U2AF1 mutations alter sequence specificity of pre-mRNA binding and splicing.

We previously identified missense mutations in the U2AF1 splicing factor affecting codons S34 (S34F and S34Y) or Q157 (Q157R and Q157P) in 11% of the patients with de novo myelodysplastic syndrome (MDS). Although the role of U2AF1 as an accessory factor in the U2 snRNP is well established, it is not yet clear how these mutations affect splicing or contribute to MDS pathophysiology. We analyzed splice junctions in RNA-seq data generated from transfected CD34+ hematopoietic cells and found significant differences in the abundance of known and novel junctions in samples expressing mutant U2AF1 (S34F). For selected transcripts, splicing alterations detected by RNA-seq were confirmed by analysis of primary de novo MDS patient samples. These effects were not due to impaired U2AF1 (S34F) localization as it co-localized normally with U2AF2 within nuclear speckles. We further found evidence in the RNA-seq data for decreased affinity of U2AF1 (S34F) for uridine (relative to cytidine) at the e-3 position immediately upstream of the splice acceptor site and corroborated this finding using affinity-binding assays. These data suggest that the S34F mutation alters U2AF1 function in the context of specific RNA sequences, leading to aberrant alternative splicing of target genes, some of which may be relevant for MDS pathogenesis.

Empyema thoracis due to actinomyces odontolyticus.

Actinomyces odontolyticus has been reported as an opportunistic pathogen causing systemic infections. A case of empyema thoracis due to this organism in a 68-year-old male is reported here. The patient did not have any underlying disease or immunosuppression. The organism was isolated from his oral flora also. Eight cases of thoracopulmonary infections due to A. odontolyticus have been reported from the western countries, but none from India.

Pleurotus Eous mushroom lectin (PEL) with mixed carbohydrate inhibition and antiproliferative activity on tumor cell lines.

A novel Pleurotus eous mushroom lectin (PEL) having mixed binding specificity was purified to homogeneity from the fruitbodies of P. eous. The lectin showed monomer weight of 16 kDa and was a glycoprotein having a 9% neutral sugar content. The antiproliferative activity of PEL was tested on four human tumor cell lines. A 50% inhibition of proliferation was demonstrated in MCF-7, K562 and Hep2 cell lines at the lowest concentration (2 microg/ml) of lectin tested. SK-N-MC cell line showed 50% inhibition at 50 microg/ml of PEL. The inhibitory activity of PEL was not associated with toxicity to cell lines.

Differential responses of staurosporine on protein kinase C activity and proliferation in two murine neuroblastoma cell lines.

We studied the effect of staurosporine (SSP), a broad-spectrum protein kinase inhibitor, on the levels of protein kinase C (PKC) activity and proliferation in two murine neuroblastoma cell lines, Neuro2a and its clone NB41A3. The PKC activity was examined in whole cell lysate, cytosolic and particulate fractions. A differential response to SSP treatment in the enzyme activity in whole cell lysate and particulate fractions was demonstrated in the two cell lines. The data on proliferation indicated that Neuro2a cells were more sensitive to the SSP treatment with significant inhibition in DNA synthesis in a time course study. Our findings suggest that the data on basal levels of PKC activity in tumors will be of significance in studies using PKC inhibitors as an approach for therapeutic intervention.

Dielectrophoretic study of human erythrocytes.

This paper is concerned with the dielectrophoretic study of human erythrocytes under cylindrical field geometry. The influence of physical variables such as the frequency and voltage of the applied electric field, conductivity of the medium in which the cells are suspended, cell concentration and exposure time of the cell to the non-uniform electric field on dielectrophoretic collection rate (DCR) is determined in a systematic manner. It is interesting to note from the DCR spectrum of human erythrocytes that the DCR is minimum at one frequency, maximum at another and there is practically no yield over a certain frequency range. This may be attributed to the variation of complex dielectric constant of the particle and medium over that frequency range. From the DCR spectrum of different groups, it is clear that DCR behaviour is different in the frequency range from 0.3-1.5 MHz, under similar conditions of temperature, conductivity and concentration of erythrocyte suspension and strength of applied AC field. The response of DCR with voltage of the applied field, concentration of cell suspension and square root of elapsed time of the cells confirms the theory of dielectrophoresis.