Morphometric and morphological appraisal of the eggs of Anopheles stephensi (Diptera: Culicidae) from India.

BACKGROUND & OBJECTIVES: Anopheles stephensi is one of the most important urban malaria vectors in India and contribute about 12% of total malaria cases. An. stephensi has three ecological variants; type, intermediate and mysorensis that can be differentiated on the basis of differences in number of ridges on egg float and on the basis of spiracular indices. Because of its anthropophilic nature the 'type' form is an efficient malaria vector. In the present study, the egg surface morphometry and morphology of An. stephensi 'type' form was studied and detail distinguish- ing characters were recorded for its correct identification. METHODS: Eggs of An. stephensi 'type' form were studied by scanning electron microscopy (SEM) after sputter- coating with gold. In total 23 egg characters were analysed morphologically and morphometrically, which included egg attributes, deck attributes, ventral tubercles, micropyle and float attributes. RESULTS: The dorsal surface of the egg of 'type' form was curved while the ventral surface was concave and both anterior and posterior ends were blunt. The average length and width of egg was 473.94 + 11.18 and 154.69 + 2.66 µm respectively. The number of float ribs observed was 20.33 ± 0.33. The maximum length of float was found to be 246.57 + 15.27 µm, whereas maximum width was 87.16 + 3.83 µm. INTERPRETATION & CONCLUSION: The present study has generated some important data which is specific to An. Stephensi 'type' form and provided significant morphological and morphometric standards for its correct identification. This information could be useful in differentiation of An. stephensi 'type' form from other ecological forms of the same species as well as other species of Anopheles.

Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology.

BACKGROUND: The malaria vector Anopheles culicifacies (sensu lato) is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E. However, only a few forms have been identified as malaria vectors in various endemic countries. Currently, for the first time egg morphometry and morphology has been used to differentiate the three known vector sibling species of Anopheles culicifacies collected from malaria endemic Madhya Pradesh state of central India. METHODS: The adult An. culicifacies (s.l.) was collected from five districts using standard mosquito collection methods. Adult female mosquitoes were allowed to lay eggs individually. The emerged mosquitoes were identified using allele specific polymerase chain reaction (AS-PCR) to sibling species. Eggs of sibling species A, D and E were studied using scanning electron microscopy (SEM) for morphometric and morphological characteristics. RESULTS: Currently AS-PCR identified four known sibling species (B, C, D and E) of An. culicifacies in the study area. The surface morphology and morphometric attributes of the sibling species A, D and E eggs considerably differed from each other. An. culicifacies E had a narrow deck as compared to A and D, while An. culicifacies A had a bigger micropyle with 6-7 sectors as compared to D and E that had 6 sectors. An. culicifacies D had the smallest float (the structure present on sides of the egg surface in which air is filled that help in floating) and the number of ribs was also fewer than for An. culicifacies A and E. CONCLUSIONS: The present study provides the first evidence that in addition to PCR assay, sibling species of An. culicifacies can also be differentiated using morphological and morphometric characteristics of the egg stage. The results also advocate that the sibling species of An. culicifacies are morphologically dissimilar and can be resolved using advanced microscopy.

Development and Validation of HPTLC Method for Estimation of Tenoxicam and its Formulations.

A simple, precise, accurate and rapid high performance thin layer chromatographic method has been developed and validated for the estimation of tenoxicam in the microemulsion gels. Tenoxicam was chromatographed on silica gel 60 F(254) TLC plate, as a stationary phase. The mobile phase was toluene: ethyl acetate: formic acid (6:4:0.3 v/v/v), which gave a dense and compact spot of tenoxicam with a R(f) value of 0.38±0.03. The quantification was carried out at 379 nm. The method was validated in terms of linearity, accuracy, precision and specificity. To justify the suitability, accuracy and precision of the proposed method, recovery studies were performed at three concentration levels. Statistical analysis proved that the proposed method is accurate and reproducible with linearity in the range of 100 to 400 ng. The limit of detection and limit of quantification for tenoxicam were 25 and 50 µg/spot, respectively. The proposed method can be employed for the routine analysis of tenoxicam as well as in pharmaceutical formulations.

Effect of calcinations temperature of CuO nanoparticle on the kinetics of decontamination and decontamination products of sulphur mustard.

