Inhibition of polo like kinase 1 in sarcomas induces apoptosis that is dependent on Mcl-1 suppression

Sarcomas are rare cancers and the current treatments in inoperable or metastatic disease have not been shown to prolong survival. In order to develop novel targeted therapies, we tested the efficacy of polo-like kinase 1 (PLK-1) inhibitor (TAK-960) in sarcoma. All the sarcoma cell lines were sensitive to TAK-960 with IC50s in the low nanomolar range. We chose MPNST, CHP100 and LS141 for our studies and of which MPNST cells exclusively underwent polyploidy after a delay in mitosis for about 18 hours; CHP100 cells, after a 24h mitotic delay, died of apoptosis; LS141, after a delay in mitosis stayed at 4N with mild apoptosis. Apoptosis induced by TAK-960 in CHP100 was associated with down-regulation of Mcl-1 and the effect was recapitulated by down-regulating PLK1 by siRNA, confirming that the effect of TAK-960 on Mcl-1 expression is target specific. With suppression of Mcl-1 by siRNA, TAK-960 induced apoptosis in MPNST cells as well. These effects were confirmed in vivo, such that TAK-960 more effectively inhibited CHP100 than MPNST xenografts. In the setting of PLK-1 inhibition, Mcl-1 down regulation is shown to be an important determinant of apoptosis. Collectively, the net effect of this is to drive cells to apoptosis, resulting in a greater anti-tumor effect in vivo. Therefore, targeting PLK-1 should have a greater impact in treating sarcomas provided there is concomitant suppression of Mcl-1. These results further indicate that Mcl-1 could be an important biomarker to predict sensitivity to the induction of apoptosis by PLK-1 targeted therapy in sarcoma.     

Chemotherapeutic sensitivity and viability of Nocardia asteroides and Nocardia brasiliensis in drugs

Procedures for testing drug sensitivity ofNocardia were standardized.Five strains each ofN. asteroides (NA) andN. brasiliensis (NB) isolated from pathogenic materials were tested for drug sensitivity in Sabouraud's glucose agar (SGA), Sabouraud's glucose broth (SGB) and SGB with addition of 10 % horse serum against sulphadiazine (S), penicillin (P), streptomycin (St), chloramphenicol (C), tetracycline (T) and 4-4-diamino diphenyl sulphone (DDS). The viability of strains at minimum inhibitory concentrations (MIC) of different drugs were also tested.Results showed that liquid medium is preferable to solid medium for drug testing as pellicle formation observed on the surface of liquid medium helps in accurate assessment of MIC. Serum addition did not appear to be necessary. Ten days incubation at 37 °C gives optimum results for determination of MIC. Taking the maximum drug concentration attainable in blood during therapy, the strains were classified as sensitive and resistant. All strains were resistant to DDS and P. One strain of NB was sensitive to St. One strain each of NA and NB was borderline sensitive to C. Four out of 5 strains in both species were sensitive to S. All strains were sensitive to T. The organisms remained viable in drugs, thus suggesting the limitations of antinocardial therapy.From Mycology Research Unit, Department of Dermatology, Seth G.S. Medical College and K.E.M. Hospital, Bombay-12.Hon. Professor Dermato-Venereology.Research Fellow.     

Food and feeding habits of the larvivorous fish Aplocheilus lineatus (Cuv. & Val.) in its natural habitat

Results are presented of a study of the food and feeding habits of Aplocheilus lineatus under natural conditions and the manner in which dietary preferences are influenced by the habitat/environment, seasons and stage of maturity of the fish is explained, based on data from montly random samples collected for a year. A qualitative assessment of the diet reveals that it is not confined to a varied range of aquatic fauna, but also encompasses allochthonous fauna. Quantitative and qualitative analyses of the diet indicate its feeding habit which does not alter with size or seasonal changes. The range of prey consumed does not differ radically, qualitatively, as a function of size, but quantitatively exhibits five levels of discrimination and differential exploitation related mainly to prey size. Seasonal fluctuations in feeding are more qualitative than quantitative and seem dependent on the occurrence of food organisms. The lack of correlation between breeding and feeding is linked to extent of the development of the gonad in the body cavity. In conclusion, the potential of the fish as an effective biological control agent of mosquito larvae is indicated by the fact that dipteran larvae are a preferred item of its diet in all size groups.     

