Crystal structures of the state 1 conformations of the GTP-bound H-Ras protein and its oncogenic G12V and Q61L mutants

GTP-bound Ras adopts two interconverting conformations, "inactive" state 1 and "active" state 2. However, the tertiary structure of wild-type (WT) state 1 remains unsolved. Here we solve the state 1 crystal structures of H-Ras WT together with its oncogenic G12V and Q61L mutants. They assume open structures characterized by impaired interactions of both Thr-35 in switch I and Gly-60 in switch II with the γ-phosphate of GTP and possess two surface pockets of mutually different shapes unseen in state 2, a potential target for selective inhibitor development. Furthermore, they provide a structural basis for the low GTPase activity of state 1.     

The TLR4-TRIF pathway protects against H5N1 influenza virus infection

Prestimulation of the TLR4 pathway with lipopolysaccharide (LPS) protects mice from lethal infection with H5N1 influenza virus. Here, we reveal that the TLR4-TRIF pathway is required for this protective effect by using mice whose TLR4-related molecules were knocked out. Microarray analysis of primary mouse lung culture cells that were LPS pretreated and infected with an H5N1 virus indicated that TLR3 mRNA was upregulated. Primary lung culture cells of TLR3 knockout mice showed no response to LPS pretreatment against H5N1 virus infection, suggesting that TLR3 is also involved in the preventive effect of LPS. Our data suggest that the TLR4-TRIF axis has an important role in stimulating protective innate immunity against H5N1 influenza A virus infection and that TLR3 signaling is involved in this pathway.     

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. SG4502 with acquired LA-polymerizing activity

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1_SG and PhaC2_SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1_SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1_SG [PhaC1_SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1_SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).     

Deuterium kinetic isotope effects in heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96: the anionic form of the substrate in the enzyme-substrate complex is a reactive species

Heterotetrameric sarcosine oxidase is a flavoprotein that catalyses the oxidative demethylation of sarcosine. It is thought that the dehydrogenated substrate is the anionic form of sarcosine. To verify this assumption, the rate of flavin-adenine dinucleotide (FAD) reduction (k(red)) was analysed using protiated and deuterated sarcosine (N-methyl-d(3)-Gly) at various pH values using stopped-flow method. By increasing the pH from 6.2 to 9.8, k(red) increased for both substrates and reached a plateau, but the pK(a) value (reflecting the ionization of the enzyme-substrate complex) was 6.8 and 7.1 for protiated and deuterated sarcosine, respectively, and the kinetic isotope effect of k(red) decreased from approximately 19 to 8, indicating deprotonation of the bound sarcosine. The k(red)/K(d) (K(d), sarcosine dissociation constant) increased with increasing pH and reached a plateau. The pK (reflecting the ionization of free enzyme or free sarcosine) was 7.0 for both substrates, suggesting deprotonation of the βLys358 residue, which has a pK(a) of 6.7, as the pK(a) of the free sarcosine amine proton was determined to be approximately 10.1. These results indicate that the amine proton of sarcosine is transferred to the unprotonated Lys residue in the enzyme-substrate complex.     

Changes in the texture and viscoelastic properties of bread containing rice porridge during storage

The objective of this study was to investigate the effects of rice porridge on the texture and viscoelastic properties of bread during storage. Three types of bread, wheat flour bread, 15% rice flour bread, and 15% rice porridge bread, were prepared. After baking and storing the bread for 24 h, 48 h, and 72 h at room temperature, we measured the texture and viscoelastic properties of the bread crumbs by texture profile analysis (TPA) and creep test. The 15% rice porridge bread showed a significantly higher specific volume and maintained softer crumbs than the other two types (p<0.05). It also had a slightly stickier texture than the others. It can be concluded that rice porridge improves the specific volume, texture, and viscoelastic properties of bread crumbs during storage.     

A successful second delivery outcome using refrozen thawed testicular sperm from an infertile male true hermaphrodite with a 46, XX/46, XY karyotype: case report

We report a successful second delivery of a healthy infant fathered using refrozen thawed testicular sperm from an infertile male chimera. We also examined sex chromosome distribution of the seminiferous tubule. Intracytoplasmic sperm injection (ICSI) was performed using the remaining refrozen testicular sperm, which had been stored during the first treatment. Biopsied testicular cells were examined by fluorescence in situ hybridization (FISH) and the peripheral lymphocyte karyotype was tested using a G-band. Following ICSI, a second pregnancy was established, and a healthy girl was successfully delivered at 40 gestational weeks without complications. Although the husband’s lymphocyte chromosomal analysis revealed a 46, XX [28]/46, XY [2] karyotype, the seminiferous tubule cells on histological examination by FISH were chimeric sex chromosome type XX [18]/XY [82]. In conclusion, this is a very rare case report of a successful subsequent delivery of a healthy infant (46, XX) from an infertile true hermaphrodite (46, XX/46, XY) using refrozen thawed testicular sperm. The seminiferous tubule cells’ karyotype ratio differed from that of the lymphocytes.