Female response to predation risk alters conspecific male behaviour during pre‐copulatory mate guarding

Mating behaviour often increases predation risk, but the vulnerability within mating pairs differs between the sexes. Such a sex difference is expected to lead to differences in responses to predation risk between the sexes. In the two‐spotted spider mite Tetranychus urticae, males engage in pre‐copulatory mate guarding because only the first mating results in fertilisation. We investigated (i) whether pre‐copulatory pairs are more conspicuous to the predatory mite Phytoseiulus persimilis than solitary females, (ii) whether the vulnerability to the predator differs between sexes within the pre‐copulatory pair, (iii) whether each sex of T. urticae responds to predation risk during pre‐copulatory mate guarding and (iv) whether T. urticae's response to predation risk affects predator behaviour. Because T. urticae females are immobile during pre‐copulatory mate guarding, we observed male behaviour to evaluate effects of predation risk. We found that the predators detect more pre‐copulatory pairs than solitary females and that more females than males of the pre‐copulatory pairs are preyed upon by the predators. The preference of spider mite males for pre‐copulatory pairs versus solitary females was affected by whether or not the female had been exposed to predators during development. Male T. urticae exposed to predation risk did not alter their behaviour. These results suggest that only the most vulnerable sex, that is the female, responds to predation risk, which modifies male behaviour. Regardless of T. urticae females’ experience, however, P. persimilis detected more T. urticae pre‐copulatory pairs than solitary females, suggesting that pre‐copulatory mate guarding itself is dangerous for T. urticae females when these predators are present. We discuss our results in the context of sex‐dependent differences in predation risk.     

Identification of CtpL as a Chromosomally Encoded Chemoreceptor for 4-Chloroaniline and Catechol in Pseudomonas aeruginosa PAO1

Bacterial chemotaxis influences the ability of bacteria to survive and thrive in most environments, including polluted ones. Despite numerous reports of the phenotypic characterization of chemotactic bacteria, only a few molecular details of chemoreceptors for aromatic pollutants have been described. In this study, the molecular basis of chemotaxis toward an environmentally toxic chlorinated aromatic pollutant, 4-chloroaniline (4CA), was evaluated. Among the three Pseudomonas spp. tested, Pseudomonas aeruginosa PAO1 exhibited positive chemotaxis both to the nonmetabolizable 4CA, where 4-chloroacetanilide was formed as a dead-end transformation product, and to the metabolizable catechol. Molecular analysis of all 26 mutants with a disrupted methyl-accepting chemotaxis gene revealed that CtpL, a chromosomally encoded chemoreceptor, was responsible for the positive chemotactic response toward 4CA. Since CtpL has previously been described to be a major chemoreceptor for inorganic phosphate at low concentrations in PAO1, this report describes a fortuitous ability of CtpL to function toward aromatic pollutants. In addition, its regulation not only was dependent on the presence of the chemoattractant inducer but also was regulated by conditions of phosphate starvation. These results expand the range of known chemotactic transducers and their function in the environmental bacterium PAO1.     

Enhanced Susceptibility to Spontaneous Seizures of Noda Epileptic Rats by Loss of Synaptic Zn2+

Zinc homeostasis in the brain is associated with the etiology and manifestation of epileptic seizures. Adult Noda epileptic rats (NER, >12-week-old) exhibit spontaneously generalized tonic-clonic convulsion about once a day. To pursue the involvement of synaptic Zn²⁺ signal in susceptibility to spontaneous seizures, in the present study, the effect of zinc chelators on epileptogenesis was examined using adult NER. Clioquinol (CQ) and TPEN are lipophilic zinc chelotors, transported into the brain and reduce the levels of synaptic Zn²⁺. The incidence of tonic-clonic convulsion was markedly increased after i.p. injection of CQ (30–100 mg/kg) and TPEN (1 mg/kg). The basal levels of extracellular Zn²⁺ measured by ZnAF-2 were decreased before tonic-clonic convulsion was induced with zinc chelators. The hippocampal electroencephalograms during CQ (30 mg/kg)-induced convulsions were similar to those during sound-induced convulsions in NER reported previously. Exocytosis of hippocampal mossy fibers, which was measured with FM4-64, was significantly increased in hippocampal slices from CQ-injected NER that did not show tonic-clonic convulsion yet. These results indicate that the abnormal excitability of mossy fibers is induced prior to epileptic seizures by injection of zinc chelators into NER. The incidence of tonic-clonic convulsion induced with CQ (30 mg/kg) was significantly reduced by co-injection with aminooxyacetic acid (5–10 mg/kg), an anticonvulsant drug enhancing GABAergic activity, which did not affect locomotor activity. The present paper demonstrates that the abnormal excitability in the brain, especially in mossy fibers, which is potentially associated with the insufficient GABAergic neuron activity, may be a factor to reduce the threshold for epileptogenesis in NER.     

