Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.     

Molecular characterization of T4-type bacteriophages in a rice field

Bacteriophages, the viruses that infect bacteria, are the most abundant biological entities in the biosphere and play a key role in global biogeochemical cycling. All T4-type bacteriophage isolates tested so far have a conserved genetic module that encodes the virion components including gene 23 (g23), the major capsid protein. Molecular analysis of the g23 sequence revealed a remarkable level of diversity of T4-type bacteriophages isolated from rice straw and surface soil in a Japanese rice field. It was found that g23 sequences obtained from the rice field were quite distinctive from those obtained in marine environments. Phylogenetic analysis showed that most of these g23 sequences belonged to two novel subgroups of T4-type bacteriophages, although some of them were related to well-studied subgroups of T4-type bacteriophages, such as marine cyanophage isolates of exoT-evens.     

Antibacterial functionalization of an experimental self-etching primer by inorganic agents: microbiological and biocompatibility evaluations

Antibacterial activities have been demonstrated on oral bacteria with inorganic antibacterial agents (ABAs) after their incorporations into an experimental self-etching primer (ESP) before curing. This study was to assess their biocompatibility and antibacterial activity after curing. Six ABAs were incorporated respectively into ESP for treating specimens. After curing, their bactericidal activities on Streptococcus mutans and influences to the early bacterial colonization were assessed by direct contact and viable count. Systemic toxicity in rats after short-term oral exposure and direct contact cytotoxicity with NIH3T3 fibroblasts were tested. Incorporation of ZnOw AT-83, Longbei antibiotic, Antim-AMS2 or IONPURE-H significantly enhanced the antibacterial effect of ESP after curing, even after 1 month aging. Specimens treated by ESP with ZnOw AT-83, Longbei antibiotic or Antim-AMS2 showed slightly less bacterial adhesion than control. Animal experiments revealed neither toxic signs nor significant differences in body weight gain between control and other groups. Cell vitality or proliferation rates were ranged from 76% to 100% with respect to controls. Basic magnesium hypochlorite, ZnOw AT-83 and ZnOw AT-88 were less toxic. Toxicity only observed in areas beneath the specimens and/or in the direct vicinity of the specimen edge. From microbiological and biocompatibility aspects, the tested ABAs can be effectively incorporated in ESP to provide antibacterial activity against S. mutans. ZnOw AT-83 was the most promising one.     

Characterization of hypothetical proteins Cpn0146, 0147, 0284 &; 0285 that are predicted to be in the <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> inclusion membrane

Background  

Although more than 100 Chlamydia pneumoniae hypothetical proteins have been predicted to be inclusion membrane proteins, only a few have been experimentally demonstrated to be in the inclusion membrane. Using antibodies raised with fusion proteins, we characterized four such hypothetical proteins encoded by two gene clusters (Cpn0146-147 and Cpn0284-285) in the C. pneumoniae genome.     

Calmodulin-dependent activation of calcineurin by chlorogenic acid

Chlorogenic acid (CGA) has been proved to be an activator of calcineurin (CN) in our previous research. In this study, the activation mechanism of CN by CGA was further explored. The results showed that although the purified CN was inactive in vitro if only Ca(2+)/calmodulin (CaM) existed without Mn(2+)/Ni(2+), CGA activated the inactive CN potently. It was found that CN's activity increased as the concentration of CGA increased and reached a plateau of 4- to 6-fold higher activity using p-nitrophenyl phosphate (pNPP) or phosphopeptide (32)P-RII as substrate. And the activation was CaM-dependent. Moreover, the fluorescent emission of CN had a 17 nm red shift in the presence of 128 muM CGA, and the quenching constant was 1.21x10(12) M(-1) . s(-1), which indicated that CGA bound to CN statically and changed its conformation. According to the kinetic analysis, CGA preferred to activate CN in a substrate noncompetitive manner. When Mn(2+) or Ni(2+) presented, CGA also activated CN with CaM-dependency by improving CN's affinity for Mn(2+) or Ni(2+). In addition, the inhibition of CN by Zn(2+) was partially eliminated by CGA chelation. Our findings suggested the activation of CN by CGA was in a CaM-dependent and substrate noncompetitive manner. This might provide the basis for the further study of CN-targeted activators.     

Novel symmetric amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) with high plasmid DNA binding affinity as a biodegradable gene carrier

