. The observed Raman bands along with their corresponding FTIR bands have been assigned based on the established assignments of the vibrational bands of the parent napthalene and quinoline molecules. Existence of both the cis and trans form of the BQ molecule in solution and in the bulk are inferred from the normal Raman and FTIR spectra, whereas SERS study reveal that in the surface adsorbed state the molecule exists in the cis form. Definite evidence of the charge transfer interaction to the overall contribution in the SER enhancement have been reported. The excitation profile also supports the CT interaction. Estimated enhancement factor of the principal SERS bands indicate that the molecule is adsorbed on the silver surface through both the nitrogen atoms with the molecular plane almost perpendicular to the surface. This preferred orientation of the molecule is in conformity with its existence in the cis form in the surface adsorbed state.

SarA represses agr operon expression in a purified in vitro Staphylococcus aureus transcription system.

Mutation and genetic complementation studies suggested that two chromosomal loci, agr and sar, are involved in the upregulation of several exotoxin genes and the downregulation of a number of surface protein genes in a growth phase-dependent manner in Staphylococcus aureus. We purified recombinant T7-tagged SarA from Escherichia coli and determined its effect on transcription from several S. aureus promoters by using purified RNA polymerase reconstituted with either sigma(A) or sigma(B) from S. aureus. Of the seven sigma(A)-dependent promoters that we tested, SarA repressed transcription from agrP2, agrP3, cna, sarP1, and sea promoters and did not affect sec and znt promoters. Furthermore, SarA had no effect on transcription from the sigma(B)-dependent sarP3 promoter. In vitro experimental data presented in this report suggest that SarA expression is autoregulated.

Energy transfer in triton-X 100 micelles: a fluorescence study.

The study of fluorescence energy transfer from the phenyl groups of the micellar triton X-100 (TX-100) to solubilised 1-pyrene butyric acid (PBA) has been carried out. Through the analysis of the donor fluorescence quenching energy transfer efficiency has been determined. The observed donor-acceptor separation suggests that pyrene molecules are distributed uniformly in the micellar core.

Characterization of a single-strand origin, ssoU, required for broad host range replication of rolling-circle plasmids.

Single-stranded DNA (ssDNA) promoters are the key components of the single-strand origins (ssos) of replication of rolling-circle (RC) replicating plasmids. The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging-strand synthesis. This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts. A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram-positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging-strand synthesis in vivo from ssoU-containing plasmids. Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram-positive bacteria, S. aureus and B. subtilis. We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology. Our data support the notion that the strength of the RNA polymerase-ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.

ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.

Cardiac myofibrillar and sarcoplasmic reticulum function are not depressed in insulin-resistant JCR:LA-cp rats.

Depressed myofibrillar Ca2+-ATPase activity and sarcoplasmic reticulum (SR) Ca2+ uptake are important mechanisms that are responsible for the cardiac dysfunction exhibited by insulin-deficient (type I) diabetic animals. The JCR:LA-cp rat is a model for type II non-insulin-dependent diabetes mellitus (NIDDM). This rat is insulin resistant, obese, and has high levels of circulating glucose, cholesterol, insulin, and triglycerides. The purpose of this study was to determine whether changes in cardiac myofibrillar, SR, and cardiomyocyte function exist in this model of type II diabetes. Myofibrils and SR were isolated from hearts by differential centrifugation. Surprisingly, we found that myofibrillar Ca2+-ATPase activities were unaltered in these animals. Ca2+ uptake in isolated SR fractions was increased in diabetic cp/cp rats, whereas Ca2+-ATPase activity and ryanodine binding were unchanged. Cardiomyocytes isolated from hearts of control and experimental animals had similar active cell shortening and intracellular Ca2+ concentration under basal conditions and in response to caffeine. Our data argue against the presence of a cardiomyopathy in this diabetic model and suggest that insulin may be an important factor in the cardiomyopathy observed in type I diabetic models.

Lagging strand replication of rolling-circle plasmids: specific recognition of the ssoA-type origins in different gram-positive bacteria.

