Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.     

N-terminal region of chitinase I of Bacillus circulans KA-304 contained new chitin-biding domain

Chitinase I (CHI1) of Bacillus circulans KA-304 forms protoplasts from Schizophyllum commune mycelia when the enzyme is combined with α-1,3-glucanase of B. circulans KA-304. CHI1 consists of an N-terminal unknown region and a C-terminal catalytic region classified into the glycoside hydrolase family-19 type. An N-terminal region-truncated mutant of CHI 1 (CatCHI1), which was expressed in Escherichia coli Rosetta-gami B (DE3), lost colloidal chitin- and powder chitin-binding activities. The colloidal chitin- and the powder chitin-hydrolyzing activities of CatCHI1 were lower than those of CHI1, and CatCHI1 was not effective in forming the protoplast. A fusion protein of the N-terminal region of CHI1 and green fluorescent protein (Nterm-GFP) was expressed in E. coli, and the fusion protein was adsorbed to colloidal chitin, powder chitin, and chitosan. Fluorescence microscopy analysis showed that Nterm-GFP bound to the S. commune cell-wall.     

Effects of struvite formation and nitratation promotion on nitrogenous emissions such as NH3, N2O and NO during swine manure composting

To reduce nitrogenous emissions from composting, two different countermeasures were applied simultaneously in swine manure composting. One was forming struvite by adding Mg and P at the start of composting, and the other was to promote nitratation (nitrite being oxidized nitrate) by adding nitrite-oxidizing bacteria after the thermophilic phase of composting. In the laboratory- and mid-scale composting experiments, 25-43% of NH3, 52-80% of N2O and 96-99% of NO emissions were reduced. From the nitrogen balance, it was revealed that the struvite formation reduced not only NH3, but also other nitrogenous emissions except N2O. The amount of total nitrogen losses was reduced by 60% by the two combined countermeasures, against 51% by the struvite formation alone. However, the nitratation promotion dissolved struvite crystals due to the pH decline, diminishing the effect of struvite as a slow-release fertilizer.     

8-Hydroxyguanine levels and repair capacity during mouse embryonic stem cell differentiation

To evaluate the defence capacities of embryonic stem (ES) cells against gene impairment, this study measured the levels of 8-hydroxyguanine (8-OH-Gua), a well-known marker of oxidative stress in DNA, and its repair capacity during differentiation. Undifferentiated ES cells (EB3) were cultured without leukaemia inhibitory factor (LIF) for 0, 4 and 7 days and are referred to as ES-D0, ES-D4 and ES-D7, respectively. These three cell lines were treated with 300 μM hydrogen peroxide (H(2)O(2)) for 48 and 72 h. After treatment, the amounts of 8-OH-Gua in the cells were determined by the high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) method. The levels of 8-OH-Gua in ES-D7 treated with H(2)O(2) were higher than those in ES-D0 and ES-D4, suggesting that the DNA in the undifferentiated cells was protected against gene impairment, as compared to that in the differentiated cells. To examine the repair capacity for 8-OH-Gua, this study analysed the expression of 8-OH-Gua repair-associated genes, 8-oxoguanine DNA glycosylase 1 (OGG1), MutY homolog (MUTYH) and Mut T homolog 1 (MTH1), in ES-D0, ES-D4 and ES-D7. The mRNA levels of MUTYH and MTH1 showed no significant change, whereas OGG1 mRNA was significantly decreased in ES-D7 treated with H(2)O(2). Moreover, it was observed that ES-D7 treated with H(2)O(2) readily underwent apoptosis, in comparison to its undifferentiated counterparts, ES-D0 and ES-D4. Taken together, ES cells are more resistant to DNA oxidative stresses than differentiated cells.     

VopV, an F-actin-binding type III secretion effector, is required for Vibrio parahaemolyticus-induced enterotoxicity

Vibrio parahaemolyticus, a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans, is characterized by two type III secretion systems (T3SS), namely T3SS1 and T3SS2. T3SS2 is indispensable for enterotoxicity but the effector(s) involved are unknown. Here, we identify VopV as a critical effector that is required to mediate V. parahaemolyticus T3SS2-dependent enterotoxicity. VopV was found to possess multiple F-actin-binding domains and the enterotoxicity caused by VopV correlated with its F-actin-binding activity. Furthermore, a T3SS2-related secretion system and a vopV homologous gene were also involved in the enterotoxicity of a non-O1/non-O139 V. cholerae strain. These results indicate that the F-actin-targeting effector VopV is involved in enterotoxic activity of T3SS2-possessing bacterial pathogens.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). III. The temperature dependence of the transient and steady-state behavior in cyclohexanone

The reversing-pulse electric birefringence (RPEB) technique was applied to the study of the temperature effect on the electrooptical and hydrodynamic properties of a fractionated [Glu(OBzl)]_n sample, which is molecularly dissolved in cyclohexanone. The aim was to develop a standard analytical method for thermal denaturation and temperature-induced conformational transitions. The field-on reverse and steady-state signal, and the field-off decay signal, were measured at 535 nm and at a constant low field strength (ca. 3 kV/cm) over a wide temperature range (5–90°C). The steady-state birefringence and the relaxation time in the decay process were also measured at two constant temperatures (5 and 70°C) over a wide field strength range (E ≤ 20 kV/cm). By the combination of these two different sets of RPEB measurements, the unwanted effect of the high pulse field on polymer conformation at elevated temperatures could be minimized. Together with the density and viscosity of cyclohexanone measured between 5 and 95°C, the following quantities could be evaluated: the weight-average permanent dipole moment and polarizability anisotropy, the reduced optical anisotropy factor (Δg/n), the weight-average length, and the degree of polydispersity. All these quantities, except for Δg/n, were found to be almost independent of temperature (5–90°C) and concentration (1.54–4.27 mM).     

