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Biomedical subjects

D Saxena

Publications and source records attributed to D Saxena.

At least 19 recordsLinked to original sources

Diversity of lactobacilli in the oral cavities of young women with dental caries.

For nearly a century, lactobacilli (LB) in the oral cavity have been generally associated with dental caries. Here, we characterized the LB isolated from the saliva of 6 women with active caries using genetic-based taxonomical identification methods. From each subject, 30 isolates growing on Rogosa medium and presumed to be LB were analyzed. Of the 180 isolates, 176 were further characterized by biotyping, DNA melting points, DNA chromosomal fingerprinting, genotyping, and phylogenetic cluster assessment. We found a total of 30 unique genotypes of LB in the saliva of caries-active women, with each woman harboring between 2 and 8 distinct genotypes. Although Lactobacillus vaginalis, L. fermentum, and L. salivarius were found in 4 of 6 of the subjects, results from other studies using comparable methods show an entirely different array of LB associated with caries. These collective observations lead us to surmise that LB associated with dental caries are likely exogenous and opportunistic colonizers, arising from food or other reservoirs outside the oral cavity.

Adolescent↗

Genetic profiling of the oral microbiota associated with severe early-childhood caries.

The determination of the composition of the microbial community in the oral cavity is usually based on cultivation methods; however, nearly half of the bacteria in the saliva and the dental plaque are not cultivable. In this study, we evaluated the difference in oral microbial diversity between children with severe early-childhood caries (S-ECC) and caries-free (CF) controls by means of a cultivation-independent approach called denaturing gradient gel electrophoresis (DGGE). Pooled dental plaque samples were collected from 20 children aged 2 to 8 years. Total microbial genomic DNA was isolated from those subjects, and a portion of the 16S rRNA gene locus was PCR amplified by using universal primers. We observed that the mean species richness of the bacterial population was greater in the CF children (n = 12) (42 +/- 3.7) than in the S-ECC children (n = 8) (35 +/- 4.3); the difference was statistically significant (P = 0.005). The overall diversity of plaque samples as measured by the Shannon index was 3.5 for the S-ECC group and 3.7 for the CF group (P = 0.004). Differences in DGGE profiles were distinguished on the basis of a cluster analysis. Sequence analysis of excised DGGE bands consisted of 2.7 phylotypes, on average. After adjusting for the number of observed bands, we estimated that the S-ECC group exhibited 94.5 total phylotypes and that the CF group exhibited 113.4. These results suggest that the microbial diversity and complexity of the microbial biota in dental plaque are significantly less in S-ECC children than in CF children.

Bacterial Typing Techniques↗

Polymerase chain reaction-based denaturing gradient gel electrophoresis in the evaluation of oral microbiota.

BACKGROUND/AIMS: Clinical evaluation of oral microbial reduction after a standard prophylactic treatment has traditionally been based on bacterial cultivation methods. However, not all microbes in saliva or dental plaque can be cultivated. Polymerase chain reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) is a cultivation-independent molecular fingerprinting technique that allows the assessment of the predominant bacterial species present in the oral cavity. This study sought to evaluate the oral microbial changes that occurred after a standard prophylactic treatment with a conventional oral care product using PCR-DGGE. METHODS: Twelve healthy adults participated in the study. Pooled plaque samples were collected at baseline, 24 h after prophylaxis (T1), and 4 days after toothbrushing with fluoride toothpaste (T4). The total microbial genomic DNA of the plaque was isolated. PCR was performed with a set of universal bacterial 16S rDNA primers. The PCR-amplified 16S rDNA fragments were separated by DGGE. The effects of the treatment and of dental brushing were assessed by comparing the PCR-DGGE fingerprinting profiles. RESULTS: The mean numbers of detected PCR amplicons were 22.3 +/- 6.1 for the baseline group, 13.0 +/- 3.1 for the T1 group, and 13.5 +/- 4.3 for the T4 group; the differences among the three groups were statistically significant (P < 0.01). The study also found a significant difference in the mean similarities of microbial profiles between the baseline and the treatment groups (P < 0.001). CONCLUSION: PCR-based DGGE has been shown to be an excellent means of rapidly and accurately assessing oral microbial changes in this clinical study.

Adult↗

Survey of oral microbial diversity using PCR-based denaturing gradient gel electrophoresis.

