Pacific oyster hemocytes undergo apoptosis following cell-adhesion mediated by integrin-like molecules

Previously, we demonstrated that in the Pacific oyster (Crassostrea gigas, a bivalve mollusc) apoptosis could be induced in hemocytes by treatment with Arg-Gly-Asp (RGD) peptides which are known to function as an integrin ligand. However, it is unclear where the RGD peptides are binding to the C. gigas hemocytes, or what mechanism or molecules are involved, e.g., integrin-ligand interactions. Therefore, this study was undertaken to investigate the binding interactions in C. gigas hemocytes. Initially, to confirm the presence of RGD-recognizing integrin-like molecule(s) on the hemocytes, we assessed the enhancement of spreading ability, and found that spreading ability was enhanced by immobilized human fibronectin, a fibronectin fragment containing the RGD motif, and C. gigas plasma in the presence of divalent cations. Interestingly, viability of the spreading hemocytes dramatically decreased 24 h later and DNA fragmentation with oligonucleosomal laddering of 180-200 bp in length was detected in the dead hemocytes by electrophoresis and TUNEL assay. These results indicated that hemocyte adhesion mediated by integrin-like molecules triggered apoptosis and suggested that integrin-activation contributes to the induction of apoptosis. This is the first report showing the possibility of an integrin functioning in the induction of apoptosis in invertebrate hemocytes.     

Description of alternative male reproductive tactics in a shell-brooding cichlid, Telmatochromis vittatus, in Lake Tanganyika

Telmatochromis vittatus (Cichlidae) is a Tanganyikan substrate brooder which spawns in the gastropod-shell nests of a cichlid, Lamprologus callipterus. We describe male reproductive tactics of T. vittatus in and around the shell nests, where males of various sizes were found. Based on utilization patterns of the shell nests, interactions among males, and spawning behaviors, males could be categorized into four types based on reproductive tactics and in order of body size: sneaker males, satellite males, territorial males and piracy males. Size range of males in tactic groups rarely overlapped. Territorial males defended shell nests harboring multiple females, but during pair-spawning they were occasionally taken over by large piracy males that visited several nests repeatedly. Small sneaker males darted to pair-spawning territorial males and might ejaculate sperm. Satellite males did not perform parasitic spawning but pair-spawned in a single shell outside the nests. Spawning of satellite males was infrequently parasitized. The largest gonado-somatic index (GSI) was found in sneaker males followed by piracy males, territorial males and satellite males, suggesting that gonadal investment of males using the four tactics may be consistent with intensity or risk of sperm competition.     

Testes Investment along a Vertical Depth Gradient in an Herbivorous Fish

Males are expected to adjust testes investment according to the varying level of sperm competition that they experience. Spatial and temporal variation in population density likely influences sperm competition. In herbivorous aquatic organisms, densities often decrease along a vertical depth gradient, because their food is photosynthetic and thus becomes less abundant in deeper regions where less light penetrates. This decrease should be dramatic on a steep slope, which allows testing of the association between density and testes investment at the within‐population level. We tested the effect in the socially monogamous herbivorous cichlid fish Variabilichromis moorii living on a steep slope in Lake Tanganyika. We examined competitor density and food abundance as ecological factors, territory defense behaviors and phenotypic traits (testes investment and somatic investment), and compared them between shallow (4–6 m depth) and deep habitats (10–13 m depth) separated by several dozen meters. We found that food availability drastically decreased with increasing depth and that V . moorii was much more abundant in shallower habitats. Males in shallower habitats were in better physical condition (based on fat and liver mass) despite experiencing greater costs in terms of territory defense. Testes investment differed in areas with different competitor density and food abundance along a vertical depth gradient, but competitor density was the most explanatory factor of the difference. This suggests that this herbivorous fish would change testes investment in response to population density.     

Active bovine selenophosphate synthetase 2, not having selenocysteine

During the course of studying selenocysteine (Sec) synthesis mechanisms in mammals, we prepared selenophosphate synthetase (SPS) from bovine liver by 4-step chromatography. In the last step of chromatography of hydroxyapatite, we found a protein band of molecular mass 33 kDa on SDS-PAGE, consistent with the pattern of SPS activity that was indirectly manifested by [⁷⁵Se]Sec production activity; however, we could not detect significant Se content in this active fraction. We also found a clear band of 33 kDa by Western blotting with antibody against a common peptide (387-401) in SPS2. We detected selenophosphate as the product of this active enzyme in the reaction mixture, composed of ATP, [⁷⁵Se]H₂Se and SPS. Chemically synthesized selenophosphate plays a role in Sec synthesis, not the addition of this enzyme. These results support that the product of SPS2 is selenophosphate itself. During this investigation, the probable sequence of bovine SPS2 not having Sec was reported in the blast information and the molecular mass was near with the protein in this report. Thus, bovine active SPS2 of molecular mass 33 kDa does not contain Sec. K. Furumiya and K. Kanaya contributed equally to this work.     

Reevaluation and Reduction of a PCR Bias Caused by Reannealing of Templates

We reevaluated the bias toward a 1:1 ratio of products in multitemplate PCR used in ecological studies and showed that the template reannealing at the annealing step would not cause the bias; however, the preferential homoduplex formation during temperature decrease from denaturation to annealing step would cause the bias.     

Rho-kinase modulates the function of STEF, a Rac GEF, through its phosphorylation

Rho family GTPases are key regulators of various physiological processes. Several recent studies indicated that the antagonistic relationship between Rho and Rac is essential for cell polarity and that the Rac activity is negatively regulated by Rho. In this study, we found that Rho-kinase, an effector of Rho, counteracted the Rac GEF STEF-induced Rac1 activation in COS7 cells. Rho-kinase phosphorylated STEF at Thr1662 in vitro, and Y-27632, a Rho-kinase inhibitor, suppressed lysophosphatidic acid-induced phosphorylation of STEF in PC12D cells. STEF interacted with specific molecules such as microtubule-associated protein 1B, and the phosphorylation of STEF by Rho-kinase diminished its interaction with these molecules. STEF promoted nerve growth factor-induced neurite outgrowth in PC12D cells, while the phosphomimic mutant of STEF had a weakened ability to enhance neurite outgrowth. Taken together, these results suggest that the phosphorylation of STEF by Rho-kinase exerts the inhibitory effect on the function of STEF.     

Mitochondrial dysfunction and increased reactive oxygen species impair insulin secretion in sphingomyelin synthase 1-null mice

Sphingomyelin synthase 1 (SMS1) catalyzes the conversion of ceramide to sphingomyelin. Here, we generated and analyzed SMS1-null mice. SMS1-null mice exhibited moderate neonatal lethality, reduced body weight, and loss of fat tissues mass, suggesting that they might have metabolic abnormality. Indeed, analysis on glucose metabolism revealed that they showed severe deficiencies in insulin secretion. Isolated mutant islets exhibited severely impaired ability to release insulin, dependent on glucose stimuli. Further analysis indicated that mitochondria in mutant islet cells cannot up-regulate ATP production in response to glucose. We also observed additional mitochondrial abnormalities, such as hyperpolarized membrane potential and increased levels of reactive oxygen species (ROS) in mutant islets. Finally, when SMS1-null mice were treated with the anti-oxidant N-acetyl cysteine, we observed partial recovery of insulin secretion, indicating that ROS overproduction underlies pancreatic β-cell dysfunction in SMS1-null mice. Altogether, our data suggest that SMS1 is important for controlling ROS generation, and that SMS1 is required for normal mitochondrial function and insulin secretion in pancreatic β-cells.