Positive selection is a general phenomenon in the evolution of abalone sperm lysin

Lysin is a 16kDa acrosomal protein used by abalone sperm to create a hole in the egg vitelline envelope (VE). The interaction of lysin with the VE is species-selective and is one step in the multistep fertilization process that restricts heterospecific (cross-species) fertilization. For this reason, the evolution of lysin could play a role in establishing prezygotic reproductive isolation between species. Previously, we sequenced sperm lysin cDNAs from seven California abalone species and showed that positive Darwinian selection promotes their divergence. In this paper an additional 13 lysin sequences are presented representing species from Japan, Taiwan, Australia, New Zealand, South Africa, and Europe. The total of 20 sequences represents the most extensive analysis of a fertilization protein to date. The phylogenetic analysis divides the sequences into two major clades, one composed of species from the northern Pacific (California and Japan) and the other composed of species from other parts of the world. Analysis of nucleotide substitution demonstrates that positive selection is a general process in the evolution of this fertilization protein. Analysis of nucleotide and codon usage bias shows that neither parameter can account for the robust data supporting positive selection. The selection pressure responsible for the positive selection on lysin remains unknown.      

Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.      

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Evaluation of the immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins

Immunological activity and safety of group B meningococcal vaccine prepared from a natural complex of specific polysaccharide and outer membrane proteins were under study. The immunological safety of the vaccine was evaluated by the absence of antibodies to denaturated and native DNA (d-DNA and n-DNA). As shown with the use of the enzyme immunoassay (EIA), the administration of the vaccine did not induce antibody formation to d-DNA and n-DNA during the observation period. The titer of bactericidal antibodies in the immune bacteriolysis assay (IBA) to the vaccine strain B:2b:P1.2 after immunization increased four-fold and greater in 80% of the vaccinated persons. The significant increase of bactericidal antibodies to heterologous strains B:2a:P1.2 and B:15:P1.7 was registered in 20-30% of the vaccinees, respectively. A month after the repeated vaccination an increase in specific IgG antibodies to the complex antigen was found to occur according to EIA results. The use of RIB made it possible to evaluate the preventive activity of group B meningococcal vaccine as a whole and to suppose that the vaccine induced mainly type-specific response.     

Water self-diffusion behavior in yeast cells studied by pulsed field gradient NMR

The water self-diffusion behavior in yeast cell water suspension was investigated by pulsed field gradient NMR techniques. Three types of water were detected, which differ according to the self-diffusion coefficients: bulk water, extracellular and intracellular water. Intracellular and extracellular water self-diffusion was restricted; the sizes of restriction regions were approximately 3 and 15-20 microm, respectively. The smallest restriction size was determined as inner cell size. This size and also cell permeability varied with the growth phase of yeast cell. Cell size increased, but permeability decreased with increasing growth time. The values of cell permeabilities P(1)(d) obtained from time dependence of water self-diffusion coefficient were in good agreement with the permeabilities obtained from the exchange rate constants P(1)(eff). The values of P(1)(eff) were 7 x 10(-6), 1.2 x 10(-6) and 1.6 x 10(-6) m/s, and P(1)(d) were 6.3 x 10(-6), 8.4 x 10(-7), 1.5 x 10(-6) m/s for yeast cells incubated for 9 h (exponential growth phase), 24 h (end of exponential growth phase), and 48 h (stationary growth phase), respectively.     

NMR-investigation of restricted self-diffusion of oil in rape seeds

The self-diffusion of oil and water in rape seeds (Brassica napus L.) was measured with the NMR pulsed field gradient technique. The self-diffusion of oil was found to be completely restricted for diffusion times > 30 ms. The experiments could be explained in terms of the model of diffusion within spherical droplets and a Gaussian mass distribution of the droplet radii. The mean droplet radius was found to be about 0.7 m; this value decreased somewhat with increasing moisture content of the seeds. The experiments could also be explained with a Gaussian number distribution of droplet radii and a fraction of immobile protons in the NMR signal of 5 ... 10%, possibly arising from lipid protons. Though the transverse nuclear magnetic relaxation decay of the oil protons is not a single exponential we observe one uniform diffusive mobility for the oil molecules. The water self-diffusion coefficient at maximum moisture content of about 40% was determined to be 4.2 · 10^-10 m² s^-1 which is typical for swollen polymer-solvent systems at such a concentration. Offprint requests to: P. W. Kuchel     

Generalized concentration dependence of globular protein self-diffusion coefficients in aqueous solutions.

The self-diffusion coefficients of globular proteins (myoglobin, bovine serum albumin, barstar, lysozyme) in aqueous solutions at different temperatures and pH values are obtained by pulsed-gradient spin-echo NMR, and their concentration dependence is analyzed. The generalized concentration dependence of globular protein self-diffusion coefficients is empirically established, and compared to the concentration dependence of diffusion coefficients of flexible polymers and rigid Brownian particles.