Bacterial Diversity and Structural Changes of Oyster Shell during 1-Year Storage

We examined the biodiversity of bacteria associated with oyster-shell waste during a 1-year storage period using 16S ribosomal DNA analysis. Temperature variation and structural changes of oyster shell were observed during storage. Initial and final temperatures were at 16-17 degrees C, but a high temperature of about 60 degrees C was recorded after approximately 6 months of storage. The crystal structure and nanograin of the oyster shell surface were sharp and large in size initially and became gradually blunter and smaller over time. Phylogenetic analysis revealed that Firmicutes were dominant in the oyster-shell waste initially, during the high-temperature stage, and after 1 year of storage (making up >65% of the biodiversity at all three sampling times). Bacillus licheniformis was presumed as the predominate Firmicutes present. These bacteria are likely to have important roles in the biodegradation of oyster shell.     

Plant PRA plays an important role in intracellular vesicular trafficking between compartments as GDF

Rab GTPases like Ras-related monomeric GTPases are well known to regulate intracellular vesicle trafficking by cycling between membrane-bound and cytosolic states. The functions of these proteins are controlled by upstream regulators and downstream effectors. Ypt/Rabs transmit signals to downstream effectors in a GTP-dependent manner. GDP-bound Rab proteins are extracted from their target membrane by cytosolic proteins known as GDP dissociation inhibitors (GDIs), and the Rab GTPase is recruited to the membrane compartment following dissociation from the GDI by GDI displacement factor (GDF). Now, we''re going to discuss the role of plant PRA concerted with Rab and GDI proteins by recycling Rab between membrane and cytosol for intracellular trafficking of cargo proteins.Key words: GDF, GDI, PRA1, Rab, vacuolar trafficking, vesicle traffickingAlthough Rabs appear to undergo multiple cycles of GDI-mediated delivery to, and extraction from membranes,¹ the mechanisms underlying Rab membrane delivery and association by GDI and other factors remain unclear. GDP-GTP exchange occurs at the target membrane, catalyzed by a guanine nucleotide exchange factor (GEF),^2,3 and the GTP-bound Rab transmits signals to downstream effectors and associates with the membrane to ensure proper docking and fusion of transport vesicles.⁴ After vesicle fusion on its target membrane, subsequently hydrolysis of GTP by the Rab is facilitated by GTPase-activating proteins (GAPs).⁴ The resulting GDP-bound Rab is subsequently retrieved from the membrane by GDI, which then maintains Rab in the cytosol to complete the cycle.Many research groups isolated PRAs, a homolog of human YIP3, in several two-hybrid screenings as interacting with multiple Rabs in their GTP- or GDP-bound form.^5,6 PRA contains two extensive hydrophobic domains which may form a membrane-spanning domain or the inner hydrophobic core of the protein.⁷ PRA1 is localized to the Golgi and late endosomes,⁸ and the related PRA2 is present in the endoplasmic reticulum.⁹Recently it has been shown that the human YIP3 stimulates the rate of nucleotide binding to Rab9 when added to prenyl Rab9-GDI complex and catalyzes the dissociation of the endosomal Rab-GDI complex, indicating that YIP3 is a GDI displacement factor that recruits Rab to membranes.¹⁰ According to the Gougeon et al. report (2002),¹¹ PRA1 inhibits the extraction of membrane-bound Rab3A by GDI1, suggesting that recycling of Rab depends on the opposing actions of PRA and GDI, with PRA favoring membrane retention but GDI favoring solubilization.Moreover, mammalian PRA1 is required for vesicle formation from the Golgi complex and might influence the recruitment of Rab effectors during cargo sequestration as well as proteins required for subsequent vesicle docking and fusion.¹¹ This is consistent with its transport function based on interaction of yeast homologue Yip3p with proteins in the secretory pathway.⁷ Yip1-Yif1p complex binds to the ER and to the Golgi SNAREs, Bos1p and Sec22p, and is required for membrane fusion machinery.⁵ In addition, a role of Yip1p had also been proposed in COPII vesicle biogenesis.¹²To our current knowledge there is no report on the physiological role of a GDF in plants. Aims to enrich the understanding of the mechanism of Rab recycling and trafficking pathways in plant, we identified and characterized OsPRA1, a rice homolog of PRA. OsPRA1, isolated by yeast two-hybrid screening using OsRab7 as bait, localized to the prevacuolar compartment as a membrane protein.¹³ Additionally, through western blot and protoplast transient assays it was confirmed that OsPRA1 has GDF activity, which dissociates the Rab7-GDI2 complex and recruits dissociated Rab from the Rab7-GDI2 complex to the donor membrane (unpublished data). When yeast two-hybrid interaction assay between OsPRA1 and OsGDI2 was performed, OsPRA1 interacted with OsGDI2 weakly (unpublished data), supporting our proposition that OsPRA1 dissociates the OsRab7-OsGDI2 complex.Furthermore, by using yeast two-hybrid and co-immunoprecipitation assays it was demonstrated that OsPRA1 interacted with dominant negative OsRab7 (T22N) which has no GTP binding activity, but not the constitutively active OsRab7 (Q67L),¹³ indicating that OsPRA1 may interact with GDP-bound OsRab7 at the donor membrane, PVC. These results support that OsPRA1 is a GDF for OsRab7.Subsequently, in order to find its interacting proteins implicated in vesicular trafficking, such as t- or v-SNAREs, yeast two-hybrid screening using OsPRA1 as bait was performed. Interestingly, a t-SNARE, OsVam3p, homolgous to AtVam3p involved in vacuolar trafficking and localizing to both PVC and vacuole membranes in Arabidopsis,¹⁴ was isolated (unpublished data). This suggests that OsPRA1 may be a component of the vesicle fusion machinery. To further strengthen our hypothesis, we examined whether or not mutant OsPRA1 (Y94A) and OsRab7 interact. Mutant OsPRA1 (Y94A) showed weak and no interaction with OsRab7 and OsVam3p, respectively, indicating that mutant OsPRA1 (Y94A) may lose its activity for recruiting Rab GTPase and Rab effector proteins and fusing vesicles to the vacuolar membrane. Actually, when OsPRA1 was mutated, its GDF activity was reduced to less than 50%, and its localization was changed from the PVC to the cytosol. These results are consistent with the assigned transport function of OsPRA1. Besides, our data from transient expression assay using vacuole markers suggested a direct involvement of OsPRA1 in the trafficking of vacuolar proteins.In summary, OsPRA1, a Yip homologous protein, may function in regulating vacuolar trafficking as a GDF dissociating OsRab7-OsGDI2 complex in plant cells.     