Present study investigates the potential of CuO nanoparticles calcined at different temperature for the decontamination of persistent chemical warfare agent sulphur mustard (HD) at room temperature (30 ± 2 °C). Nanoparticles were synthesized by precipitation method and characterized by using SEM, EDAX, XRD, and Raman Spectroscopy. Synthesized nanoparticles were tested as destructive adsorbents for the degradation of HD. Reactions were monitored by GC-FID technique and the reaction products characterized by GC-MS. It was observed that the rate of degradation of HD decreases with the increase in calcination temperature and there is a change in the percentage of product of HD degradation. GC-MS data indicated that the elimination product increases with increase in calcination temperature whereas the hydrolysis product decreases.

Photocatalytic inactivation of spores of Bacillus anthracis using titania nanomaterials.

Studies on photocatalytic inactivation of spores of Bacillus anthracis have been carried out using nanosized titania materials and UVA light or sun light. Results demonstrated pseudo first order behaviour of spore inactivation kinetics. The value of kinetic rate constant increased from 0.4h(-1) to 1.4h(-1) indicating photocatalysis facilitated by addition of nanosized titania. Nanosized titania exhibited superior inactivation kinetics on par with large sized titania. The value of kinetic rate constant increased from 0.02 h(-1) to 0.26 h(-1) on reduction of size from 1000 nm to 16 nm depicting the enhanced rate of inactivation of Bacillus anthracis Sterne spores on the decrease of particle size.

Decontamination of Yperite using mesoporous mixed metal oxide nanocrystals.

Mixed metal oxide nanocrystals of AP-Al(2)O(3), AP-Al(2)O(3)-Fe(2)O(3), AP-Al(2)O(3)-V(2)O(5) and AP-Al(2)O(3)-CuO have been prepared by aerogel process. XRD data of prepared materials revealed the formation of nanocrystals with a size range of 3-15 nm diameters. N(2) BET investigations on these materials revealed larger values of surface area ranging from 350 to 540 m(2)/g. Reactivity of these nanocrystalline materials against Yperite was examined by gas chromatography, gas chromatography-mass spectrometry and infrared spectroscopy techniques. AP-Al(2)O(3)-Fe(2)O(3), AP-Al(2)O(3)-V(2)O(5) and AP-Al(2)O(3)-CuO nanocrystals exhibited superior decontamination properties against Yperite than AP-Al(2)O(3). The reactions exhibited pseudo first order behaviour. 100% of Yperite was found to be decontaminated on Al(2)O(3)-Fe(2)O(3), Al(2)O(3)-V(2)O(5) and Al(2)O(3)-CuO where only 75% of the same was found to be decontaminated on AP-Al(2)O(3) within 40 h.

Reactions of sulphur mustard and sarin on V 1.02 O 2.98 nanotubes.

Reactions of sulphur mustard and sarin were studied on the surface of V(1.02)O(2.98) nanotubes by gas chromatography and gas chromatography-mass spectrometry techniques. The V(1.02)O(2.98) nanotube samples were made by using hydrothermal method and characterized by scanning electron microscopy, nitrogen adsorption, X-ray diffractometry and thermogravimetry. Later, they were exposed to sulphur mustard and sarin separately at ambient temperature (30+/-2 degrees C). The data explored the formation of sulphoxide of sulphur mustard, thiodiglycol for sulphur mustard and isopropyl methyl phosphonic acid for sarin on V(1.02)O(2.98) nanotubes illustrating the role of oxidation and hydrolysis reactions in the decontamination.

Nanocrystalline zinc oxide for the decontamination of sarin.

Nanocrystalline zinc oxide materials were prepared by sol-gel method and were characterized by X-ray diffraction, scanning electron microscopy, thermogravimetry, nitrogen adsorption and infrared spectroscopy techniques. The data confirmed the formation of zinc oxide materials of zincite phase with an average crystallite size of approximately 55 nm. Obtained material was tested as destructive adsorbent for the decontamination of sarin and the reaction was followed by GC-NPD and GC-MS techniques. The reaction products were characterized by GC-MS and the data explored the role of hydrolysis reaction in the detoxification of sarin. Sarin was hydrolyzed to form surface bound non-toxic phosphonate on the surface of nano-zinc oxide. The data also revealed the values of rate constant and half-life to be 4.12h(-1) and 0.16 h in the initial stages of the reaction and 0.361 h(-1) and 1.9h at the final stages of the reaction for the decontamination reaction on nanocrystalline ZnO.

Antisense DNA-targeting protein kinase A-RIA subunit: a novel approach to cancer treatment.

