Heterospecific pollen deposition: does diversity alter the consequences?

? In natural communities, plants can receive pollen from multiple heterospecifics as well as conspecifics. However, studies on the effects of interspecific pollen transfer have focused on interactions between species pairs. The potential exists for diverse interactions among heterospecific pollen (HP) grains on the stigma, and for these to affect plant reproduction, alone or in combination with conspecific pollen (CP) loss, but these interactions have not yet been explored. ? We used hand-pollinations to simulate increasing community diversity and CP loss on Mimulus guttatus stigmas. We used pollen mixes of one to three heterospecific donors to determine how species composition and CP load size affect seed production and to characterize the mechanisms underlying fertilization failure. ? Heterospecific pollen deposition reduced M. guttatus seed production and while the effect increased with the number of heterospecific donors, the strength depended on species composition and was independent of conspecific load size. Different types of interactions (additive and synergistic) are hypothesized to underlie the diverse effects on M. guttatus reproductive success. ? Our results suggest that an increase in the diversity of heterospecific donors will not always lead to a greater decrease in fitness because multispecies effects depend on the interacting species.     

A comparison of two in vitro maturation media for use with adult porcine oocytes for adult somatic cell nuclear transfer

Two media used to mature adult porcine oocytes for somatic cell nuclear transfer were compared. In the first experiment, parthenogenetic embryos were produced using a maturation medium used by us previously to clone pigs (OMM199) and that described by Kühholzer et al. (2001) to transport oocytes overnight (BOMED). There was no difference in maturation rates between the two different media. However, BOMED medium increased the percentage of parthenogenetic embryos that developed to the blastocyst stage compared with OMM199 (49% vs. 29%, respectively). In a second experiment, BOMED medium increased the percentage of SCNT embryos that developed to the blastocyst stage compared with OMM199 (22% vs. 8%, respectively). The efficiency of our cloning protocol using adult oocytes matured in BOMED medium was then determined by transferring SCNT embryos reconstructed using adult fibroblasts to synchronized recipients. Primary cultures of adult fibroblasts were obtained from two adult male pigs and used for SCNT (passages 2-4). Between 82 and 146 fused couplets were transferred to seven recipients synchronized 1 day behind the embryos. Five recipients (71% pregnancy rate) subsequently farrowed a total of 23 piglets (4.4 average litter size). Overall efficiencies (liveborn/embryos transferred) were 3.2% for all transfers and 4.3% for animals that gave birth.     

Actin polymerization in murine B lymphocytes is stimulated by cytochalasin D but not by anti-immunoglobulin.

One might predict that cytochalasin D, which slows polymerization of actin in solution and which inhibits actin-containing microfilament function in live B lymphocytes, would also prevent actin polymerization in these cells. However, we have used the NBD-Phallacidin flow cytometric assay for F-actin and the DNase I inhibition assay for G-actin to demonstrate that cytochalasin D (at 20 micrograms/ml and higher) stimulates actin polymerization in murine B lymphocytes within the first 30 sec of exposure. A similar response was seen in human neutrophils. Actin polymerization induced in neutrophils by chemotactic peptides has been linked to activation of the polyphosphoinositide-calcium increase-protein kinase C signal transduction pathway. As B lymphocytes also transduce signals using this pathway, we investigated whether cytochalasin D induced actin polymerization by activating this pathway. Cytochalasin D and ionomycin both stimulated a rapid increase in internal calcium (by 1 min) in the B cell which was inhibitable by EGTA, implicating calcium influx. Ionomycin also induced actin polymerization, detectable later, by 10 min. EGTA blocked the ionomycin-induced actin polymerization, but not that induced by cytochalasin D. Cytochalasin D-induced actin polymerization was not associated with detectable hydrolysis of polyphosphoinositides, nor was it inhibited by H7 (a protein kinase C inhibitor) or by HA1004 (an inhibitor of cyclic nucleotide-dependent kinases). Furthermore, anti-immunoglobulin antibodies, which stimulate B lymphocytes through the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, failed to induce actin polymerization in these cells. These antibodies did, however, stimulate the cells to perform activities that involve actin-containing microfilaments. Other primary activators of B lymphocytes (dextran sulfate, PMA, and LPS) and a panel of lymphokines previously shown to enhance B lymphocyte activation (IL-1, IL-2, IL-4, IL-5) were also screened in the F-actin assay and no evidence for actin polymerization was found. We conclude that the actin polymerization response to cytochalasin D in the B cell does not involve the polyphosphoinositide hydrolysis-calcium increase-protein kinase C pathway, nor does it depend on cyclic nucleotide-dependent kinases. Furthermore, our studies failed to provide any evidence that early actin polymerization occurs in murine B lymphocyte activation.     

A kinetic approach to defining the role of chemically modifiable residues at the active sites of enzymes.

Initial velocity studies can be used to define the function of chemically modifiable residues in the active site of a multisubstrate enzyme. Since the method relies on measuring biological activity, it has the advantage that it can be used with small amounts of relatively impure enzyme, but requires that modified enzyme molecules have some residual catalytic activity. The kinetic analysis of the modified enzyme can be carried out in the presence of some unmodified enzyme molecules. Consideration of examples of single- and multisubstrate enzymic reactions shows that interpretation of changes in apparent Km and apparent V values following chemical modification, in terms of effects on substrate binding or catalysis or both, requires a detailed knowledge of the kinetic reaction sequence. In the case of multisubstrate enzymes, it is necessary to ensure that, during the kinetic investigation, the concentrations of the non-varied substrates remain at near-saturating levels. For this reason, and because modification may induce changes in the rate-limiting step, a full kinetic analysis of the modified enzyme is advisable.