Molecular characterization of mungbean peroxisomal alanine glyoxylate aminotransferase gene induced by low temperature stress

Photorespiration reduces carbon fixation rate, but it is an essential process in plant. Photorespiration involves reactions in chloroplasts, peroxisomes, and mitochondria. In photorepiratory peroxisome, alanine glyoxylate aminotransferase (AGT) catalyzes the conversion of alanine and glyoxylate into glycine and pyruvate, respectively. We isolated a low temperature-inducible cDNA encoding AGT from mungbean leaves. The full-length cDNA, designated asMLT92, contains an open reading frame of 1,203 nucleotides coding for a protein of 401 amino acids. Genomic DNA blotting showed that the mungbean genome has one copy ofMLT92. MLT92 mRNA was induced by low temperature and its RNA expression was not much increased by drought stress. ABA and NaCl did not induce RNA expression ofMLT92. Based on GFP/RFP targeting experiment, GFP-MLT92 fusion protein and SKL-RFP, a peroxisome marker, were colocalized to peroxisome in tobacco protoplasts. This suggests that peroxisomalMLT92 is involved in molecular response to low temperature stress.     

The inhibition of the T-cell immunoglobulin and mucin domain 3 (Tim3) pathway enhances the efficacy of tumor vaccine

T-cell immunoglobulin and mucin domain 3 (Tim3) plays an important role in the Th1-mediated immune response; however, its effect on the efficacy of tumor vaccines has not been fully evaluated. Here, we demonstrate the effect of Tim3 pathway inhibition on tumor growth in mice. Lewis lung carcinoma (3LL) cells expressing a Tim3 pathway inhibitor, when injected into mice, showed suppressed tumor growth and a reduced frequency of CD4⁺CD25⁺Foxp3⁺ T-cells. Furthermore, Tim3 pathway inhibition significantly enhanced the efficacy of a prophylactic tumor vaccine and marginally enhanced the efficacy of a therapeutic tumor vaccine. However, when given in combination with the chemotherapeutic agent, 5-fluorouracil, the therapeutic tumor vaccine capable of Tim3 pathway inhibition had no additional anti-tumor effect. Our results show that Tim3 pathway inhibition can enhance tumor vaccine efficacy.     

