Rotenone inhibits mammalian cell proliferation by inhibiting microtubule assembly through tubulin binding

Rotenone, a widely used insecticide, has been shown to inhibit mammalian cell proliferation and to depolymerize cellular microtubules. In the present study, the effects of rotenone on the assembly of microtubules in relation to its ability to inhibit cell proliferation and mitosis were analyzed. We found that rotenone inhibited the proliferation of HeLa and MCF-7 cells with half maximal inhibitory concentrations of 0.2 +/- 0.1 microm and 0.4 +/- 0.1 microm, respectively. At its effective inhibitory concentration range, rotenone depolymerized spindle microtubules of both cell types. However, it had a much stronger effect on the interphase microtubules of MCF-7 cells compared to that of the HeLa cells. Rotenone suppressed the reassembly of microtubules in living HeLa cells, suggesting that it can suppress microtubule growth rates. Furthermore, it reduced the intercentrosomal distance in HeLa cells at its lower effective concentration range and induced multipolar-spindle formation at a relatively higher concentration range. It also increased the level of checkpoint protein BubR1 at the kinetochore region. Rotenone inhibited both the assembly and the GTP hydrolysis rate of microtubules in vitro. It also inhibited the binding of colchicine to tubulin, perturbed the secondary structure of tubulin, and reduced the intrinsic tryptophan fluorescence of tubulin and the extrinsic fluorescence of tubulin-1-anilinonaphthalene-8-sulfonic acid complex, suggesting that it binds to tubulin. A dissociation constant of 3 +/- 0.6 microm was estimated for tubulin-rotenone complex. The data presented suggest that rotenone blocks mitosis and inhibits cell proliferation by perturbing microtubule assembly dynamics.     

A Randomized Controlled Trial of Increased Dose and Frequency of Albendazole with Standard Dose DEC for Treatment of Wuchereria bancrofti Microfilaremics in Odisha,India

Although current programmes to eliminate lymphatic filariasis have made significant progress it may be necessary to use different approaches to achieve the global goal, especially where compliance has been poor and ‘hot spots’ of continued infection exist. In the absence of alternative drugs, the use of higher or more frequent dosing with the existing drugs needs to be explored. We examined the effect of higher and/or more frequent dosing with albendazole with a fixed 300mg dose of diethylcarbamazine in a Wuchereria bancrofti endemic area in Odisha, India. Following screening, 104 consenting adults were randomly assigned to treatment with the standard regimen annually for 24 months (S1), or annually with increased dose (800mg albendazole)(H1) or with increased frequency (6 monthly) with either standard (S2) or increased (H2) dose. Pre-treatment microfilaria counts (GM) ranged from 348 to 459 mf/ml. Subjects were followed using microfilaria counts, OG4C3 antigen levels and ultrasound scanning for adult worm nests. Microfilarial counts tended to decrease more rapidly with higher or more frequent dosing at all time points. At 12 months, Mf clearance was marginally greater with the high dose regimens, while by 24 months, there was a trend to higher Mf clearance in the arm with increased frequency and 800mg of albendazole (76.9%) compared to other arms, (S1:64%, S2:69.2% & H1:73.1%). Although higher and/or more frequent dosing showed a trend towards a greater decline in antigenemia and clearance of “nests”, all regimens demonstrated the potential macrofilaricidal effect of the combination. The higher doses of albendazole did not result in a greater number or more severe side effects. The alternative regimens could be useful in the later stages of existing elimination programmes or achieving elimination more rapidly in areas where programmes have yet to start.     

Characterization of Calmodulin-Free Murine Inducible Nitric-Oxide Synthase

Nitric-Oxide Synthase (NOS), that produces the biological signal molecule Nitric-Oxide (NO), exists in three different isoforms called, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All NOS isoforms require post-translational interaction with the calcium-binding protein, calmodulin (CaM) for manifesting their catalytic activity. However, CaM has been suggested to control the translational assembly of the enzyme as well, particularly in helping its inducible isoform, iNOS assume a stable, heme-replete, dimeric and active form. Expression of recombinant murine iNOS in E.coli in the absence of CaM has been previously shown to give extremely poor yield of the enzyme which was claimed to be absolutely heme-free, devoid of flavins, completely monomeric and catalytically inactive when compared to the heme-replete, active, dimeric iNOS, generated through co-expression with CaM. In contrast, we found that although iNOS expressed without CaM does produce significantly low amounts of the CaM-free enzyme, the iNOS thus produced, is not completely devoid of heme and is neither entirely monomeric nor absolutely bereft of catalytic activity as reported before. In fact, iNOS synthesized in the absence of CaM undergoes compromised heme incorporation resulting in extremely poor dimerization and activity compared to its counterpart co-expressed with CaM. Moreover, such CaM-free iNOS has similar flavin content and reductase activity as iNOS co-expressed with CaM, suggesting that CaM may not be as much required for the functional assembly of the iNOS reductase domain as its oxygenase domain. LC-MS/MS-based peptide mapping of the CaM-free iNOS confirmed that it had the same full-length sequence as the CaM-replete iNOS. Isothermal calorimetric measurements also revealed high affinity for CaM binding in the CaM-free iNOS and thus the possible presence of a CaM-binding domain. Thus CaM is essential but not indispensible for the assembly of iNOS and such CaM-free iNOS may help in elucidating the role of CaM on iNOS catalysis.     