Expression of nerve growth factor (NGF), and its receptors TrkA and p75 in the reproductive organs of the adult male rats

Immunolocalization of nerve growth factor (NGF) and its receptors, TrkA and p75 in the reproductive organs of adult male rats was investigated. Sections of the testis, efferent duct, epididymis, deferent duct, seminal vesicle, coagulating gland and prostate of adult male rats were immunostained by the avidin-biotin-peroxidase complex methods (ABC). NGF was expressed in Leydig cells, primary spermatocytes and pachytene spermatocytes in the testis. TrkA only immunoreacted to elongate spermatids and p75 showed positive immunostaining in the Sertoli cells, Leydig cells, the pachytene spermatocytes and elongate spermatids. Immunoreactions for NGF and its two receptors were detected in epithelial cells of efferent duct, deferent duct and epididymis. In addition, immunoreactions for NGF and its two receptors were also observed in columnar secretory epithelium lines of the seminal vesicles, prostate and coagulating gland. These results suggest that NGF is an important growth factor in gonadal function of adult male rats.     

Ancestry of American polyploid Hordeum species with the I genome inferred from 5S and 18S-25S rDNA

* BACKGROUND AND AIMS: The genus Hordeum exists at three ploidy levels (2x, 4x and 6x) and presents excellent material for investigating the patterns of polyploid evolution in plants. Here the aim was to clarify the ancestry of American polyploid species with the I genome. * METHODS: Chromosomal locations of 5S and 18S-25S ribosomal RNA genes were determined by fluorescence in situ hybridization (FISH). In both polyploid and diploid species, variation in 18S-25S rDNA repeated sequences was analysed by the RFLP technique. * KEY RESULTS: Six American tetraploid species were divided into two types that differed in the number of rDNA sites and RFLP profiles. Four hexaploid species were similar in number and location of both types of rDNA sites, but the RFLP profiles of 18S-25S rDNA revealed one species, H. arizonicum, with a different ancestry. * CONCLUSIONS: Five American perennial tetraploid species appear to be alloploids having the genomes of an Asian diploid H. roshevitzii and an American diploid species. The North American annual tetraploid H. depressum is probably a segmental alloploid combining the two closely related genomes of American diploid species. A hexaploid species, H. arizonicum, involves a diploid species, H. pusillum, in its ancestry; both species share the annual growth habit and are distributed in North America. Polymorphisms of rDNA sites detected by FISH and RFLP analyses provide useful information to infer the phylogenetic relationships of I-genome Hordeum species because of their highly conserved nature during polyploid evolution.     

Nitrite is an alternative source of NO in vivo

In this study, we investigated whether orally administered nitrite is changed to NO and whether nitrite attenuates hypertension in a dose-dependent manner. We utilized a stable isotope of [15N]nitrite (15NO2-) as a source of nitrite to distinguish between endogenous nitrite and that exogenously administered and measured hemoglobin (Hb)-NO as an index of circulating NO in whole blood using electron paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg Na15NO2 was orally administered to rats, an apparent EPR signal derived from Hb15NO (A(Z) = 23.4 gauss) appeared in the blood. The peak blood HbNO concentration occurred at the first measurement after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 +/- 0.52 and 10.58 +/- 0.40 microM, respectively) and at 15 min with 10 mg/kg (HbNO: 38.27 +/- 9.23 microM). In addition, coadministration of nitrite (100 mg/l drinking water) with N(omega)-nitro-L-arginine methyl ester (L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAME-induced hypertension (149 +/- 10 mmHg) compared with L-NAME alone (170 +/- 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly indicate that orally ingested nitrite can be an alternative to L-arginine as a source of NO in vivo and may explain, at least in part, the mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop Hypertension diet-induced hypotensive effects.     

Novel Repetitive Structures, Deviant Protein-Encoding Sequences andUnidentified ORFs in the Mitochondrial Genome of the BrachiopodLingula anatina

Complete sequence determination of the brachiopod Lingula anatina mtDNA (28,818 bp) revealed an organization that is remarkably atypical for an animal mt-genome. In addition to the usual set of 37 animal mitochondrial genes, which make up only 57% (16,555 bp) of the entire sequence, the genome contains lengthy unassigned sequences. All the genes are encoded in the same DNA strand, generally in a compact way, whereas the overall gene order is highly divergent in comparison with known animal mtDNA. Individual genes are generally longer and deviate considerably in sequence from their homologues in other animals. The genome contains two major repeat regions, in which 11 units of unassigned sequences and six genes (atp8, trnM, trnQ, trnV, and part of cox2 and nad2) are found in repetition, in the form of nested direct repeats of unparalleled complexity. One of the repeat regions contains unassigned repeat units dispersed among several unique sequences, novel repetitive structure for animal mtDNAs. Each of those unique sequences contains an open reading frame for a polypeptide between 80 and 357 amino acids long, potentially encoding a functional molecule, but none of them has been identified with known proteins. In both repeat regions, tRNA genes or tRNA gene-like sequences flank major repeated units, supporting the view that those structures play a role in the mitochondrial gene rearrangements. Although the intricate repeated organization of this genome can be explained by recurrent tandem duplications and subsequent deletions mediated by replication errors, other mechanisms, such as nonhomologous recombinations, appear to explain certain structures more easily.