A novel microtubule-modulating noscapinoid triggers apoptosis by inducing spindle multipolarity via centrosome amplification and declustering

We have previously shown that a non-toxic noscapinoid, EM011 binds tubulin without altering its monomer/polymer ratio. EM011 is more active than the parent molecule, noscapine, in inducing G2/M arrest, inhibiting cellular proliferation and tumor growth in various human xenograft models. However, the mechanisms of mitotic-block and subsequent cell death have remained elusive. Here, we show that EM011-induced attenuation of microtubule dynamics was associated with impaired association of microtubule plus-end tracking proteins, such as EB1 and CLIP-170. EM011 treatment then led to the formation of multipolar spindles containing 'real' centrioles indicating drug-induced centrosome amplification and persistent centrosome declustering. Centrosome amplification was accompanied by an upregulation of Aurora A and Plk4 protein levels, as well as a surge in the kinase activity of Aurora A, suggesting a deregulation of the centrosome duplication cycle. Cell-cycle phase-specific experiments showed that the 'cytotoxicity-window' of the drug encompasses the late S-G2 period. Drug-treatment, excluding S-phase, not only resulted in lower sub-G1 population but also attenuated centrosome amplification and spindle multipolarity, suggesting that drug-induced centrosome amplification is essential for maximal cell death. Subsequent to a robust mitotic arrest, EM011-treated cells displayed diverse cellular fates suggesting a high degree of intraline variation. Some 'apoptosis-evasive' cells underwent aberrant cytokinesis to generate rampant aneuploidy that perhaps contributed to drug-induced cell death. These data indicate that spindle multipolarity induction by means of centrosome amplification has an exciting chemotherapeutic potential that merits further investigation.     

CEP70 protein interacts with γ-tubulin to localize at the centrosome and is critical for mitotic spindle assembly

Deregulation of the mitotic spindle has been implicated in genomic instability, an important aspect of tumorigenesis and malignant transformation. To ensure the fidelity of chromosome transmission, the mitotic spindle is assembled by exquisite mechanisms and orchestrated by centrosomes in animal cells. Centrosomal proteins especially are thought to act coordinately to ensure accurate spindle formation, but the molecular details remain to be investigated. In this study, we report the molecular characterization and functional analysis of a novel centrosomal protein, Cep70. Our data show that Cep70 localizes to the centrosome throughout the cell cycle and binds to the key centrosomal component, γ-tubulin, through the peptide fragments that contain the coiled-coil domains. Our data further reveal that the centrosomal localization pattern of Cep70 is dependent on its interaction with γ-tubulin. Strikingly, Cep70 plays a significant role in the organization of both preexisting and nascent microtubules in interphase cells. In addition, Cep70 is necessary for the organization and orientation of the bipolar spindle during mitosis. These results thus report for the first time the identification of Cep70 as an important centrosomal protein that interacts with γ-tubulin and underscore its critical role in the regulation of mitotic spindle assembly.     

Origin of sexual isolation in <Emphasis Type="Italic">Drosophila ananassae</Emphasis> due to founder effects

The origin of sexual isolation is the central event in the evolution of biological species and plays a key role in maintaining biological diversity. Three mass culture stocks of D. ananassae originating from different geographic localities showing no isolation with each other were subjected to different degrees of bottlenecks i.e. one pair, five pairs and ten pairs. These drift lines were passed through flush-crash cycle at every generation with same initial number of founders, and maintained for twenty-seven generations and then the pattern of matings was tested among these nine drift lines involving 36 crosses in total. In 23 of 36 crosses, the difference between homogamic and heterogamic matings was significant and isolation indices were significantly more than zero in one direction only providing evidence for asymmetrical sexual isolation. Further, when Bonferroni test for pair-wise analysis was employed, significant differences between homogamic and heterogamic matings were found in 25 crosses. These findings provide evidence for origin of sexual isolation by founder effects in D. ananassae.     

Genomic profiling identifies novel mutations and SNPs in ABCD1 gene: a molecular, biochemical and clinical analysis of X-ALD cases in India

X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encode the peroxisomal membrane protein. We conducted a genomic and protein expression study of susceptibility gene with its clinical and biochemical analysis. To the best of our knowledge this is the first preliminary comprehensive study in Indian population that identified novel mutations and SNPs in a relatively large group. We screened 17 Indian indigenous X-linked adrenoleukodystrophy cases and 70 controls for mutations and SNPs in the exonic regions (including flanking regions) of ABCD1 gene by direct sequencing with ABI automated sequencer along with Western blot analysis of its endogenous protein, ALDP, levels in peripheral blood mononuclear cells. Single germ line mutation was identified in each index case in ABCD1 gene. We detected 4 novel mutations (2 missense and 2 deletion/insertion) and 3 novel single nucleotide polymorphisms. We observed a variable protein expression in different patients. These findings were further extended to biochemical and clinical observations as it occurs with great clinical expression variability. This is the first major study in this population that presents a different molecular genetic spectrum as compared to Caucasian population due to geographical distributions of ethnicity of patients. It enhances our knowledge of the causative mutations of X-ALD that grants holistic base to develop effective medicine against X-ALD.     

Altered composition of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> poly(hydroxyalkanoate) through expression of PHA synthase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 35206

The class III poly(hydroxyalkanoate) synthase (PHAS) genes (phaC and phaE) of a photosynthetic bacterium, Allochromatium vinosum ATCC 35206, were cloned, sequenced and expressed in a heterologous host. PCR coupled with a chromosomal gene-walking method was used to clone and subsequently sequence the contiguous phaC (1,068 bps) and phaE (1,065 bps) genes of A. vinosum ATCC 35206. BLASTP search of protein databases showed that the gene-products of phaC and phaE are different (<66% identities) from the previously reported class III PHASs such as those of A. vinosum DSM180. Domain analysis revealed the presence of a conserved α/β-hydrolase fold in PhaC, the putative gene-product of phaC. Upon electroporation of a poly(hydroxybutanoate) (PHB)-negative mutant of Ralstonia eutropha PHB⁻4 with a shuttle plasmid pBHR1 containing the newly cloned phaC and phaE genes, the bacteria resumed the synthesis of PHB, albeit at a low level (4–5% of the cell dry wt) due to kanamycin selection pressure. We further showed that the recombinant strain grown in kanamycin-containing culture medium synthesized a blend of PHA that also contains a high content of 3-hydroxyoctanoate and 3-hydroxydecanoate as its repeat-unit monomers. Genomic analysis suggested the existence of two PHA synthase genes in R. eutropha. The results of this study not only make available a phylogenetically diverse type III phaC and phaE genes, but also confirm through kanamycin selection pressure the existence of multiple PHA biosynthesis systems in R. eutropha.     

Micropropagation of Sesbania aculeata (Pers.) by adventitious organogenesis

Multiple shoots differentiated from hypocotyl explants of Sesbania aculeata (Pers.) syn S. cannabina (Retz.) Pers., a leguminous woody shrub, when cultured on Murashige and Skoog's basal medium supplemented with auxin (IBA, NAA) or auxin and cytokinin (IBA + BAP, NAA + BAP). Shoot budding occurred directly from the explant as well as from callus. Differentiation of shoot and root occurred in one step in the same concentration of auxin or auxin and cytokinin. Elongation of shoots occurred in the shoot induction medium.