Production of Aujeszky's disease (pseudorabies) virus envelope glycoproteins gB and gC as recombinant polyhedra in baculovirus-infected silkworm larvae

The expression of viral antigens in baculovirus-infected insect cells is often ineffective. As an alternative approach, therefore, we developed the recombinant polyhedra technology, which is an efficient strategy for the production of viral subunit vaccine. Here, we report a strategy for the large-scale production of a pseudorabies virus (PRV) gB or gC in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the epitope regions of PRV gB or heparin-binding domains of PRV gC. Recombinant BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae expressed native polyhedrin and fusion protein that was detected using both anti-polyhedrin and anti-PRV gB or anti-PRV-gC antibodies. Electron and confocal microscopy demonstrated that the recombinant polyhedra contained both the fusion protein and native polyhedrin with a normal morphology and that the recombinant polyhedra contained PRV gB or gC. The yield of gB or gC antigen produced in BmNPV-PRV-gB or BmNPV-PRV-gC-infected silkworm larvae reached 0.69 or 0.46 mg per larva, respectively, at 6 days post-infection. These results demonstrate that the recombinant polyhedra strategy can be used for the large-scale production of PRV gB or gC antigen.     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.     

Synthesis and antibacterial activities of novel oxazolidinones having cyclic sulfonamide moieties

The synthesis of a new series of oxazolidinones having cyclic sulfonamide moieties is described. Their in vitro antibacterial activities against both Gram-positive and Gram-negative bacteria were tested and the effect of substituents on the oxazolidinone ring was investigated. A particular compound 15g having [1,2,5]thiadiazolidin-1,1-dioxide moiety showed the most potent antibacterial activity.     

Past,current, and future trends of red spiny lobster based on PCA with MaxEnt model in Galapagos Islands,Ecuador

In order to enhance in terms of accuracy and predict the modeling of the potential distribution of species, the integration of using principal components of environmental variables as input of maximum entropy (MaxEnt) has been proposed in this study. Principal components selected previously from the principal component analysis results performed in ArcGIS in the environmental variables was used as an input data of MaxEnt instead of raw data to model the potential distribution of red spiny lobster from the year 1997 to 2015 and for three different future scenarios 2020, 2050, and 2070. One set of six original environmental variables pertaining to the years 1997–2015 and one set of four variables for future scenarios were transformed independently into a single multiband raster in ArcGIS in order to select the variables whose eigenvalues explains more than 5% of the total variance with the purpose to use in the modeling prediction in MaxEnt. The years 1997 and 1998 were chosen to compare the accuracy of the model, showing better results using principal components instead of raw data in terms of area under the curve and partial receiver operating characteristic as well as better predictions of suitable areas. Using principal components as input of MaxEnt enhances the prediction of good habitat suitability for red spiny lobster; however, future scenarios suggest an adequate management by researches to elaborate appropriate guidelines for the conservation of the habitat for this valuable specie with face to the climate change.     

Distal NF-kB binding motif functions as an enhancer for nontypeable H. influenzae-induced DEFB4 regulation in epithelial cells

Among the antimicrobial molecules produced by epithelial cells, DEFB4 is inducible in response to proinflammatory signals such as cytokines and bacterial molecules. Nontypeable Haemophilus influenzae (NTHi) is an important human pathogen that exacerbates chronic obstructive pulmonary disease in adult and causes otitis media and sinusitis in children. Previously, we have demonstrated that DEFB4 effectively kills NTHi and is induced by NTHi via TLR2 signaling. The 5′-flanking region of DEFB4 contains several NF-κB binding motifs, but their NTHi-specific activity remains unclear. In this study, we aimed to elucidate molecular mechanism involved in DEFB4 regulation, focusing on the role of the distal NF-κB binding motif of DEFB4 responding to NTHi. Here, we show that the human middle ear epithelial cells up-regulate DEFB4 expression in response to NTHi via NF-κB activation mediated by IκKα/β−IκBα signaling. Deletion of the distal NF-κB binding motif led to a significant reduction in NTHi-induced DEFB4 up-regulation. A heterologous construct containing the distal NF-κB binding motif was found to increase the promoter activity in response to NTHi, indicating a NTHi-responding enhancer activity of the distal NF-κB binding motif. Furthermore, electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that the p65 domain of NF-κB binds to the distal NF-κB binding motif in response to NTHi. Taken together, our results suggest that NTHi-induced binding of p65 NF-κB to the distal NF-κB binding motif of DEFB4 enhances NTHi-induced DEFB4 regulation in epithelial cells.     

Orexinergic Neurotransmission in Temperature Responses to Methamphetamine and Stress: Mathematical Modeling as a Data Assimilation Approach

Experimental Data

Orexinergic neurotransmission is involved in mediating temperature responses to methamphetamine (Meth). In experiments in rats, SB-334867 (SB), an antagonist of orexin receptors (OX1R), at a dose of 10 mg/kg decreases late temperature responses (t>60 min) to an intermediate dose of Meth (5 mg/kg). A higher dose of SB (30 mg/kg) attenuates temperature responses to low dose (1 mg/kg) of Meth and to stress. In contrast, it significantly exaggerates early responses (t<60 min) to intermediate and high doses (5 and 10 mg/kg) of Meth. As pretreatment with SB also inhibits temperature response to the stress of injection, traditional statistical analysis of temperature responses is difficult.

Mathematical Modeling

We have developed a mathematical model that explains the complexity of temperature responses to Meth as the interplay between excitatory and inhibitory nodes. We have extended the developed model to include the stress of manipulations and the effects of SB. Stress is synergistic with Meth on the action on excitatory node. Orexin receptors mediate an activation of on both excitatory and inhibitory nodes by low doses of Meth, but not on the node activated by high doses (HD). Exaggeration of early responses to high doses of Meth involves disinhibition: low dose of SB decreases tonic inhibition of HD and lowers the activation threshold, while the higher dose suppresses the inhibitory component. Using a modeling approach to data assimilation appears efficient in separating individual components of complex response with statistical analysis unachievable by traditional data processing methods.     

Construction of malate-sensing <Emphasis Type="Italic">Escherichia coli</Emphasis> by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.     

Junctional membrane inositol 1,4,5-trisphosphate receptor complex coordinates sensitization of the silent EGF-induced Ca2+ signaling

Ca(2+) is a highly versatile intracellular signal that regulates many different cellular processes, and cells have developed mechanisms to have exquisite control over Ca(2+) signaling. Epidermal growth factor (EGF), which fails to mobilize intracellular Ca(2+) when administrated alone, becomes capable of evoking [Ca(2+)](i) increase and exocytosis after bradykinin (BK) stimulation in chromaffin cells. Here, we provide evidence that this sensitization process is coordinated by a macromolecular signaling complex comprised of inositol 1,4,5-trisphosphate receptor type I (IP(3)R1), cAMP-dependent protein kinase (PKA), EGF receptor (EGFR), and an A-kinase anchoring protein, yotiao. The IP(3)R complex functions as a focal point to promote Ca(2+) release in two ways: (1) it facilitates PKA-dependent phosphorylation of IP(3)R1 in response to BK-induced elevation of cAMP, and (2) it couples the plasmalemmal EGFR with IP(3)R1 at the Ca(2+) store located juxtaposed to the plasma membrane. Our study illustrates how the junctional membrane IP(3)R complex connects different signaling pathways to define the fidelity and specificity of Ca(2+) signaling.