Repeated evolution and reversibility of self‐fertilization in the volvocine green algae*

Outcrossing and self‐fertilization are fundamental strategies of sexual reproduction, each with different evolutionary costs and benefits. Self‐fertilization is thought to be an evolutionary “dead‐end” strategy, beneficial in the short term but costly in the long term, resulting in self‐fertilizing species that occupy only the tips of phylogenetic trees. Here, we use volvocine green algae to investigate the evolution of self‐fertilization. We use ancestral‐state reconstructions to show that self‐fertilization has repeatedly evolved from outcrossing ancestors and that multiple reversals from selfing to outcrossing have occurred. We use three phylogenetic metrics to show that self‐fertilization is not restricted to the tips of the phylogenetic tree, a finding inconsistent with the view of self‐fertilization as a dead‐end strategy. We also find no evidence for higher extinction rates or lower speciation rates in selfing lineages. We find that self‐fertilizing species have significantly larger colonies than outcrossing species, suggesting the benefits of selfing may counteract the costs of increased size. We speculate that our macroevolutionary results on self‐fertilization (i.e., non‐tippy distribution, no decreased diversification rates) may be explained by the haploid‐dominant life cycle that occurs in volvocine algae, which may alter the costs and benefits of selfing.     

EVIDENCE FOR TUBULAR MATING STRUCTURES INDUCED IN EACH MATING TYPE OF HETEROTHALLIC GONIUM PECTORALE (VOLVOCALES,CHLOROPHYTA)1

Gametes were induced separately in cultures of each mating type of the heterothallic, isogamous colonial volvocalean Gonium pectorale O. F. Müll. to examine the tubular mating structure (TMS) of both mating types plus and minus (plus and minus), referred to as “bilateral mating papillae.” Addition of dibutyryl cyclic adenosine monophosphate (DcAMP or db‐cAMP) and 3‐isobutyl‐1‐methylxanthine (IBMX) to approximately 3‐week‐old cultures of each mating type induced immediate release of naked gametes from the cell walls. Both plus and minus gametes formed a TMS in the anterior region of the protoplasts. Accumulation of actin was visualized by antibody staining in the TMS of both mating types as occurs in the TMS (fertilization tubule) of the plus gametes of the unicellular volvocalean Chlamydomonas reinhardtii P. A. Dang. Induction of naked gametes with a TMS in each mating type will be useful for future cell biological and evolutionary studies of the isogametes of colonial volvocalean algae.     

Ezrin Is Required for the Functional Regulation of the Epithelial Sodium Proton Exchanger,NHE3

The sodium hydrogen exchanger isoform 3 (NHE3) mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2^kd/kd) and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin) proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP) and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.     

Conformational analysis of chitobiose and chitosan

The relaxed potential energy surfaces of chitobiose were calculated based on the MM3-force field by optimizing dimer structures on a 10° grid spacing of the torsional angles about the glycosidic bonds (Φ,Ψ). The 36 conformations; the four combinations of the hydroxymethyl group orientations coupled with the nine of the secondary group ones— were assumed for each Φ,Ψ conformation. The four conformations, each differing in the hydroxymethyl group orientations, were considered for the whole Φ,Ψ space, and all the 36 conformations, for the restricted space of low energy. While the resulting energy map and the structures of the energy minima were similar to those proposed for cellobiose in many respects, more restricted energy profile was suggested for the relaxed map of chitobiose where differences in the energy level between the global minimum and the local minima were within 5.4 kcal/mol, compared with the equivalent value of 3.6 kcal/mol for cellobiose. Further depression of the global minimum occurred when the acidic residue was used. The Monte Carlo samples of the chitosan chain were generated based on the relaxed map to predict the unperturbed coil dimension in solution. The chitosan chains showed Gaussian behavior at x = 500 (x, degree of polymerization) and gave the characteristic ratio C_x, of about 70, which was much larger than the experimental values observed for the chitosan and cellulosic chains. © 1994 John Wiley & Sons, Inc.     

