Detection of β-glucosidase as saprotrophic ability from an ectomycorrhizal mushroom, Tricholoma matsutake

We investigated extracellular carbohydrase production in the medium of an ectomycorrhizal fungus, Tricholoma matsutake, to reveal its ability to utilize carbohydrates such as starch as a growth substrate and to survey the saprotrophic aspects. We found β-glucosidase activity in the static culture filtrate of this fungus. The β-glucosidase was purified and characterized. The purified enzyme was obtained from about 2.1 l static culture filtrate, with 9.0% recovery, and showed a single protein band on SDS-PAGE. Molecular mass was about 160 kDa. The enzyme was most active around 60°C and pH 5.0, and stable over a pH of 4.0–8.0 for 30 min at 37°C. The purified enzyme was activated by the presence of Ca²⁺ and Mn²⁺ ions (about 2–3 times that of the control). The enzyme readily hydrolyzed oligosaccharides having a β-1,4-glucosidic linkage such as cellobiose and cellotriose. However, it did not hydrolyze polysaccharides such as avicel and CM-cellulose or oligosaccharides having an α-glucosidic linkage. Moreover, cellotriose was hydrolyzed by the enzyme for various durations, and the resultant products were analyzed by TLC. We concluded that the enzyme from T. matsutake seems to be a β-glucosidase because cellotriose with a β-1,4-glucosidic linkage decomposed to glucose during the enzyme reaction.     

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.     

Crystal structure of the RUN domain of the RAP2-interacting protein x

Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 A of resolution by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel alpha-helices, which form an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

Lipid polarity is maintained in absence of tight junctions

The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.     

Association of major histocompatibility complex class I haplotypes with disease progression after simian immunodeficiency virus challenge in burmese rhesus macaques

Nonhuman primate AIDS models are essential for the analysis of AIDS pathogenesis and the evaluation of vaccine efficacy. Multiple studies on human immunodeficiency virus and simian immunodeficiency virus (SIV) infection have indicated the association of major histocompatibility complex class I (MHC-I) genotypes with rapid or slow AIDS progression. The accumulation of macaque groups that share not only a single MHC-I allele but also an MHC-I haplotype consisting of multiple polymorphic MHC-I loci would greatly contribute to the progress of AIDS research. Here, we investigated SIVmac239 infections in four groups of Burmese rhesus macaques sharing individual MHC-I haplotypes, referred to as A, E, B, and J. Out of 20 macaques belonging to A(+) (n = 6), E(+) (n = 6), B(+) (n = 4), and J(+) (n = 4) groups, 18 showed persistent viremia. Fifteen of them developed AIDS in 0.5 to 4 years, with the remaining three at 1 or 2 years under observation. A(+) animals, including two controllers, showed slower disease progression, whereas J(+) animals exhibited rapid progression. E(+) and B(+) animals showed intermediate plasma viral loads and survival periods. Gag-specific CD8(+) T-cell responses were efficiently induced in A(+) animals, while Nef-specific CD8(+) T-cell responses were in A(+), E(+), and B(+) animals. Multiple comparisons among these groups revealed significant differences in survival periods, peripheral CD4(+) T-cell decline, and SIV-specific CD4(+) T-cell polyfunctionality in the chronic phase. This study indicates the association of MHC-I haplotypes with AIDS progression and presents an AIDS model facilitating the analysis of virus-host immune interaction.     

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.     

Effect of temperature on gelatinization and retrogradation in high hydrostatic pressure treatment of potato starch-water mixtures

The effect of temperature (20-70 °C) on the gelatinization and retrogradation of potato starch-water mixtures (10-70%, w/w) treated with high hydrostatic pressure (HHP) (400-1000 MPa) was investigated. Gelatinization enthalpy change (ΔH_gel) and re-gelatinization enthalpy change of retrograded crystalline part (ΔH_retro) of the HHP-treated starch were evaluated using differential scanning calorimetry. The value of ΔH_gel of 10-20% (w/w) mixtures decreased with increased pressure and temperature, while ΔH_gel of 30-50% (w/w) mixtures decreased to certain values with increased pressure and the values depended on treatment temperature. With higher temperature and pressure conditions, ΔH_gel of 10-40% (w/w) mixtures reached zero, but ΔH_gel of 50-70% (w/w) mixtures did not. Retrogradation was observed with HHP-treated 20-60% (w/w) mixtures and the value of ΔH_retro depended on the starch content, pressure, and temperature. The value of ΔH_retro trended to increase with increase in starch content. In addition, retrogradation was promoted by HHP treatment at low temperature. Gelatinizaiton and retrogradation behaviors of HHP-treated (400-1000 MPa) potato starch-water mixtures (10-70%, w/w) at 20-70 °C were summerized in a series of state diagrams.