Efficient selection intensity in early generation index selection in groundnut (Arachis hypogaea L.)

Summary The F₂ potential of single and three-way crosses was evaluated using a set of physiological and yield components. Results were based on an index of selection using (a) only yield components and (b) both physiological and yield components. The indices were constructed using the percentage improvement of F₂ over the better parent of the corresponding F₁ cross for every character. The performance of F₂ plants assessed by the expected value of the regression index was ranked in descending order to provide a ranked F₂ distribution (FRD). The FRD was divided into four equal parts, T₂₅ (top 25%), T₅₀ (26–50%), T₇₅ (51–75%) and T₁₀₀ (76–100%). F₃ families derived from F₂ plants in T₂₅ were found to provide a higher frequency of selections for pod number than T₅₀, T₇₅ and T₁₀₀. The frequency of selections was higher in three-way than single crosses. Selection index based on physiological and yield components was more efficient in trapping F₂ plants providing selections in F₃ than the index based on yield components only. The results brought out the importance of bunch x bunch crosses as a complement to the usually advocated bunch x runner ones.     

Multiple myosin II heavy chain kinases: roles in filament assembly control and proper cytokinesis in Dictyostelium

Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B(-)/C(-) triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.     

Isolation and characterization of hyaluronidase from scorpion (Heterometrus fulvipes) venom

Hyaluronidase (Hyaluronate lyase, E.C. 3.2.1.35) has been isolated from Heterometrus fulvipes scorpion venom by a combination of gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE-cellulose. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and a molecular weight of 82,000. The final preparation was purified 27-fold. The optimum pH for enzyme activity was 4.0. No loss of activity was observed up to 30 degrees C and showed a sharp decrease in activity at 50 degrees C. Heparin inhibited the enzyme activity.     

Purification and characterization of Cu-Zn superoxide dismutases from mungbean (Vigna radiata) seedlings

Mungbean contains three isoenzymes of superoxide dismutase designated isoenzyme I, II and III. The two cytosolic superoxide dismutases (I and II) were purified to homogeneity by ammonium sulphate fractionation, ion exchange chromatography on diethylaminoethyl cellulose, gel filtration and preparative polyacrylamide.gel electrophoresis. The molecular weights of isoenzyme I and isoenzyme II were determined to be 33,000 and 31,600 respectively. The subunit molecular weight was approximately 16,000 indicating that the isoenzymes contained two identical subunits. The ultra-violet absorption spectra revealed a maximum at 258–264 nm for the two isoenzymes. Superoxide dismutase I and II were inhibited to different extents by metal chelators. Isoenzyme I was more sensitive to inhibition by cyanide and azide, while isoenzyme II was more susceptible to inhibition by diethyldithiocarbamate ando-phenanthroline. Both the isoenzymes exhibited similar denaturation profiles with heat, guanidinium chloride and urea. The denaturation with urea and guanidinium chloride was reversible. The two copper-zinc enzymes were more stable towards thermal inactivation compared to manganese and iron superoxide dismutases from other sources. The results indicate that the two isoenzymes differ from each other only with respect to charge and sensitivity towards metal chelators.     

Myosin-IIA heavy-chain phosphorylation regulates the motility of MDA-MB-231 carcinoma cells

In mammalian nonmuscle cells, the mechanisms controlling the localized formation of myosin-II filaments are not well defined. To investigate the mechanisms mediating filament assembly and disassembly during generalized motility and chemotaxis, we examined the EGF-dependent phosphorylation of the myosin-IIA heavy chain in human breast cancer cells. EGF stimulation of MDA-MB-231 cells resulted in transient increases in both the assembly and phosphorylation of the myosin-IIA heavy chains. In EGF-stimulated cells, the myosin-IIA heavy chain is phosphorylated on the casein kinase 2 site (S1943). Cells expressing green fluorescent protein-myosin-IIA heavy-chain S1943E and S1943D mutants displayed increased migration into a wound and enhanced EGF-stimulated lamellipod extension compared with cells expressing wild-type myosin-IIA. In contrast, cells expressing the S1943A mutant exhibited reduced migration and lamellipod extension. These observations support a direct role for myosin-IIA heavy-chain phosphorylation in mediating motility and chemotaxis.     

Haemolytic effect of the scorpion Heterometrus fulvipes venom

The haemolytic effect of the venom from scorpion Heterometrus fulvipes was studied using sheep erythrocytes. While there was no haemolytic effect seen directly by the erythrocytes, erythrocytes sensitized with homologous haemolysin were lysed by the venom. This factor in Heterometrus fulvipes venom having a 'complement like' haemolytic effect was found to be thermolabile and dialysable and sensitive to the action of 2-mercaptoethanol. It was identified as a low molecular weight peptide containing disulphide groups.     

Generation of double gene disruptions in Dictyostelium discoideum using a single antibiotic marker selection

Gene targeting is a powerful molecular genetic technique that has been widely used to understand specific gene function in vivo. This technique allows the ablation of an endogenous gene by recombination between an introduced DNA fragment and the homologous target gene. However, when multiple gene disruptions are needed, the availability of only a limited number of marker genes becomes a complication. Here we describe a new approach to perform double gene disruptions in Dictyostelium discoideum by simultaneous transfection of two gene targeting cassettes followed by performing clonal selection against only one marker gene. The subsequent PCR-based screens of blasticidin-resistant clones revealed the integration of both the selected and the nonselected targeting cassettes at their original respective loci creating complete gene disruptions. For the genes we have tested in these studies (myosin heavy chain kinases B and C), the efficiency of the double gene targeting event is found in the range of 2%-5% of all blasticidin-resistant colonies following the transfection step. This approach for the simultaneous disruptions of multiple genes should prove to be a valuable tool for other laboratories interested in creating multiple gene disruptants in Dictyostelium or other organisms where a limited number of selectable markers are available.     

A proteomic study of myosin II motor proteins during tumor cell migration

Myosin II motor proteins play important roles in cell migration. Although myosin II filament assembly plays a key role in the stabilization of focal contacts at the leading edge of migrating cells, the mechanisms and signaling pathways regulating the localized assembly of lamellipodial myosin II filaments are poorly understood. We performed a proteomic analysis of myosin heavy chain (MHC) phosphorylation sites in MDA-MB 231 breast cancer cells to identify MHC phosphorylation sites that are activated during integrin engagement and lamellar extension on fibronectin. Fibronectin-activated MHC phosphorylation was identified on novel and previously recognized consensus sites for phosphorylation by protein kinase C and casein kinase II (CK-II). S1943, a CK-II consensus site, was highly phosphorylated in response to matrix engagement, and phosphoantibody staining revealed phosphorylation on myosin II assembled into leading-edge lamellae. Surprisingly, neither pharmacological reduction nor small inhibitory RNA reduction in CK-II activity reduced this stimulated S1943 phosphorylation. Our data demonstrate that S1943 phosphorylation is upregulated during lamellar protrusion, and that CK-II does not appear to be the kinase responsible for this matrix-induced phosphorylation event.