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses

Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ～330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ～128 kDa (～32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.     

Morphological and biochemical characterization of basal and starvation-induced autophagy in isolated adult rat cardiomyocytes

Autophagy is simultaneously a mode of programmed cell death and an important physiological process for cell survival, but its pathophysiological significance in cardiac myocytes remains largely unknown. We induced autophagy in isolated adult rat ventricular cardiomyocytes (ARVCs) by incubating them in glucose-free, mannitol-supplemented medium for up to 4 days. Ultrastructurally, intracellular vacuoles containing degenerated subcellular organelles (e.g., mitochondria) were markedly apparent in the glucose-starved cells. Microtubule-associated protein-1 light chain 3 was significantly upregulated among the glucose-starved ARVCs than among the controls. After 4 days, glucose-starved ARVCs showed a significantly worse survival rate (19+/-5.2%) than the controls (55+/-8.3%, P<0.005). Most dead ARVCs in both groups showed features of necrosis, and the rate of apoptosis did not differ between the groups. Two inhibitors of autophagy, 3-methyladenine (3-MA) and leupeptin, significantly and dose-dependently reduced the viability of both control and glucose-starved ARVCs and caused specific morphological alterations; 3-MA reduced autophagic findings, whereas leupeptin greatly increased the numbers and the sizes of vacuoles that contained incompletely digested organelles. The knockdown of the autophagy-related genes with small interfering RNA also reduced the glucose-starved ARVCs viability, but rapamycin, an autophagy enhancer, improved it. Reductions in the ATP content of ARVCs caused by glucose depletion were exacerbated by the inhibitors while attenuated by rapamycin, suggesting that autophagy inhibition might accelerate energy depletion, leading to necrosis. Taken together, our findings suggest that autophagy in cardiomyocytes reflects a prosurvival, compensatory response to stress and that autophagic cardiomyocyte death represents an unsuccessful outcome due to necrosis.     

The Simplest Integrated Multicellular Organism Unveiled

Volvocine green algae represent the “evolutionary time machine” model lineage for studying multicellularity, because they encompass the whole range of evolutionary transition of multicellularity from unicellular Chlamydomonas to >500-celled Volvox. Multicellular volvocalean species including Gonium pectorale and Volvox carteri generally have several common morphological features to survive as integrated multicellular organisms such as “rotational asymmetry of cells” so that the cells become components of the individual and “cytoplasmic bridges between protoplasts in developing embryos” to maintain the species-specific form of the multicellular individual before secretion of new extracellular matrix (ECM). However, these morphological features have not been studied in the four-celled colonial volvocine species Tetrabaena socialis that is positioned in the most basal lineage within the colonial or multicellular volvocine greens. Here we established synchronous cultures of T. socialis and carried out immunofluorescence microscopic and ultrastructural observations to elucidate these two morphological attributes. Based on immunofluorescence microscopy, four cells of the mature T. socialis colony were identical in morphology but had rotational asymmetry in arrangement of microtubular rootlets and separation of basal bodies like G. pectorale and V. carteri. Ultrastructural observations clearly confirmed the presence of cytoplasmic bridges between protoplasts in developing embryos of T. socialis even after the formation of new flagella in each daughter protoplast within the parental ECM. Therefore, these two morphological attributes might have evolved in the common four-celled ancestor of the colonial volvocine algae and contributed to the further increase in cell number and complexity of the multicellular individuals of this model lineage. T. socialis is one of the simplest integrated multicellular organisms in which four identical cells constitute the individual.     

Loureirin C and Xanthoceraside Prevent Abnormal Behaviors Associated with Downregulation of Brain Derived Neurotrophic Factor and AKT/mTOR/CREB Signaling in the Prefrontal Cortex Induced by Chronic Corticosterone Exposure in Mice

Neurochemical Research - Brain derived neurotrophic factor (BDNF) is one of the most abundant neurotrophic factors, and its deficits are involved in the pathogenesis of major depressive disorders...