Polymicrobial biofilms in the human oral cavity exhibit marked diversity. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) surveys microbial diversity by displaying PCR-generated 16S rDNA fragments that migrate at different distances, reflecting the differences in the base-pair (i.e., % G+C) composition of the fragment. This study examined DGGE-generated diversity profiles of cultivable bacteria from individuals with different caries status. Initially, we developed a set of PCR-DGGE running conditions appropriate to oral bacteria. Next, we assessed migration standards from known oral bacterial reference strains. To test the methods, we profiled 20 bacterial saliva samples cultivated from young adults. The study produced a battery of species-specific 16S rDNA amplicons that could be used as a migration distance standard necessary for computer-assisted profile analysis. From the clinical samples, we found a significantly greater diversity of oral microbes in caries-free individuals compared with caries-active individuals (P = 0.01). These findings suggest thtat a portion of oral microbiota of caries-active individuals may be absent, suppressed, or replaced.

Actinomyces↗

HPLC and HPTLC assays for the antimalarial agents chloroquine, primaquine and bulaquine.

A combination Kit for antirelapse treatment of P. vivax malaria, consisting of Chloroquine phosphate tablets and Bulaquine capsules has been recently developed, and marketed under the trade name Aablaquine. Bulaquine is prepared from Primaquine. Several methods of analysis are reported for each drug separately as well two drugs in combination but no method for simultaneous estimation of these three drugs is known. Therefore, the present study was undertaken to develop a sensitive and reproducible high performance liquid chromatographic as well as high performance thin layer chromatographic assay method for the simultaneous estimation of Chloroquine, Primaquine and Bulaquine.

Antimalarials↗

Assay method for quality control and stability studies of a new CVS disorder agent (compound 93/478).

1-[4-(4-fluorophenyl)-piperazine-1-yl]-3-(2-oxopyrrolidin-1-yl)-propane hydrochloride, (I), (CDRI code No. 93/478) is a new potent anti-ischemic and anti-hypertensive agent, being developed at the Central Drug Research Institute (CDRI), Lucknow, India. A sensitive high performance liquid chromatographic assay method has been developed and validated for in process quality control and for stability studies. HPLC separation was achieved on a C(18) Purospher (Merck) column using a gradient of 0.02% tetra-methyl ammonium hydroxide (pH 7.5) and acetonitrile as mobile phase. The eluents were monitored by diode array detector at 240 and 290 nm. The lower limit of detection of I was 0.62 microg/ml, while the lower limit of quantitation was set to be 1.5 microg/ml. The calibration curves were linear in the range 1.5-62 microg/ml. Reproducibility of the method was determined by inter and intra assay variation, which were <10%.

Calibration↗

Effect of accessory proteins P19 and P20 on cytolytic activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis in Escherichia coli.

The gene coding for the accessory protein P19 of Bacillus thuringiensis subsp. israelensis was expressed in Escherichia coli and its product was characterized. To investigate its putative role in delta-endotoxin crystallization as a P20-like polypeptide, each of the two encoding genes, p20 and p19, was cloned for inducible expression coordinatively with cyt1Aa. The latter is known to kill its transgenic host. P20 but not P19 stabilized Cyt1Aa and protected the host cells from its lethal effect. Neither GroEL nor GroES, expressed in trans, affected Cyt1Aa as did P20. The function of P20 is thus more specific than that of the chaperones, but that of P19 remains enigmatic. The correct sequence of p19, confirmed in all five isolates of B. thuringiensis subsp. israelensis, does not explain the slow electrophoretic mobility of its 179 amino acids product.

Amino Acid Sequence↗

Inhalable microparticles containing drug combinations to target alveolar macrophages for treatment of pulmonary tuberculosis.

PURPOSE: Drug therapy of tuberculosis (TB) requires long-term oral administration of multiple drugs for curing as well as preventing and/ or combating multi-drug resistance. Persistent, high blood levels of antitubercular drugs resulting from prolonged oral administration of anti-TB drugs may be neither necessary nor sufficient to kill mycobacteria residing in macrophages (M4). Inhalable biodegradable microparticles containing two of the first-line anti-TB drugs, isoniazid (H), and rifampicin (R), were prepared and tested for (i) phagocytosis by mouse Mphi. (ii) administration as a dry powder inhalation to rats, and (iii) targeting alveolar Mphi with high drug doses when administered to rats. METHODS: poly(D-L lactic acid) microparticles were prepared by emulsion methods and their drug content and size distribution determined. These were tested for uptake by murine Mphi in culture and resultant intracellular drug concentrations determined by high performance thin-layer chromatography (HPTLC). Rats were administered an inhalation of microparticles using an inhalation chamber developed in the lab. The extent of microparticle delivery in vivo was examined by flow-cytometry. Drug concentrations in the blood and in alveolar Mphi were estimated by high-performance liquid chromatography after oral, vascular. intratracheal, and inhalation administration. RESULTS: Inhalable microparticles could be prepared and were taken up by cultured Mphi. Large numbers of particles could be delivered to the bronchiopulmonary system through a 2-min exposure to fluidized particles. The intracellular drug concentrations resulting from vascular delivery of soluble drugs were found to be lower than those resulting from particle inhalation. CONCLUSIONS: Inhalable microparticles containing multiple anti-TB drugs offer promises of dose and dosing-frequency reduction, toxicity alleviation, and targeting Mphi-resident persistent mycobacteria.