Enhanced Active Targeting via Cooperative Binding of Ligands on Liposomes to Target Receptors

To achieve effective active targeting in a drug delivery system, we previously developed dual-targeting (DT) liposomes decorated with both vascular endothelial growth factor receptor-1 (VEGFR-1)-targeted APRPG and CD13-targeted GNGRG peptide ligands for tumor neovessels, and observed the enhanced suppression of tumor growth in Colon26 NL-17 tumor-bearing mice by the treatment with the DT liposomes encapsulating doxorubicin. In this present study, we examined the binding characteristics of DT liposomes having a different couple of ligands, namely, APRPG and integrin α_vβ₃-targeted GRGDS peptides. These DT liposomes synergistically associated to stimulated human umbilical vein endothelial cells compared with single-targeting (ST) liposomes decorated with APRPG or GRGDS. The results of a surface plasmon resonance assay showed that ST liposomes modified with APRPG or GRGDS peptide selectively bound to immobilized VEGFR-1 or integrin α_vβ₃, respectively. DT liposomes showed a higher affinity for a mixture of VEGFR-1 and integrin α_vβ₃ compared with ST liposomes, suggesting the cooperative binding of these 2 kinds of ligand on the liposomal surface. In a biodistribution assay, the DT liposomes accumulated to a significantly greater extent in the tumors of Colon26 NL-17 tumor-bearing mice compared with other liposomes. Moreover, the intratumoral distribution of the liposomes examined by confocal microscopy suggested that the DT liposomes targeted not only angiogenic endothelial cells but also tumor cells due to GRGDS-decoration. These findings suggest that "dual-targeting" augmented the affinity of the liposomes for the target cells and would thus be useful for active-targeting drug delivery for cancer treatment.     

Identification of a novel endochitinase from a marine bacterium Vibrio proteolyticus strain No. 442

Chitin binding proteins prepared from Vibrio proteolyticus were purified and the N-terminal amino-acid sequence of a protein from a 110-kDa band on SDS-PAGE was found to be 85-90% identical to the 22nd-41st residues of the N-termini of chitinase A precursor proteins from other vibrios. We cloned the corresponding gene, which encodes a putative protein of 850 amino acids containing a 26-residue signal sequence. The chitinase precursor from V. proteolyticus was 78-80% identical to those from Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio carchariae. However, the proteolytic cleavage site for C-terminal processing between R597 and K598 in the chitinase precursor of other vibrios was not observed in the amino acid sequence of V. proteolyticus, which instead had the sequence R600 and A601. Subsequently, full-length and truncated chitinases were generated in Escherichia coli. The specific activity of full-length chitinase expressed in E. coli was 17- and 20-folds higher for colloidal and alpha-chitins (insoluble substrate), respectively, than that of the C-terminal truncated enzyme. However, both recombinants showed similar hydrolysis patterns of hexa-N-acetyl-chitohexaose (soluble substrate), producing di-N-acetyl-chitobiose as major product on TLC analysis. We showed that the C-terminus of the V. proteolyticus chitinase A was important for expression of high specific activity against insoluble chitins.     

Systemic Simvastatin Rescues Retinal Ganglion Cells from Optic Nerve Injury Possibly through Suppression of Astroglial NF-κB Activation

Neuroinflammation is involved in the death of retinal ganglion cells (RGCs) after optic nerve injury. The purpose of this study was to determine whether systemic simvastatin can suppress neuroinflammation in the optic nerve and rescue RGCs after the optic nerve is crushed. Simvastatin or its vehicle was given through an osmotic minipump beginning one week prior to the crushing. Immunohistochemistry and real-time PCR were used to determine the degree of neuroinflammation on day 3 after the crushing. The density of RGCs was determined in Tuj-1 stained retinal flat mounts on day 7. The effect of simvastain on the TNF-α-induced NF-κB activation was determined in cultured optic nerve astrocytes. On day 3, CD68-positive cells, most likely microglia/macrophages, were accumulated at the crushed site. Phosphorylated NF-κB was detected in some astrocytes at the border of the lesion where the immunoreactivity to MCP-1 was intensified. There was an increase in the mRNA levels of the CD68 (11.4-fold), MCP-1 (22.6-fold), ET-1 (2.3-fold), GFAP (1.6-fold), TNF-α (7.0-fold), and iNOS (14.8-fold) genes on day 3. Systemic simvastatin significantly reduced these changes. The mean ± SD number of RGCs was 1816.3±232.6/mm² (n = 6) in the sham controls which was significantly reduced to 831.4±202.5/mm² (n = 9) on day 7 after the optic nerve was crushed. This reduction was significantly suppressed to 1169.2±201.3/mm² (P = 0.01, Scheffe; n = 9) after systemic simvastatin. Simvastatin (1.0 µM) significantly reduced the TNF-α-induced NF-κB activation in cultured optic nerve astrocytes. We conclude that systemic simvastatin can reduce the death of RGCs induced by crushing the optic nerve possibly by suppressing astroglial NF-κB activation.     