This study communicates the molecular design, preparation, and biological application of novel symmetric amphiphilic polycationic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) D2-LLA15-D2 bearing two two-generation poly(L-lysine) PLL dendrons D2 and a central hydrophobic biodegradable poly(L-lactide) block LLA15. First, an amino-protected precursor of L1-OH was designed and synthesized and was further employed to prepare L1-LLA15 with an organic 4-(dimethylamino)-pyridine-mediated living-ring-opening polymerization of l-lactide. Subsequently, the hydroxy end-capped L1-LLA15 was coupled to synthesize a new triblock L1-LLA15-L1 with two one-generation amino-protected PLL dendrons L1. Furthermore, with a repeated trifluoroacetic-acid-mediated amino deprotection-protection cycle, new amphiphilic triblock D2-LLA15-D2 was successfully prepared. By means of NMR, mass spectrometry, and gel permeation chromatography, these synthetic precursors and final amphiphilic product were characterized to bear well-defined triblock structures. In addition, this synthesized amphiphilic triblock polycationic macromolecule was applied as a new polycationic plasmid DNA carrier, and its DNA binding affinity was examined via an agarose electrophoresis and a fluorescence titration assay along with two important references of hydrophilic dendritic D2-HEX-D2 and double-hydrophilic D2-PEG-4K-D2 bearing the same two D2 dendrons; much enhanced DNA binding affinity was interestingly revealed for the new amphiphilic structural D2-LLA15-D2. Moreover, the assembled polyplex microparticles of plasmid DNA/polycationic carrier were further analyzed by dynamic light scattering and transmission electron microscopy, indicating their averaged nanoparticle size around 150-200 nm. As for the cytotoxicity of the new D2-LLA15-D2, MTT assays were conducted with a human hepatocellular carcinoma cell line (SMMC-7721), indicating a very low cytotoxicity as compared with commercial linear PLL-23K and PEI-2K, and a DNase I degradation of the assembled polyplex particles was also done in the HBS buffer solution to evaluate their stabilities. Finally, employing the new amphiphilic D2-LLA15-D2 as gene carrier, in vitro gene transfection experiments were conducted with the SMMC-7721 cell line, indicating a transfection efficiency increase of at least 10 times higher than that of the naked plasmid DNA under a N/P charge ratio of 10. Therefore, these interesting results may provide a new possible way to construct efficient polycationic macromolecular gene carriers with low toxicity and less expensive low-generation PLL dendrons.     

Bioaccumulation and ROS generation in liver of Carassius auratus, exposed to phenanthrene

In the present study, the bioaccumulation and reactive oxygen species (ROS) generation were studied after fish (Carassius auratus) were exposed to different concentrations (0.01, 0.02, 0.05, 0.07 and 0.1 mg/L) of phenanthrene for 4 days. The accumulation of phenanthrene in liver increased with the exposure concentration (R(2)=0.88). A secondary spin trapping technique was used followed by electron paramagnetic resonance (EPR) analysis, to study the ROS production. The ROS generated in fish liver after exposure to phenanthrene was identified as hydroxyl radical ((*)OH). The (*)OH signal intensity of the EPR spectrum showed a significant increase (p<0.05) compared to the control when the phenanthrene concentration was as low as 0.05 mg/L. A good positive relationship (R(2)=0.97) was found between the (*)OH formation and exposure concentrations. The changes of the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-S-transferase (GST), and contents of reduced glutathione (GSH) also were detected. The results clearly indicated that phenanthrene could induce (*)OH generation and result in oxidative stress in liver of fish.     

Pharmacometabonomic phenotyping reveals different responses to xenobiotic intervention in rats

In conventional pharmacological studies, intersubject differences within an animal strain are normally neglected, leading to variations in pharmacological outcomes in response to the same stimulus. Using two classical experimental models, the Streptozotocin (STZ)-induced diabetic model of Wistar rats and the high-energy, diet-induced obesity model of Sprague-Dawley rats, we demonstrate that the different outcomes of STZ or diet intervention are closely associated with variation in predose (baseline) urinary metabolic profiles of the rats. The pharmacometabonomic analysis of predose metabolic profiles indicates that the intersubject difference is, to a great extent, associated with gut-microbiota, which predisposes different pathophysiological outcomes upon diet alteration or chemical stimulus. We hypothesize that there may exist an important association between observations from these two models and the obese/diabetic human population in that subtle variations in metabolic phenotype may predetermine different systems' responses to xenobiotic perturbation, ultimately leading to varied pathophysiological processes. Results from two independent models also suggest that the pharmacometabonomics approach is of great importance in the study of pharmacology and clinical drug evaluations, where endogenous metabolite signatures of predose individuals should be taken into consideration to minimize intersubject difference and the resulting variation in the postdose pharmacological outcomes.     

Glycoprotein microarrays with multi-lectin detection: unique lectin binding patterns as a tool for classifying normal, chronic pancreatitis and pancreatic cancer sera

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 4%. Effective early detection and screening are currently not available, and tumors are typically diagnosed at a late stage, frequently after metastasis. Existing clinical markers of pancreatic cancer lack specificity, as they are also found in inflammatory diseases of the pancreas and biliary tract. In the work described here, naturally occurring glycoproteins were enriched by using lectin affinity chromatography and then further resolved by nonporous reversed-phase chromatography. Glycoprotein microarrays were then printed and probed with a variety of lectins to screen glycosylation patterns in sera from normal, chronic pancreatitis, and pancreatic cancer patients. Ten normal, 8 chronic pancreatitis, and 6 pancreatic cancer sera were investigated. Data from the glycoprotein microarrays were analyzed using bioinformatics approaches including principal component analysis (PCA) and hierarchical clustering (HC). Both normal and chronic pancreatitis sera were found to cluster close together, although in two distinct groups, whereas pancreatic cancer sera were significantly different from the other two groups. Both sialylation and fucosylation increased as a function of cancer on several proteins including Hemopexin, Kininogen-1, Antithrombin-III, and Haptoglobin-related protein, whereas decreased sialylation was detected on plasma protease C1 inhibitor. Target alterations on glycosylations were verified by lectin blotting experiments and peptide mapping experiments using microLC-ESI-TOF. These altered glycan structures may have utility for the differential diagnosis of pancreatic cancer and chronic pancreatitis and identify critical differences between biological samples from patients with different clinical conditions.