Administration, Inhalation↗

Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.

Base Sequence↗

Removal of copper by Pseudomonas putida strain S4 isolated from copper mines.

A bacterial strain, Pseudomonas putida S4, was isolated from smelter drainage of copper mines. The strain exhibited resistance to several heavy metals, like aluminium (Al), zinc (Zn), nickel (Ni), cobalt (Co) besides copper (Cu). Strain S4 could accumulate Cu from the Cu-supplemented growth medium. In the present study, we have demonstrated the Cu2+ removal capacity of this strain from various samples such as mine effluent, low-grade ore and ore-tailings, collected from the mining site. Moreover, approximately 80% of the accumulated Cu2+ could be recovered from the loaded biomass by a simple desorption procedure.

Biodegradation, Environmental↗

Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ.

The insecticidal toxin encoded by the cry1Ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a plant growth room and from plants grown to maturity in the field. The presence of the toxin in rhizosphere soil was determined by immunological and larvicidal assays. No toxin was detected in any soils from isogenic non-Bt corn or without plants. Persistence of the toxin was apparently the result of its binding on surface-active particles in the soils, which reduced the biodegradation of the toxin. The release of the toxin could enhance the control of insect pests or constitute a hazard to nontarget organisms, including the microbiota of soil, and increase the selection of toxin-resistant target insects.

Journal Article↗

NADPH dependent superoxide generation in the ovary and uterus of mice during estrous cycle and early pregnancy.

NADPH dependent O2- production in the ovary and uterus of cycling and pregnant mice was estimated employing a novel technique of time-lapse electron spin resonance enhancement of a superoxide-trapping spin trap. The NADPH dependent O2- generation in the mouse ovary increased during the early pre-ovulatory phase in cycling females and during extended luteal phase in pregnant animals. A peak of uterine NADPH dependent O2- production at proestrous in the cycling animals at pre-implantation phase in pregnant animals suggested a contribution of this enzyme towards generating high levels of superoxide anion radical during the respective stages. Both ovarian and uterine NADPH dependent O2- production appeared to be LH-inducible.

Animals↗

Altered molecular dynamics and antioxidant status in the spermatozoa in testosterone-induced oligospermia in mouse.

Though supraphysiological doses testosterone (T) and its derivatives are known to suppress spermatogenesis in mammals by interfering with the hypothalamus-pituitary axis leading to oligozoospermia, no study has been performed to evaluate the integrity of the sperm cells produced by such individuals. In T-induced oligozoospermia in the mouse, the spermatozoa showed suppressed zona-binding ability though the motility and viability remained unchanged. In order to assess whether this decreased zona-binding ability is due to perturbations in the mechanical properties of the sperm membranes, we attempted to examine the molecular dynamics employing a lipophilic spin label (16-doxyl stearate) and a protein-binding label (Mal-Net) in two sets of independent experiments. The results showed that the rotational freedom of lipophilic molecules reduced significantly within the first week of T-treatment. During weeks 1 through 4, the protein rotation was found to be retarded significantly. We observed a sharp increase in the ascorbyl radical associated with the cauda epididymal spermatozoa and epididymal fluid of testosterone-treated mice. Moreover, the glutathione (GSH) content in the spermatozoa and the epididymal fluid increased significantly after testosterone-treatment. Further, there was a elevation in the superoxide dismutase (SOD) activity and suppression in the superoxide anion radical generated by the cauda epididymal spermatozoa of testosterone-treated animals. A change in the mechanical properties of a bilayer could modify both the mechanical properties and the function of incorporated proteins. In many instances, a liquid-crystalline bilayer is necessary for protein function. It is likely that the change in the physical properties of sperm membranes might cause the inhibition of enzymes associated with spermatozoa after T-treatment. The alterations in the sperm membrane structure and the antioxidant potentials of both the spermatozoa and the cauda epididymal fluid could also account for the decrease in the zona-binding index of the spermatozoa in T-treated animals. Thus, this study demonstrates for the first time that supraphysiological doses of testosterone could modify the mechano-dynamic properties of sperm membranes and could perturb the redox status of both spermatozoa and the epididymal fluid.