7,3′,4′-Trihydroxyisoflavone Inhibits Epidermal Growth Factor-induced Proliferation and Transformation of JB6 P+ Mouse Epidermal Cells by Suppressing Cyclin-dependent Kinases and Phosphatidylinositol 3-Kinase

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3′,4′-Trihydroxyisoflavone (7,3′,4′-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3′,4′-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G₁ phase. As shown by Western blot, 7,3′,4′-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G₁ phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3′,4′-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3β/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3′,4′-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3′,4′-THIF provide a molecular basis for the possible development of new chemoprotective agents.     

Overexpression and Inactivation of <Emphasis Type="Italic">UGT73B2</Emphasis> Modulate Tolerance to Oxidative Stress in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Glycosyltransferases (GTs) play an important role in modulating solubility, stability, bioavailability, and bioactivity of secondary metabolites, such as flavonoids. In Arabidopsis thaliana, at least 120 family 1 uridine diphosphate (UDP)-glycosyltransferases (UGTs) have been predicted. However, little is known about their substrates or their physiological roles in planta. To define the role of UGT73B2 in planta, we first characterized its expression pattern using transgenic Arabidopsis plants carrying the cis-elements of UGT73B2 fused to the GUS reporter. During vegetative phase, its expression was high in embryonic and postembryonic roots, where it may play a physiological role in the glycosylation of flavonoids. Loss of function of UGT73B2 alone or in conjunction with its closest homologs, UGT73B1 and UGT73B3, confers greater tolerance to oxidative stress, whereas overexpression of UGT73B2 increases sensitivity to oxidative stress. In addition, growth phenotypes of mutant and transgenic seedlings correlate well with ROS levels in planta. Our results suggest that the glycosylation of flavonoids by UGT73B2—and/or its closest homologs—modulate the response of plants to oxidative stress.     

Secretion of EGF-like domain of heregulinβ promotes axonal growth and functional recovery of injured sciatic nerve

Neuregulin 1 (NRG1) and epidermal growth factor receptor (ErbB) signaling pathways control Schwann cells during axonal regeneration in an injured peripheral nervous system. We investigated whether a persistent supply of recombinant NRG1 to the injury site could improve axonal growth and recovery of sensory and motor functions in rats during nerve regeneration. We generated a recombinant adenovirus expressing a secreted form of EGF-like domain from Heregulinβ (sHRGβE-Ad). This virus, sHRGβE-Ad allowed for the secretion of 30-50 ng of small sHRGβE peptides per 10^7–8 virus particle outside cells and was able to phosphorylate ErbB receptors. Transduction of the concentrated sHRGβE-Ad into an axotomy model of sciatic nerve damage caused an effective promotion of nerve regeneration, as shown by histological features of the axons and Schwann cells, as well as increased expression of neurofilaments, GAP43 and S100 in the distal stump of the injury site. This result is consistent with longer axon lengths and thicker calibers observed in the sHRGβE-Ad treated animals. Furthermore, sensory and motor functions were significantly improved in sHRGβE-Ad treated animals when evaluated by a behavioral test. These results suggest a therapeutic potential for sHRGβE-Ad in treatment of peripheral nerve injury.     

Immune response and expression analysis of cathepsin K in goldfish during Aeromonas hydrophila infection

The innate immunity and expression profiles of cathepsins D were determined in the goldfish (Carassius auratus) tissues after challenge with a fish pathogen Aeromonas hydrophila. The innate immunity of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were determined by peripheral blood leucocytes. Blood and tissue samples of the muscle, gills, liver, kidney, heart, spleen, and intestine were sampled at 1, 3, 6 and 12 h post-infection for cathepsin D expression by semi-quantitative RT-PCR. The ROS and RNS production did not significantly increase at 1 h post-challenged goldfish. However, the ROS and RNS production was significantly increased after 3 h post-challenged fish compared to the control. The cathepsin D expression was found very low in muscle and kidney of the control fish, other tissues was not found the expression. A similar pattern was found in goldfish at 1 h post-challenge with A. hydrophila. However, at 3 h post-challenge goldfish, the cathepsin D expression was high only in the heart. At 6 h post-challenge goldfish, the cathepsin D expression was seen high all the tissues, except in the spleen. However, the expression was decreased at 12 h post-infection samples. This result was suggested that the goldfish infected with A. hydrophila decreased the innate immunity level in peripheral blood and expressed the cathepsin D in tissues.