Quantitative assessment of the genotoxicity of fecapentaenes

Fecapentaenes are a group of fecal mutagens of microbial origin isolated from human stools. Fecapentaene-12 (F-12) and fecapentaene-14 (F-14), differing only in two carbon atoms in the side chain, are glyceryl ethers with a highly reactive chromophoric aliphatic side chain incorporating a conjugated pentaene moiety. Although these compounds are known for their genotoxicity, no test systems have been developed to precisely assess their relative genotoxicity. In this study F-12 and F-14 were assayed for their genotoxicity using the SOS Chromotest in which the induction of beta-galactosidase in E. coli PQ37 was used as a quantitative measure of biological activity. The activity obtained with F-12 and F-14 was compared with that of 4-nitroquinoline oxide (4-NQO) as the reference standard of a direct acting mutagen. While F-14 was almost as active as 4-NQO, F-12 was only about 25% as active as F-14, the higher analog.     

Functional Analysis of the Kv1.1 N255D Mutation Associated with Autosomal Dominant Hypomagnesemia

Mutations in the voltage-gated K⁺ channel Kv1.1 have been linked with a mixed phenotype of episodic ataxia and/or myokymia. Recently, we presented autosomal dominant hypomagnesemia as a new phenotypic characteristic associated with a mutation in Kv1.1 (N255D) (Glaudemans, B., van der Wijst, J., Scola, R. H., Lorenzoni, P. J., Heister, A., van der Kemp, A. W., Knoers, N. V., Hoenderop, J. G., and Bindels, R. J. (2009) J. Clin. Invest. 119, 936–942). A conserved asparagine at position 255 in the third transmembrane segment was converted into an aspartic acid, resulting in a non-functional channel. In this study, we explored the functional consequence of this conserved residue by substitution with other hydrophobic, polar, or charged amino acids (N255E, N255Q, N255A, N255V, N255T, and N255H). Upon overexpression in human embryonic kidney (HEK293) cells, cell surface biotinylation revealed plasma membrane expression of all mutant channels. Next, we used the whole-cell patch clamp technique to demonstrate that the N255E and N255Q mutants were non-functional. Substitution of Asn-255 with other amino acids (N255A, N255V, N255T, and N255H) did not prevent ion conduction, and these mutant channels activated at more negative potentials when compared with wild-type channels, −41.5 ± 1.6, −45.5 ± 2.0, −50.5 ± 1.9, and −33.8 ± 1.3 mV to −29.4 ± 1.1 mV, respectively. The time constant of activation was significantly faster for the two most hydrophobic mutations, N255A (6.2 ± 0.2 ms) and N255V (5.2 ± 0.3 ms), and the hydrophilic mutant N255T (9.8 ± 0.4 ms) in comparison with wild type (13.0 ± 0.9 ms). Furthermore, the voltage dependence of inactivation was shifted ∼13 mV to more negative potentials in all mutant channels except for N255H. Taken together, our data showed that an asparagine at position 255 in Kv1.1 is required for normal voltage dependence and kinetics of channel gating.     

Statistical optimization of medium components for improved synthesis of pectinase by Aspergillus niger

The synthesis of pectinase using Aspergillus niger, NCIM 548, has been enhanced by optimizing the carbon and nitrogen sources present in the medium. Enzyme synthesis was about 40% more using the optimized medium than using the unoptimized medium. The study provided information on the appropriate carbon source as well as the optimum ratio of carbon to nitrogen, yielding the highest enzyme levels.