Animals↗

Increased appearance of inducible nitric oxide synthase in the uterus and embryo at implantation.

The aim of this study was to investigate the presence of iNOS in the murine uterus and embryo at implantation. Western blot analysis showed the presence of a 130-kDa band with strong reactivity to anti-iNOS antibody in the pre- and peri-implantation stage uteri. This band was faint in the postimplantation uteri. Immunocytochemical studies showed a heavy localization of iNOS specifically on the apical cells of the uterine endometrium in the pre- and peri-implantation stages. But the postimplantation uteri showed resorbed endometrium showing weaker expression of iNOS. The iNOS was induced by estrogen and the induction was intensified when progesterone was given along with estrogen. This truly mimics the in vivo situation since implantation in mice occurs when an estrogen surge occurs on a background of progesterone. The embryos too express iNOS at the peri-implantation stage. We suggest that iNOS expressed at peri-implantation would lead to enhanced NO production, which could act as a vasodilator and an angiogenic mediator. These effects could promote the attachment of the blastocyst to the uterus.

Animals↗

Trends and determinants of immunisation coverage in India.

The 20th century has witnessed many important events in the control of infectious diseases that mostly affect children. In addition to the eradication of smallpox, the interruption of poliomyelitis transmission in many countries with a distinct possibility of its eradication by the turn of this century are some of the major achievements. Also, the rates of other vaccine preventable diseases such as measles, pertussis and diphtheria have gone down significantly. The discovery and use of vaccines have made it possible to save approximately 8 million deaths, annually. This is in addition to the reduction in millions of children's suffering and disability. It is now important to build on these gains through adequate utilisation of other vaccines e.g., hepatitis B, typhoid and Haemophilus influenzae type b that are currently available, but in limited use. But, a high level of coverage for any vaccination programme is a pre-requisite to witness the effective reduction of the specific disease against which child population is vaccinated. This paper reviews the coverage levels by surveys in the last 3 years. It has been observed that vaccination coverage levels are falling. Keeping the promises of immunising every child to fulfill his/her right is the need of the hour. To achieve this the major action points are: (a) The need for organising fixed immunisation sessions at the community, where low proportion of sessions are held; and (b) The need to improve demand generation activities where the coverage is poor despite better service availability at the community level. Therefore, the challenge for the next century is to make sure that the enormous impact of vaccines on the health and well-being of the population is maintained as well as expanded. Vaccines that effectively prevent rotavirus diarrhoea, pneumococcal pneumonia, menigococcal meningitis, if made available, could prevent deaths up to two million a year. Research efforts are currently under progress to develop new vaccines against malaria, tuberculosis, shigella-induced dysentery, and Esch coli-induced diarrhoea.

Child, Preschool↗

Effect of estradiol and selected antiestrogens on pro- and antioxidant pathways in mammalian uterus.

In this study, we examined the effect of 17 beta-estradiol and selected antiestrogens on uterine NADPH-oxidase activity, superoxide dismutase (SOD) activity, hydride (H.-), dienyl radical and O2 -radical generation, and membrane fluidity. NADPH oxidase activity was positively modulated in estradiol-treated animals and negatively regulated in animals that received injections of AF-45, RU-39411, tamoxifen, or ICI-182780. The SOD activity was markedly reduced in estradiol-treated animals when compared with the control animals. A positive modulation of SOD activity was observed upon treatment with AF45, RU39411, tamoxifen, and ICI 182780, though the potency varied among the individual test compounds. We observed detectable H(.-)-radical generation as evidenced from MNP H.- adduct formation in the uterine cell preparations from untreated control animals. Estradiol produced a tremendous augmentation in the superoxide radical profiles in uterine cell preparations compared to the control levels. All the other compounds that were tested significantly lowered the superoxide levels in the test set-up. AF-45, RU-39411, tamoxifen, and ICI-182780 induced varying orders of suppression of H(.-)-radical generation in the test subjects. There was a significant enhancement in membrane fluidity, hydride radical levels, and dienyl radical generation in the estradiol-treated group. All the antiestrogens did not exhibit a similar action on these parameters. RU-39411 exhibited antiestrogen-like activity in modulating hydride levels and membrane fluidity, whereas it stimulated dienyl radical generation. Thus our tests showed that the selected antiestrogens failed to show estrogen-like activity in these assays. It appears that estradiol exerts feedback control over pro- and antioxidant pathways and that markers of oxidative status could be used as a measure to evaluate the antiestrogenic activity of estradiol agonists/antagonists.

Animals↗