Enhanced expression of the RIa subunit of cAMP-dependent protein kinase type I (PKA-I) has been shown during carcinogenesis, in human cancer cell lines and in primary tumors. We demonstrate that the sequence-specific inhibition of RIa gene expression by antisense oligonucleotides results in the differentiation of leukemia cells and growth arrest of cancer cells of epithelial origin and tumors in mice. The loss of RI by the antisense results in rapid increase in the half-life of the competitor molecule, RII protein, via its stabilization in a holoenzyme complex (PKA-II) that insures depletion of PKA-I and sustained inhibition of tumor growth. RI antisense, which restrains tumor cell growth by turning on the signals for blockade of tumor cell survival, namely blockade of the tyrosine kinase signaling, cell cycle deregulation and apoptosis, provides a single gene-targeting approach to treatment of cancer.

Synergistic effects of retinoic acid and 8-chloro-adenosine 3',5'-cyclic monophosphate on the regulation of retinoic acid receptor beta and apoptosis: involvement of mitochondria.

In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective, even at doses that are toxic to the host. In late stages of breast cancer, patients do not respond to RA because the expression of RA receptor beta (RARbeta) is lost. In the present study, the intracellular mechanism(s) of synergistic effects of RA and a site-selective cyclic AMP (cAMP) analogue, 8-chloro-adenosine 3',5'-cyclic monophosphate (8-Cl-cAMP), on growth inhibition and apoptosis in breast cancer cells was examined. Our data demonstrated that hormone-dependent MCF-7 cells, but not hormone-independent MDA-MB-231 cells, are sensitive to RA-induced growth inhibition and apoptosis. Introduction of the RARbeta gene into MDA-MB-231 cells resulted in a gain of RA sensitivity. 8-Cl-cAMP acted synergistically with all-trans-RA in inducing and activating RARbeta gene expression that correlates with the reduction in mitochondrial membrane potential, redistribution of cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase and DNA-dependent protein kinase (catalytic subunit), and induction of apoptosis. Mutations in the cAMP response element-related motif within the RARbeta promoter resulted in loss of synergy in RARbeta transcription. In addition, inhibition of RARbeta expression by an antisense construct also blocked the antitumor effects of RA + 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis. Therefore, loss of RARbeta expression may contribute to the tumorigenicity of human mammary epithelial cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion and may have potential for combination biotherapy for the treatment of malignant diseases.

Growth arrest and induction of apoptosis in breast cancer cells by antisense depletion of protein kinase A-RI alpha subunit: p53-independent mechanism of action.

The enhanced expression of the RI alpha subunit of cyclic AMP-dependent protein kinase type 1 (PKA-1) has been correlated with cancer cell growth. We have investigated the effects of sequence-specific inhibition of RI alpha gene expression on the growth of MCF-7 human breast cancer cells. We report that RI alpha antisense treatment results in a reduction in RI alpha expression at both mRNA and protein levels and inhibition of cell growth. The growth inhibition was accompanied by changes in cell morphology, cleavage of poly(ADP-ribose) polymerase (PARP) and appearance of apoptotic nuclei. In addition, bcl-2 protein level was reduced and p53 expression increased in growth arrested cells. Interestingly, RI alpha antisense inhibited cell viability and induced apoptosis in the absence of p53, suggesting that these actions of RI alpha antisense are exerted independent of p53. In contrast, two- and four-base mismatched control oligonucleotides had no effect on either cell growth or morphology. These results demonstrate that the RI alpha antisense, which efficiently depletes the growth stimulatory molecule RI alpha, induces cell differentiation and apoptosis, providing a new approach to combat breast cancer cell growth.

Synergistic effects of retinoic acid and 8-Cl-cAMP on apoptosis require caspase-3 activation in human ovarian cancer cells.