Changes in Expression of Aquaporin-4 and Aquaporin-9 in Optic Nerve after Crushing in Rats

The purpose of this study was to determine the temporal and spatial changes in the expression of AQP4 and AQP9 in the optic nerve after it is crushed. The left optic nerves of rats were either crushed (crushed group) or sham operated (sham group), and they were excised before, and at 1, 2, 4, 7, and 14 days later. Four optic nerves were pooled for each time point in both groups. The expression of AQP4 and AQP9 was determined by western blot analyses. Immunohistochemistry was used to determine the spatial expression of AQP4, AQP9, and GFAP in the optic nerve. Optic nerve edema was determined by measuring the water content in the optic nerve. The barrier function of the optic nerve vessels was determined by the extravasated Evans blue dye on days 7 and 14. The results showed that the expression of AQP4 was increased on day 1 but the level was significantly lower than that in the sham group on days 4 and 7 (P<0.05). In contrast, the expression of AQP9 gradually increased, and the level was significantly higher than that in the sham group on days 7 and 14 (P<0.05, Tukey-Kramer). The down-regulation of AQP4 was associated with crush-induced optic nerve edema, and the water content of the nerve was significantly increased by 4.3% in the crushed optic nerve from that of the untouched fellow nerve on day 7. The expression of AQP4 and GFAP was reduced at the crushed site where AQP4-negative and AQP9-positive astrocytes were present. The barrier function was impaired at the crushed site on days 7 and 14, restrictedly where AQP4-negative and AQP9-positive astrocytes were present. The presence of AQP9-positive astrocytes at the crushed site may counteract the metabolic damage but this change did not fully compensate for the barrier function defect.     

Trichinella nativa and Trichinella T9 in the Hokkaido island, Japan

Trichinella sp. muscle larvae were isolated from the thigh muscle of two red foxes (Vulpes vulpes) captured in Sapporo and Otofuke, Hokkaido, Japan, in 2003. Multiplex PCR designed for genotyping the genus Trichinella revealed that the Sapporo isolate showed a specific pattern to T. britovi complex (T. britovi, Trichinella T8 and Trichinella T9) and the Otofuke isolate showed that to T. nativa. Nucleotide sequences of a part of the mitochondrial cytochrome oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) of the Sapporo isolate showed the highest similarity to those of Trichinella T9, a species detected in the mainland of Japan. This study shows that both T. nativa and Trichinella T9 are circulating in wildlife of the Hokkaido island.     

Identification of an actin-binding site in p47phox an organizer protein of NADPH oxidase

Actin has been reported to enhance the superoxide-generating activity of neutrophil NADPH oxidase in a cell-free system and to interact with p47phox, a regulatory subunit of the oxidase. In the present study, we searched for an actin-binding site in p47phox by far-western blotting and blot-binding assays using truncated forms of p47phox. The amino-acid sequence 319-337 was identified as an actin-binding site, and a synthetic peptide of this sequence bound to actin. The sequence shows no homology to other known actin-binding motifs. It is located in the autoinhibitory region of p47phox and includes Ser-328, a phosphorylation site essential for unmasking. Although a phosphorylation-mimetic p47phox mutant bound to actin with a lower affinity than the wild type, the same mutant interacted with filamentous actin more efficiently than the wild type. A mutant peptide p47phox (319-337, Ser328Glu) bound to filamentous actin more tightly than to monomer actin. These results suggest that p47phox moves to cortical actin when it becomes unmasked in the cells.     

Role of Vac8 in formation of the vacuolar sequestering membrane during micropexophagy

Vac8 is a yeast vacuolar membrane protein involved in vacuolar membrane dynamics, e.g., vacuole inheritance and vacuolar membrane fusion. This protein is also necessary for a subset of autophagic pathways that deliver specific cellular components to the vacuole. In this study, we show that the micropexohagy and vacuole inheritance required distinct domain structures of Pichia pastoris Vac8 (PpVac8). Whereas vacuole inheritance required the Armadillo repeat (ARM) region that resides in the middle part of the protein, micropexophagy did not. Deletion of both the ARM and C-terminal domains inhibited a characteristic of vacuolar dynamics during micropexophagy, i.e., formation of the vacuolar sequestering membrane (VSM). Subsequent analyses indicated that PpVAC8 disruption abolished recruitment of PpAtg11, another protein required for formation of the VSM, to the vacuolar membrane. These results present a novel molecular function of PpVac8 in micropexophagy.