Nimbolide a limonoid from Azadirachta indica inhibits proliferation and induces apoptosis of human choriocarcinoma (BeWo) cells

We investigated the cytotoxic effects of nimbolide, a limonoid present in leaves and flowers of the neem tree (Azadirachta indica) on human choriocarcinoma (BeWo) cells. Treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC50 values of 2.01 and 1.19 μM for 7 and 24 h respectively, accompanied by downregulation of proliferating cell nuclear antigen. Examination of nuclear morphology revealed fragmentation and condensation indicating apoptosis. Increase in the generation of reactive oxygen species (ROS) that was reversed by addition of reduced glutathione suggested ROS involvement in the cytotoxicity of nimbolide. A decrease in Bcl-2/Bax ratio with increased expression of Apaf-1 and caspase-3, and cleavage of poly(ADP-ribose) polymerase provide compelling evidence that nimbolide-induced apoptosis is mediated by the mitochondrial pathway. The results of the present study suggest that nimbolide has immense potential in cancer prevention and therapy based on its antiproliferative and apoptosis inducing effects.

Cholinergic Muscarinic Receptors in Human Fetal Brain: Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Corpus Striatum, Brainstem, and Cerebellum

The ontogeny of muscarinic receptors was studied in human fetal striatum, brainstem, and cerebellum to investigate general principles of synaptogenesis as well as the physiological balance between various chemical synapses during development in a given region of the brain. [3H]Quinuclidinyl benzilate ([-'H]QNB) binding was assayed in total particulate fraction (TPF) from various parts of brain. In the corpus striatum, QNB binding sites are present at 16 weeks of gestation (average concentration 180 fmol/mg protein of TPF), slowly increase up to 24 weeks (average concentration 217 fmol/mg protein), and rapidly increase during the third trimester to 480 fmol/mg protein of TPF. In contrast, dopaminergic receptors exist as two subpopulations. one with low affinity and the other with high affinity up to the 24th week of gestation; all of them acquire the highaffinity characteristic during the third trimester. In brainstem, the muscarinic receptors show maximum concentration by 16 weeks of age (360 fmolimg protein of TPF). Subsequently the muscarinic receptor concentration shows a gradual decline in the brainstem. In cerebellum, except for a slight increase at 24 weeks (average concentration 90 fmol/mg protein of TPF), the receptor concentration remained nearly constant at about 60-70 fmolimg protein of TPF throughout fetal life. This study demonstrates that the ontogeny of muscarinic receptors varies among the different regions, and the patterns observed suggest that receptor formation occurs principally in the third trimester. Also noteworthy is the finding that the QNB binding sites decreased in all regions of the human brain during adult life. Key Words: Cholinergic muscarinic receptors-Quinuclidinyl benzilate-Corpus striaturn-Brainstem-Cerebellum. Ravikumar B. V. and Sastry P. S. Cholinergic muscarinic receptors in human fetal brain: Ontogeny of [3H]quinuclidinyl benzilate binding sites in corpus striatum, brainstem, and cerebellum. J. Neurochem. 45, 1948- 1950 (1985).

Modeling the role of competition and cooperation in the evolution of katydid acoustic synchrony

The precise timing of individual signals in response to those of signaling neighbors is seen in many animal species. Synchrony is the most striking of the resultant timing patterns. One of the best examples of acoustic synchrony is in katydid choruses where males produce chirps with a high degree of temporal overlap. Cooperative hypotheses that speculate on the evolutionary origins of acousti synchrony include the preservation of the species-specific call pattern, reduced predation risks, and increased call intensity. An alternative suggestion is that synchrony evolved as an epiphenomenon of competition between males in response to a female preference for chirps that lead other chirps. Previous models investigating the evolutionary origins of synchrony focused only on intrasexual competitive interactions. We investigated both competitive and cooperative hypotheses for the evolution of synchrony in the katydid Mecopoda ``Chirper'' using physiologically and ecologically realistic simulation models incorporating the natural variation in call features, ecology, female preferences, and spacing patterns, specifically aggregation. We found that although a female preference for leading chirps enables synchronous males to have some selective advantage, it is the female preference for the increased intensity of aggregations of synchronous males that enables synchrony to evolve as an evolutionarily stable strategy.

Effect of Temperature Variation on Sister Chromatid Exchange Frequency in Cultured Human Lymphocytes

The effect of temperature variation on sister chromatid exchange (SCE) frequencies in human lymphocytes was studied. An increase as well as decrease in incubation temperature of cells leads to a higher frequency of sister chromatid exchanges than in cultures grown at 37°C. In addition, it was observed that mitotic: index and cell cycle duration were affected by low temperature.

Expression Profiling of Nucleotide Metabolism-Related Genes in Human Breast Cancer Cells After Treatment with 5-Fluorouracil

5-Fluorouracil (5-FU) is one of the most widely used drugs for treatment of cancers, including breast cancer that exhibits its anticancer activity by inhibiting DNA synthesis and also incorporated into DNA and RNA. The objective of this investigation was to find out the total nucleotide metabolism genes regulated by 5-FU in breast cancer cell line. The breast cancer cell line MCF-7 was treated with the drug 5-FU. To analyze the expression of genes, we have conducted the experiment using 1.7k and 19k human microarray slide and confirmed the expression of genes by semiquantitative reverse transcription-polymerase chain reaction. The expression of 44 genes involved in the nucleotide metabolism pathway was quantified. Of these 44 genes analyzed, transcription of 6 genes were upregulated and 9 genes were downregulated. Earlier studies revealed that the transcription of genes for key enzymes like thymidylate synthase, thymidinekinase, and dihydropyrimidine dehydrogenase are regulated by 5-FU. This study identified some novel genes like thioredoxin reductase, ectonucleotide triphosphate dephosphorylase, and CTP synthase are regulated by 5-FU. The data also reveal large-scale perturbation in transcription of genes not involved directly in the known mechanism of action of 5-FU.

