Tetrameric ring formation of Epstein-Barr virus polymerase processivity factor is crucial for viral replication

The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.     

Isolation,cDNA Cloning,and Structure-based Functional Characterization of Oryctin,a Hemolymph Protein from the Coconut Rhinoceros Beetle,Oryctes rhinoceros,as a Novel Serine Protease Inhibitor

We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant ¹³C,¹⁵N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K_i values of 3.9 × 10⁻¹⁰ m, 6.2 × 10⁻¹⁰ m, 1.4 × 10⁻⁹ m, and 1.2 × 10⁻⁸ m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.     

AAA+ Chaperone ClpX Regulates Dynamics of Prokaryotic Cytoskeletal Protein FtsZ

AAA⁺ chaperone ClpX has been suggested to be a modulator of prokaryotic cytoskeletal protein FtsZ, but the details of recognition and remodeling of FtsZ by ClpX are largely unknown. In this study, we have extensively investigated the nature of FtsZ polymers and mechanisms of ClpX-regulated FtsZ polymer dynamics. We found that FtsZ polymerization is inhibited by ClpX in an ATP-independent manner and that the N-terminal domain of ClpX plays a crucial role for the inhibition of FtsZ polymerization. Single molecule analysis with high speed atomic force microscopy directly revealed that FtsZ polymer is in a dynamic equilibrium between polymerization and depolymerization on a time scale of several seconds. ClpX disassembles FtsZ polymers presumably by blocking reassembly of FtsZ. Furthermore, Escherichia coli cells overproducing ClpX and N-terminal domain of ClpX show filamentous morphology with abnormal localization of FtsZ. These data together suggest that ClpX modulates FtsZ polymer dynamics in an ATP-independent fashion, which is achieved by interaction between the N-terminal domain of ClpX and FtsZ monomers or oligomers.     

Micropatterning contractile C2C12 myotubes embedded in a fibrin gel

Contractile C₂C₁₂ myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C₂C₁₂ myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010;105: 1161–1167. © 2009 Wiley Periodicals, Inc.     

Outbreak of gastroenteritis caused by the pandemic Vibrio parahaemolyticus O3:K6 in Mexico

During 2003 and during late September of 2004, more than 1230 cases of gastroenteritis were reported in the south of Sinaloa State, north-western Mexico. All cases were attributed to the consumption of raw or undercooked shrimp collected at the Huizache-Caimanero lagunary system. Vibrio parahaemolyticus was identified by standard biochemical methods, and many strains were positive for PCR amplifications of the tlh and tdh genes and negative for the trh gene. A representative strain belonged to the O3:K6 serogroup. This is the first outbreak of gastroenteritis caused by the pandemic strains of O3:K6 V. parahaemolyticus in México.     

Tactile stimulation-induced rapid elevation of the synaptophysin mRNA expression level in rat somatosensory cortex

Synaptophysin is an integral membrane protein abundant in the synaptic vesicle and is found in nerve terminals throughout the brain. It was recently suggested that synaptophysin is also involved in the modulation of activity-dependent synapse formation. In this study, we examined at the individual level whether tactile stimulation selectively influenced the synaptophysin mRNA expression level in the somatosensory cortex of rats. Anesthetized rats were caressed on the back by an experimenter's palms for 20 min and the mRNA expression levels in the somatosensory and the visual cortices 5 min afterwards were determined using quantitative PCR methodology. The synaptophysin mRNA expression level was selectively higher in the experimental group than in the control group in the somatosensory cortex but not in the visual cortex. This suggests that the mRNA expression level of synaptophysin induced by neuronal activity is related to the regulation of synapse formation or remodeling or both.     

Aquaporin subfamily with unusual NPA boxes

Aquaporins have been identified based on highly conserved two asparagine-proline-alanine (NPA) boxes that are important for the formation of a water-permeating pore. Some aquaporin-like sequences, however, have less conserved NPA boxes. Although they have lower homology with conventional aquaporins, they should be included in aquaporin family based on their conserved six transmembrane domains and hydrophobic NPA box-like repeats. They are widely distributed in multicellular organisms. Only SIPs from plants and AQP11/12 from mammals were examined previously and found to be localized inside the cell. Intracellular localization will be a common feature of these aquaporin-like proteins since most of them have positively charged amino acid clusters at the carboxy-termini similar to di-lysine motif (-KKXX) for an endoplasmic reticulum retention signal. Accordingly, they are tentatively named subcellular-aquaporins in this review. Currently, studies on their functions and biological roles are limited. SIPs were shown to function as water channels and the disruption of AQP11 produced neonatally fatal polycystic kidneys. Further works on subcellular-aquaporins will reveal new insights into the roles of aquaporins.     

Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system

Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the β -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specificity were found in the culture fluid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k(cat)) (k(cat)) of the recombinant EGCase I to be 22- and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K(m) of both enzymes was almost the same for these substrates. Comparison of the three-dimensional structure of EGCase I (model) and EGCase II (crystal) indicated that a flexible loop hangs over the catalytic cleft of EGCase II but not EGCase I. Deletion of the loop from EGCase II increased the k(cat) of the mutant enzyme, suggesting that the loop is a critical factor affecting the turnover of substrates and products in the catalytic region. Recombinant EGCase I exhibited broad specificity and good reaction efficiency compared with EGCase II, making EGCase I well-suited to a comprehensive analysis of GSLs.