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.      

Sodium alterations in isolated rat heart during cardioplegic arrest

Schepkin, V. D., I. O. Choy, and T. F. Budinger. Sodiumalterations in isolated rat heart during cardioplegic arrest. J. Appl. Physiol. 81(6):2696-2702, 1996.Triple-quantum-filtered (TQF) Na nuclearmagnetic resonance (NMR) without chemical shift reagent is used toinvestigate Na derangement in isolated crystalloid perfused rat heartsduring St. Thomas cardioplegic (CP) arrest. Theextracellular Na contribution to the NMR TQF signal of a rat heart isfound to be 73 ± 5%, as determined by wash-out experiments atdifferent moments of ischemia and reperfusion. With the use of thiscontribution factor, the estimated intracellular Na([Na⁺]_i)TQF signal is 222 ± 13% of preischemic level after 40 min of CParrest and 30 min of reperfusion, and the heart rate pressure productrecovery is 71 ± 8%. These parameters aresignificantly better than for stop-flow ischemia: 340 ± 20% and 6 ± 3%, respectively. At 37°C, the initial delay of 15 min in[Na⁺]_igrowth occurs during CP arrest along with reduced growth later (~4.0%/min) in comparison with stop-flow ischemia (~6.7%/min). The hypothermia (21°C, 40 min) for the stop-flow ischemia and CPdramatically decreases the[Na⁺]_igain with the highest heart recovery for CP (~100%). These studiesconfirm the enhanced sensitivity of TQF NMR to[Na⁺]_iand demonstrate the potential of NMR without chemical shift reagent tomonitor[Na⁺]_iderangements.

     

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.      

2D NMR of the Metabolic Antioxidant Dihydrolipoic Acid and its Derivatives

Dihydroplipoate and lipoate are physiological thiols which in addition to their coenzyme functions exhibit antioxidant activity. For NMR investigations of their protective mechanism in biological and model systems it is very important to know the full assignment of proton and carbon spectra of these molecules in water (D₂O). An unambiguous assignment of proton and carbon NMR spectra has been made for dihydrolipoate and its short chain derivatives bisnor-and tetranor-lipoic acid in D₂O and CDCl₃ solutions using 2D NMR methods.

Oxidation of dihydrolipoic acid produces substantial electron density deshielding of the carbons nearest to the SH groups with the largest shift found at the inner SH group (17.79 ppm in D₂O, 16.93 in CDCl₃) and almost no changes in the tail portion of the molecule. However, bisnor-dihydrolipoic acid and especially tetranor-dihydrolipoic acid have more carbon deshielding near the outer SH group of the molecule which correlates with their known diminished ion chelating activity.

Moreover, the proton triplet at position 2 of lipoic acid has strong pH dependence (pK = 4.58) due to the close proximity to the carboxylic group and this feature may be used for monitoring pH.     

A conjugate of a tumor-targeting ligand and a T cell costimulatory antibody to treat brain tumors

T cell immunotherapy is a potential strategy for the treatment of brain tumors because it offers a high degree of specificity, the ability to extravasate into solid tumors, and the potential for eliciting a long-term protective immune response. Various approaches have been developed to overcome T cell immune tolerance to cancer, including the use of cytokines and bispecific antibodies. T cell stimulation with the proinflammatory cytokine IL-12 can elicit antitumor immunity. T cell activation can be increased using bispecific antibodies against activating molecules on the surface of T cells and a tumor antigen. We studied the effects of systemic IL-12 administration in combination with a conjugate of an anti-CD28 antibody and a ligand for the folate receptor. The high affinity folate receptor is expressed on endogenously arising choroid plexus tumors of SV11 mice, which are transgenic for large T antigen under the control of the SV40 promoter. SV11 mice are immunocompetent, yet immunologically tolerant to large T antigen expressed by choroid plexus tumors. MRI analysis showed that the administration of IL-12 and anti-CD28 Fab/folate significantly slowed tumor growth. Proliferating CD8(+) T cells were found in choroid plexus tumors of treated animals. Treatment of animals with IL-12 + anti-CD28 Fab/folate prolonged survival compared to IL-12 alone. Cytokine treatment combined with tumor-targeted costimulation may be a useful adjunct treatment.