Effect of recombinant boar beta-acrosin on sperm binding to intact zona pellucida during in vitro fertilization

In a previous paper we demonstrated that boar beta-acrosin recombinant proteins were able to bind non-enzymatically to solubilized pig zona pellucida (ZP) glycoproteins. Here we report the participation of boar beta-acrosin in the secondary binding of sperm to intact pig ZP. This was achieved by using two boar recombinant proteins: beta-acrosin and a mutant of the catalytic site, beta-acrosin Ser/Ala(222). Assays of binding between the iodinated recombinant beta-acrosin and whole ZP showed that this binding could be saturated, was specific, and was stable over time. Using autoradiography, we determined that recombinant beta-acrosin bound on the entire surface of the ZP but initially was distributed heterogeneously. This suggests that the ligands for beta-acrosin may not be homogeneously distributed on the ZP. To study the contribution of acrosin in sperm secondary binding to the ZP, we preincubated in vitro-matured oocytes with these recombinant proteins and then performed in vitro fertilization assays. Under the experimental conditions used, binding of beta-acrosin recombinant proteins did not block sperm penetration. These results suggest that there may be other proteins that participate in the secondary binding, and that these proteins may recognize ligands that are different from those blocked by beta-acrosin recombinant proteins.     

A high-throughput system for two-hybrid screening based on growth curve analysis in microtiter plates

The yeast two-hybrid system is a powerful tool for identifying novel protein-protein interactions. In general, biochemical marker genes such as lacZ are exploited for indirect quantification of the interaction, and commonly involve the conduct of rather laborious beta-galactosidase assays. This paper describes a simple alternative method based on growth curve analysis of yeast cultures that is amenable to microtiter plate format, and therefore allows the quantification of large numbers of yeast two-hybrid combinations. The analyzed results of yeast cultures grown in microtiter plates were compared with those obtained from the classical beta-galactosidase assay. We conclude that the method presented here is reproducible, of equal or greater sensitivity than the beta-galactosidase assay, and can be further adapted for application to the conduct of large-scale, automated yeast two-hybrid experiments.     

Physical and functional interaction between protein kinase C delta and Fyn tyrosine kinase in human platelets

An increasing number of tyrosine kinases have been shown to associate with isoforms of the protein kinase C (PKC) family. Here, we show evidence for physical and functional interaction between PKCdelta and the Src family kinase Fyn in human platelets activated by alboaggregin-A, a snake venom capable of activating both GPIb-V-IX and GPVI adhesion receptors. This interaction involves phosphorylation of PKCdelta on tyrosine and is specific in that other isoforms of PKC, PKCepsilon and lambda, which also become tyrosine-phosphorylated, do not interact with Fyn. In addition, PKCdelta does not interact with other platelet-expressed tyrosine kinases Syk, Src, or Btk. Stimulation also leads to activation of both Fyn and PKCdelta and to serine phosphorylation of Fyn within a PKC consensus sequence. Alboaggregin-A-dependent activation of Fyn is blocked by bisindolylmaleimide I, suggesting a role for PKC isoforms in regulating Fyn activity. Platelet activation with alboaggregin-A induces translocation of the two kinases from cytoplasm to the plasma membrane of platelets, as observed by confocal immunofluorescence microscopy. Translocation of Fyn and PKCdelta are blocked by PP1 and bisindolylmaleimide I, showing a dependence upon Src and PKC kinase activities. Although PKC activity is required for translocation, it is not required for association between the two kinases, because this was not blocked by bisindolylmaleimide I. Rottlerin, which inhibited PKCdelta activity, did not block translocation of either PKCdelta or Fyn but potentiated platelet aggregation, 5-hydroxytryptamine secretion, and the calcium response induced by alboaggregin-A, indicating that this kinase plays a negative role in the control of these processes.     

Apollo: a sequence annotation editor

The well-established inaccuracy of purely computational methods for annotating genome sequences necessitates an interactive tool to allow biological experts to refine these approximations by viewing and independently evaluating the data supporting each annotation. Apollo was developed to meet this need, enabling curators to inspect genome annotations closely and edit them. FlyBase biologists successfully used Apollo to annotate the Drosophila melanogaster genome and it is increasingly being used as a starting point for the development of customized annotation editing tools for other genome projects.     

Respiratory effects in humans of a 5-day elevation of end-tidal PCO2 by 8 Torr.

The aims of this study were to determine 1) whether ventilatory adaptation occurred over a 5-day exposure to a constant elevation in end-tidal PCO2 and 2) whether such an exposure altered the sensitivity of the chemoreflexes to acute hypoxia and hypercapnia. Ten healthy human subjects were studied over a period of 13 days. Their ventilation, chemoreflex sensitivities, and acid-base status were measured daily before, during, and after 5 days of elevated end-tidal PCO2 at 8 Torr above normal. There was no major adaptation of ventilation during the 5 days of hypercapnic exposure. There was an increase in ventilatory chemosensitivity to acute hypoxia (from 1.35 +/- 0.08 to 1.70 +/- 0.07 l/min/%; P < 0.01) but no change in ventilatory chemosensitivity to acute hypercapnia. There was a degree of compensatory metabolic alkalosis. The results do not support the hypothesis that the ventilatory adaptation to chronic hypercapnia would be much greater with constant elevation of alveolar PCO2 than with constant elevation of inspired PCO2, as has been used in previous studies and in which the feedback loop between ventilation and alveolar PCO2 is left intact.     

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross-reacted with the anti-ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.     

Benzylidene ketal derivatives as M2 muscarinic receptor antagonists

Benzylidene ketal derivatives were investigated as selective M₂ receptor antagonists for the treatment of Alzheimer's disease. Compound 10 was discovered to have subnanomolar M₂ receptor affinity and 100-fold selectivity against other muscarinic receptors. Also, 10 demonstrated in vivo efficacy in rodent models of muscarinic activity and cognition.     

The Silver syndrome variant of hereditary spastic paraplegia maps to chromosome 11q12-q14, with evidence for genetic heterogeneity within this subtype

The hereditary spastic paraplegias (HSPs) are a complex group of neurodegenerative disorders characterized by lower-limb spasticity and weakness. Silver syndrome (SS) is a particularly disabling dominantly inherited form of HSP, complicated by amyotrophy of the hand muscles. Having excluded the multiple known HSP loci, we undertook a genomewide screen for linkage of SS in one large multigenerational family, which revealed evidence for linkage of the SS locus, which we have designated "SPG17," to chromosome 11q12-q14. Haplotype construction and analysis of recombination events permitted the minimal interval defining SPG17 to be refined to approximately 13 cM, flanked by markers D11S1765 and D11S4136. SS in a second family was not linked to SPG17, demonstrating further genetic heterogeneity in HSP, even within this clinically distinct subtype.