Many bacterial plasmids replicate by a rolling-circle mechanism that involves the generation of single-stranded DNA (ssDNA) intermediates. Replication of the lagging strand of such plasmids initiates from their single strand origin (sso). Many different types of ssos have been identified. One group of ssos, termed ssoA, which have conserved sequence and structural features, function efficiently only in their natural hosts in vivo. To study the host specificity of sso sequences, we have analyzed the functions of two closely related ssoAs belonging to the staphylococcal plasmid pE194 and the streptococcal plasmid pLS1 in Staphylococcus aureus. The pLS1 ssoA functioned poorly in vivo in S. aureus as evidenced by accumulation of high levels of ssDNA but supported efficient replication in vitro in staphylococcal extracts. These results suggest that one or more host factors that are present in sufficient quantities in S. aureus cell-free extracts may be limiting in vivo. Mapping of the initiation points of lagging strand synthesis in vivo and in vitro showed that DNA synthesis initiates from specific sites within the pLS1 ssoA. These results demonstrate that specific initiation of replication can occur from the pLS1 ssoA in S. aureus although it plays a minimal role in lagging strand synthesis in vivo. Therefore, the poor functionality of the pLS1 in vivo in a nonnative host is caused by the low efficiency rather than a lack of specificity of the initiation process. We also have identified ssDNA promoters and mapped the primer RNAs synthesized by the S. aureus and Bacillus subtilis RNA polymerases from the pE194 and pLS1 ssoAs. The S. aureus RNA polymerase bound more efficiently to the native pE194 ssoA as compared with the pLS1 ssoA, suggesting that the strength of RNA polymerase-ssoA interaction may play a major role in the functionality of the ssoA sequences in Gram-positive bacteria.

Alternative transcription factor sigmaSB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar.

A homolog of the multiple-stress-responsive transcription factor sigmaB of Bacillus subtilis was predicted from the DNA sequence analysis of a region of the Staphylococcus aureus chromosome. A hybrid between the coding sequence of the first 11 amino acids of the gene 10 leader peptide of phage T7 (T7.Tag) and the putative sigB gene of S. aureus was constructed and cloned into Escherichia coli BL21(DE3)pLysS for overexpression from a T7 promoter. A homogeneous preparation of the overproduced protein was obtained by affinity chromatography with a T7.Tag monoclonal antibody coupled to agarose. The amino-terminal amino acid sequence of the first 22 residues of the purified protein matched that deduced from the nucleotide sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein, designated sigmaSB, indicated that it migrated as an approximately 39-kDa polypeptide. Promoter-specific transcription from the B. subtilis sigmaB-dependent PB promoter of the sigB operon was stimulated by sigmaSB in a concentration-dependent fashion when reconstituted with the S. aureus core RNA polymerase (RNAP). Specific transcript from the predicted sigmaB-dependent PB promoter of the sigB operon of S. aureus was obtained by the reconstituted RNAP in a runoff transcription reaction. The sar operon of S. aureus contains three promoter elements (P1, P2, and P3) and is known to partly control the synthesis of a number of extracellular toxins and several cell wall proteins. Our in vitro studies revealed that transcription from the P1 promoter is dependent on the primary sigma factor sigmaSA, while that of the P3 promoter is dependent on sigmaSB. As determined by primer extension studies, the 5' end of the sigmaSB-initiated mRNA synthesized in vitro from the sar P3 promoter is in agreement with the 5' end of the cellular RNA.

Characterization of the primary sigma factor of Staphylococcus aureus.

RNA polymerase (RNAP) isolated from Staphylococcus aureus is deficient in sigma factor and is poorly active in transcription assays. Based on amino acid sequence homology of the Bacillus subtilis vegetative sigma factor sigmaA and the predicted product of the chromosomally located plaC gene of S. aureus, it was hypothesized that plaC could encode the vegetative sigma factor. We cloned plaC under a T7 promoter and overexpressed it in Escherichia coli strain BL21(DE3)pLysE. The overproduced protein, present in inclusion bodies, was solubilized with guanidine hydrochloride, renatured, and purified by DEAE-Sephacel and Sephadex G-75 chromatography. The purified protein, designated sigmaSA, cross-reacted with the B. subtilis anti-sigmaA antibody. E. coli core RNAP, reconstituted with sigmaSA, initiated promoter-specific transcription from the S. aureus promoters hla, sea, and sec and from the E. coli promoters rpoH P1, rpoH P4, and ColE1 RNA-1, which are recognized by the E. coli sigma70. sigmaSA, when added to the purified RNAP from S. aureus, stimulated transcriptional activity of the RNAP up to 72-fold. As determined by primer extension studies, the 5'-ends of the sigmaSA-initiated mRNAs synthesized in vitro from the agr P2 and sea promoters are in general agreement with the 5'-ends of the cellular RNAs. Disruption of the plaC gene on the S. aureus chromosome was lethal. We conclude that plaC encodes the primary sigma factor in S. aureus.