We investigated the intracellular mechanisms of retinoic acid (9-cis-RA, 13-cis-RA or all-trans-RA) and a cyclic AMP analog 8-Cl-cAMP on growth-inhibition and apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. The cyclic AMP analog, 8-Cl-cAMP, acted synergistically with RA in inducing and activating retinoic acid receptor beta (RARbeta) which correlated with the growth inhibition, cell cycle arrest, and apoptosis in both cell types. In addition, combined treatment of cells with RA plus 8-Cl-cAMP resulted in the release of cytochrome c, loss in mitochondrial membrane potential and activation of caspase-3 followed by cleavage of anti-poly(ADP-ribose)polymerase and DNA-dependent protein kinase (catalytic subunit). Interestingly, inhibition of caspase-3 activation blocked RA plus 8-Cl-cAMP induced apoptosis. Furthermore, mutations in a CRE-related motif within the RARbeta promoter resulted in loss of both transcriptional activation of RARbeta and synergy between RA and 8-Cl-cAMP. Thus, RARbeta can mediate RA and/or cyclic AMP action in ovarian cancer cells by promoting apoptosis. Loss of RARbeta expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion in inducing apoptosis via caspase-3 activation, and may have potential for combination biotherapy for the treatment of malignant disease such as ovarian cancer.

Antisense depletion of RIalpha subunit of protein kinase A induces apoptosis and growth arrest in human breast cancer cells.

In recent years, several laboratories have explored the possibility of using antisense oligodeoxynucleotides for specific manipulation of gene expression leading to cancer treatment. The enhanced expression of the RIalpha subunit of cyclic AMP-dependent protein kinase type I (PKA-I) has been correlated with cancer cell growth. In the present study, the effects of an antisense oligodeoxynucleotide targeted against RIalpha subunit of PKA-I on growth inhibition and apoptosis in MDA-MB-231 human breast cancer cells were investigated. The growth inhibitory effects of RIalpha antisense oligodeoxynucleotide correlated with a decrease in the RIalpha mRNA and protein levels. The growth inhibition was accompanied by changes in the cell cycle phase distribution, cell morphology, cleavage of poly (ADP-ribose) polymerase (PARP), and appearance of apoptotic nuclei. By comparison, mismatched control oligodeoxynucleotide had no effect. On the basis of these results, it can be suggested that the RIalpha antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RIalpha and induces apoptosis/differentiation, could be used as a therapeutic agent for breast cancer treatment.

Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase.

The Bcl2 family of proteins plays a significant role in regulation of apoptosis. In this study, the microtubule-damaging drugs paclitaxel, vincristine, and vinblastine induced Bcl2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells and reduced Bcl2-Bax dimerization. Paclitaxel or vincristine induced increased expression of Bax, while overexpression of Bcl2 in these cell lines counteracted the effects of low doses of these drugs. In addition, paclitaxel- and vincristine-induced activation of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) induced Bcl2 hyperphosphorylation and apoptosis, which were blocked by the PKA inhibitor Rp diastereomers of cAMP (Rp-cAMP). This finding suggests that activation of PKA due to microtubule damage is an important event in Bcl2 hyperphosphorylation and induction of apoptosis. These microtubule-damaging drugs caused growth arrest in G2-M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation mediated inactivation of Bcl2 and apoptosis without the involvement of p53. By comparison, the DNA-damaging drugs methotrexate and doxorubicin had no effect on Bcl2 hyperphosphorylation but induced p53 expression. Interestingly, paclitaxel or vincristine induced activation of caspase 3 and cleavage of poly(ADP-ribose) polymerase downstream of Bcl2 hyperphosphorylation. These data suggest that there may be a signaling cascade induced by agents that disrupt or damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to apoptosis), the cellular response to DNA damage.

Synergistic effects of 8-chlorocyclic-AMP and retinoic acid on induction of apoptosis in Ewing's sarcoma CHP-100 cells.

The enhanced expression of the regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase type I, RIalpha, has been correlated with cancer cell growth. Retinoic acid (RA) has been shown to play an important role in the regulation of proliferation and differentiation in neoplastic cells. In the present study, the effects of cAMP analogue 8-chlorocyclic-AMP (8-Cl-cAMP) and RA (both singly and combined) on growth inhibition and apoptosis in Ewing's sarcoma CHP-100 cells were evaluated. The inhibitory effects of 8-Cl-cAMP and RA (9-cis-RA, 13-cis-RA, and all-trans-RA) on cell viability were time and dose related. The degree of growth inhibition induced by 9-cis-RA was the greatest among all of the RA analogues (13-cis-RA and all-trans-RA) examined. The combined effects of 8-Cl-cAMP and RA on the induction of growth arrest at the G0-G1 stage of the cell cycle, apoptosis, down-regulation of RIalpha, and cleavage of poly(ADP-ribose) polymerase were synergistic. In conclusion, it is clear that RA and 8-Cl-cAMP act in a synergistic fashion and have potential for combination chemotherapy for the treatment of malignant disease.