Predictive Estimation of Finite Population Mean Using a Product Estimator for Two-Stage Sampling

In an attempt to estimate a finite population mean under the predictive approach described in Basu (1971) through the product method of estimation, we created a new product-type estimator for a two-stage sampling procedure. We also report a simulation study that is made in order to understand better the performance of the new estimator compared to the classical product estimator.     

Comparison of dosimetric data of bone marrow between standard IMRT and bone marrow sparing IMRT in carcinoma cervix

BackgroundThe aim of the study was to assess the dosimetric comparison of bone marrow between standard IMRT(SD-IMRT) and bone marrow sparing IMRT (BMS-IMRT) among carcinoma cervix patients who underwent radical or adjuvant chemoradiation in a tertiary cancer center.Materials and methodsForty eligible patients of histo-pathologically proven carcinoma cervix were enrolled in the study that was randomized on a 1:1 basis between SD-IMRT and BMS-IMRT from July 2018 to October 2019. The whole pelvis, bilateral femoral heads, and upper 1/3^rd femur were contoured using the whole bone technique as a surrogate marker for the bone marrow. In both arms, V10, V20, and V40, bone marrow was noted along with mean, maximum, minimum dose, and total volume. DVH for the bone marrow in both arms was compared using the unpaired student t-test.ResultsWe found no significant difference in the mean of various parameters in SD-IMRT arm vs. BMS IMRT arm — for the bone marrow: V10 (89 ± 4.3% vs. 86.7 ± 3.7%), V20 (73.2 ± 5.3% vs. 73.1 ± 4.5%), V40 (23.9 ± 5.4% vs. 26.6 ± 7.4%) and, similarly, for mean dose (28.1 ± 3.5% vs. 28.1 ± 1.8%), maximum dose (53.4 ± 0.58% vs. 53.2 ± 0.58%), minimum dose (0.33 ± 0.18% vs. 0.38 ± 0.38%), total volume (961 ± 110 cc vs. 901 ± 152 cc).ConclusionThis study shows no statistically significant difference in dosimetry between the two groups, which suggests that SD-IMRT spares the bone marrow adequately. Therefore, the need for BMS-IMRT using the present contouring technique does not give any added advantage over SD-IMRT. However, large sample size, other novel contouring technique, and multivariate analysis are needed to reach a definite conclusion.     

Monoclonal antibodies specific against tumors and viral infections

Monoclonal antibodies are monospecific antibodies. They are generated also by hybridoma technology. They are useful in target specific therapy against dangerous diseases. This property of monoclonal antibody offers us a new hope to combat deadly diseases where chemotherapy fails to produce specific treatment without adverse effects. This review article deals with the use of monoclonal antibodies specific against tumors and viral infections.     

Molecular physiology of aluminum toxicity and tolerance in plants

Aluminum being the third most abundant metal in the earth’s crust poses a serious threat to crop productivity in acid soils, which comprise almost half of the arable land. This review travels across time and updates research done on aluminum stress in plants. In its phytotoxic forms, aluminum affects root growth by acting in the root apical zone, resulting in growth inhibition in a very short time at micromolar concentrations. The mechanisms of aluminum toxicity in plants may proceed by growth inhibition, callose accumulation, cytoskeletal distortion, disturbance of plasma membrane surface charge, and H⁺-ATPase activity, lipid peroxidation of membranes, production of reactive oxygen species in cytosol and mitochondria, respiratory dysfunction, opening of mitochondrial permeability transition pores, collapsing of inner mitochondrial membrane potential, activation of mitochondrial protease, and induction of nuclear apoptosis, resulting ultimately in programmed cell death. In contrast, the mechanism of tolerance involves the exudation of organic acid anions, complexation of aluminum with organic acids, and subsequent detoxification. Many oxidative stress genes and other metabolically important genes have also been found to be induced under aluminum stress and overexpression analyses have also shown some plants to develop some degree of tolerance. In the future, researchers in the area of aluminum research should investigate more basic mechanisms of aluminum toxicity and discover and study more aluminum-responsive genes that confer resistance against this toxic metal, to ensure food security for ever-increasing human populations in the future.     

Pretreatment of indian cane molasses for increased production of citric acid

Surface culture citric acid fermentation was carried out by Aspergillus niger T55, a strain isolated from its natural source, using cane molasses, either untreated or treated by various methods. Citric acid biosynthesis is seriously impaired by both organic and inorganic inhibitors. A combined treatment of molasses with tricalcium phosphate, hydrochloric acid, and Sephadex fractionation minimizes the level of inorganic and organic inhibitors in molasses and increases the production of citric acid (65% weight yield based on total reducing sugar). The optimum level of individual metal ions for citric acid production depends on the concentration of other metals in the medium.