An avidin-biotin microELISA for rapid measurement of total and allergen-specific human IgE

his paper describes an improved microtiter solid-phase enzyme immunoassay for the determination of total and allergen-specific human IgE. This assay technique is unique in its use of the avidin-biotin interaction to increase sensitivity. The avidin-biotin microtiter enzyme-linked immunosorbant assay (AB-microELISA) was performed in polyvinyl chloride microtiter plates using biotinylated anti-IgE and horseradish peroxidase (HRP)-avidin conjugate. This AB-microELISA technique enabled the quantitation of human serum IgE in the range of 0.1–5 ng/ml (10–500 pg/test) in less than 3 h. Total serum IgE, whether measured by the AB-microELISA or the paper radioimmunosorbant test (PRIST) was similar (correlation coefficient, r = 0.92). Further, the presence or absence of positive skin tests to 7 specific allergens determined in serum donors generally agreed with the presence or absence of allergen-specific IgE in their sera as measured by the AB-microELISA. The quantity of short ragweed allergen-specific IgE as determined by the AB-microELISA agreed with values obtained by the radioimmunosorbant test (RAST) (correlation coefficient, r = 0.89). No significant interference by ragweed-specific IgG (blocking antibody) was observed in the quantitation of allergen-specific IgE. The AB-microELISA is not only rapid and inexpensive, but also more sensitive than other published ELISA procedures and comparable to solid-phase radioimmunoassays in the quantitation of total and allergen-specific IgE.

Effect of human telomerase reverse transcriptase transfection on differentiation in BeWo choriocarcinoma cells

Arrest of proliferation is one of the prerequisites for differentiation of cytotrophoblasts into syncytiotrophoblasts, and thus during differentiation telomerase activity, as well as human telomerase reverse transcriptase (hTERT) expression, is down-regulated. Considering this, it is of interest to investigate whether syncytium formation can be delayed by prolonging the expression of telomerase in cytotrophoblasts. BeWo cells were transfected with pLPC-hTERT retroviral vector and the reverse transcription-polymerase chain reaction analysis for hTERT mRNA concentrations in the transfected cells revealed a several-fold increase in hTERT mRNA compared with the cells transfected with empty vector, and this confirmed that the transfection was successful. An increase in the proliferation, as assessed by bromodeoxyuridine incorporation assay, as well as an increase in mRNA and protein concentration of various cyclins and proliferating cell nuclear antigen, was noticed. The effect of hTERT transfection was also assessed after the addition of forskolin to induce differentiation and it was observed that cell–cell fusion was delayed and differentiation did not occur in hTERT-transfected cells. However, the effects seen were only transient as stable transfection was not possible and the cells were undergoing apoptosis after 72 h, which suggested that apart from hTERT other factors might be important for immortalization of BeWo cells.

Evolution of cooperation by reciprocation within structured demes

The iterative two-person Prisoners’ Dilemma game has been generalised to theN-person case. The evolution of cooperation is explored by matching the Tit For Tat (TFT) strategy (Axelrod and Hamilton 1981) against the selfish strategy. Extension of TFT toN-person situations yields a graded set of strategies from the softest TFT, which continues cooperation even if only one of the opponents reciprocates it, to the hardest, which would do so only when all the remaining opponents cooperate. The hardest TFT can go to fixation against the selfish strategy provided it crosses a threshold frequencypc. All the other TFT are invadable by the selfish (D) or the pure defector strategy, while none can invadeD. Yet, provided a thresholdpc is crossed, they can coexist stably withD. AsN, the size of the group increases, the threshold pc also increases, indicating that the evolution of cooperation is more difficult for larger groups. Under certain conditions, only the soft TFT can coexist stably against the selfish strategyD, while the harder ones cannot. An interesting possibility of a complete takeover of the selfish population by successive invasions by harder and harder TFT strategies is also presented.

Synthesis and in vitro cytotoxic evaluation of novel diazaspiro bicyclo hydantoin derivatives in human leukemia cells: A SAR study

To study the structure activity relationship (SAR) on the cytotoxic activity and probe the structural requirement for the potent antitumor activity, a series of novel diazaspiro bicyclo hydantoin derivatives were designed and synthesized. Their structures were confirmed by H-1 NMR, LCMS and IR analyses. The antiproliferative effect of these compounds were determined against human leukemia, K562 (chronic myelogenous leukemia) and CEM (T-cell leukemia) cells using trypan blue and MTT assay, and the SAR associated with the position of N-terminal substituents in diazaspiro bicyclo hydantoin have also been discussed. It has been observed that these compounds displayed strong, moderate and weak cytotoxic activities. Interestingly, compounds having electron withdrawing groups at third and fourth position of the phenyl ring displayed selectively cytotoxic activities to both the cell lines tested with IC50 value lower than 50 mu M. In addition, the cytotoxic activities of the compounds 7(a-o) bearing the substituents at N-3 position of diazaspiro bicyclo hydantoin increases in the order alkene > ester > ether and plays an important role in determining their antitumor activities. The position and number of substituents in benzyl group attached to N-8 of diazaspiro bicyclo hydantoin nucleus interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.