SPARC overexpression inhibits cell proliferation in neuroblastoma and is partly mediated by tumor suppressor protein PTEN and AKT

Secreted protein acidic and rich in cysteine (SPARC) is also known as BM-40 or Osteonectin, a multi-functional protein modulating cell-cell and cell-matrix interactions. In cancer, SPARC is not only linked with a highly aggressive phenotype, but it also acts as a tumor suppressor. In the present study, we sought to characterize the function of SPARC and its role in sensitizing neuroblastoma cells to radio-therapy. SPARC overexpression in neuroblastoma cells inhibited cell proliferation in vitro. Additionally, SPARC overexpression significantly suppressed the activity of AKT and this suppression was accompanied by an increase in the tumor suppressor protein PTEN both in vitro and in vivo. Restoration of neuroblastoma cell radio-sensitivity was achieved by overexpression of SPARC in neuroblastoma cells in vitro and in vivo. To confirm the role of the AKT in proliferation inhibited by SPARC overexpression, we transfected neuroblastoma cells with a plasmid vector carrying myr-AKT. Myr-AKT overexpression reversed SPARC-mediated PTEN and increased proliferation of neuroblastoma cells in vitro. PTEN overexpression in parallel with SPARC siRNA resulted in decreased AKT phosphorylation and proliferation in vitro. Taken together, these results establish SPARC as an effector of AKT-PTEN-mediated inhibition of proliferation in neuroblastoma in vitro and in vivo.     

A familial deletion 4q syndrome: An outcome of a paracentric inversion

Chromosome inversions are intra-chromosomal rearrangements formed when the chromosome breaks occur at two places, and in the process of repair the intervening segments are joined in an inverted or opposite manner. Inversions themselves do not appear to cause clinical anomalies, if balanced. Abnormal phenotypes can occur due to gene disruption at the point of breakage and reunion or due to duplication/deficiency recombinants formed during crossover at meiosis. We report a case with familial deletion 4q syndrome in a 1-year-old female child with dysmorphism and congenital abnormalities. The deletion was an outcome of a paracentric inversion 4q31.2q35.2. The deletion was confirmed by fluorescence in situ hybridization using telomeric DNA probes for chromosome No. 4. An attempt was made to correlate the genotype with the phenotype. The father had the same rearrangement with a milder phenotype. The recurrence risk in such cases is high.     

Superior quality agar from <Emphasis Type="Italic">Gracilaria</Emphasis> species (Gracilariales,Rhodophyta) collected from the Gulf of Mannar,India

Gracilaria edulis, G. crassa, G. foliifera, and G. corticata are naturally occurring agarophytes of Indian waters. These agarophytes were evaluated for their agar contents using an improved process recently reported by us (US Patent 2005/0267296A1). The effect of different concentrations of NaOH in the alkali treatment was studied for optimizing the extraction conditions. These Gracilaria species of Indian waters produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera, as one would expect. Among these, G. edulis and G. crassa produced agar polymers having high gel strengths of 490 ± 8.16 and 800 ± 15.4 g cm⁻², respectively, with 8% NaOH treatment as opposed the low gel strength agars that have been reported in the literature to date.     

The acidic motif of WNK4 is crucial for its interaction with the K channel ROMK

WNK kinases have rapidly emerged as important regulators of Na⁺ and K⁺ homoeostasis in the mammalian kidney where they regulate the trafficking of proteins such as the NaCl-cotransporter (NCCT) and K⁺ channel, ROMK. However, an increasing number of WNK effects are kinase-independent, including their interaction with ROMK, and involve instead protein-protein interactions. Outside of their kinase domain all WNKs contain a unique run of predominantly negatively charged amino acids dubbed the acidic motif, where the WNK4 disease mutations causing Gordon’s syndrome also cluster. To look further at the role of this motif we studied the effects of WNK4 fragments, including one with a deleted acidic motif (ΔAM) and a 10-mer acidic motif peptide on ROMK expression in Xenopus oocytes. We found that an N-terminal fragment of WNK4 (1-620 WNK4) containing the acidic motif retains full activity in inhibiting ROMK currents. However, ΔAM WNK4 is completely inactive and the effect of WNK4 or 1-620 WNK4 can be completely blocked by co-injection of the 10-mer acidic motif peptide. The blocking action of the peptide was sequence specific as a peptide with a randomised sequence was inactive. These results on ROMK currents were paralleled by changes in membrane expression of fluorescent EGFP-ROMK. Finally, we show that 1-620 WNK4 can pull down ROMK and this interaction can be blocked with the acidic motif peptide. These results confirm the important role of the acidic motif of WNK4 in its protein-protein interaction with the ROMK channel.     

Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 (<Emphasis Type="Italic">NF1</Emphasis>) gene

The identification of mutations in the NF1 gene causing type 1 neurofibromatosis (NF1) has presented a considerable challenge because of the large size of the gene, the lack of significant mutational clustering, the diversity of the underlying pathological lesions and the presence of NF1 pseudogenes. Denaturing high performance liquid chromatography (DHPLC), a high throughput, non-hazardous and largely automated heteroduplex-based technique, is in many ways ideally suited to mutation detection in this condition. DHPLC was therefore optimised for the rapid screening of the 60 exons and splice junctions of the NF1 gene in patients with NF1. The sensitivity of DHPLC was evaluated in a retrospective study of a cohort of 111 unrelated NF1 patients with known germline mutations; 97% of mutations were detected. In a subsequent prospective analysis of 50 unrelated NF1 patients, germline mutations were identified in 34 individuals (68%), 22 of these alterations being novel. This represents the highest rate of mutation detection so far reported for the NF1 gene with a single screening technique and genomic DNA as a target.     

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer

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