Reversal by dithiothreitol treatment of the block in murine leukemia virus maturation induced by disulfide cross-linking

We previously reported that if murine leukemia virus particles are produced in the presence of the mild oxidizing agent disulfide-substituted benzamide-2, they fail to undergo the normal process of virus maturation. We now show that treatment of these immature particles with a reducing agent (dithiothreitol) induces their maturation in vitro, as evidenced by proteolytic cleavage of Gag, Gag-Pol, and Env proteins and by their morphology. The identification of partial cleavage products in these particles suggests the sequence with which the cleavages occur under these conditions. This may be a useful experimental system for further analysis of retroviral maturation under controlled conditions in vitro.     

Hydrotropic response and expression pattern of auxin-inducible gene,CS-IAA1, in the primary roots of clinorotated cucumber seedlings

Primary roots of cucumber seedlings showed positive hydrotropism when exposed to a moisture gradient and rotated on a two-axis clinostat. To examine the role of auxin in the differential growth of the hydrotropically responding roots, we first examined the expression of auxin-inducible genes, CS-AUX/IAAs, in cucumber roots. After auxin starvation, mRNA levels of CS-IAA1 and CS-IAA3 decreased in the roots. Applying auxin to the auxin-starved roots resulted in accumulation of CS-IAA1 and CS-IAA3 mRNA. The level of expression of these genes increased when the auxin concentration was increased. CS-IAA1 mRNA accumulated in response to 10(-8) M auxin, and the level increased further, depending on the dose. Auxin starvation did not result in a decrease in the level of CS-IAA2 mRNA; however, adding exogenous auxin at concentrations higher than 10(-7) M increased its accumulation. In the primary roots responding hydrotropically or gravitropically, CS-IAA1 expression was greater on the concave side of the curving roots than on the convex side. The difference could be detected 30 min following stimulation by gravity or a moisture gradient, and that difference increased with time. These results support the idea that asymmetry of localization of auxin is associated with differential growth in hydrotropically responding roots.     

Hironoia fusiformis gen. et sp. nov.; a cornalean fruit from the Kamikitaba locality (Upper Cretaceous,Lower Coniacian) in northeastern Japan

The application of sieving techniques to bulk samples from the Ashizawa Formation, Futaba Group (Lower Coniacian) of northeastern Honshu, Japan, has yielded well-preserved mesofossil assemblages comparable with those recently described from eastern North America, Europe, and central Asia. Among the most abundant and distinctive components of these assemblages are fusiform fruits that are assigned here to a new genus and species, Hironoia fusiformis gen. et sp. nov. The fruits developed from an epigynous ovary with three to four locules. Each locule bears one seed and has a distinctive dorsal germination valve. These features of the fruit, along with the adnate calyx, indicate an affinity to extant Cornales and specifically the Cornaceae sensu lato. The recognition of an unequivocal cornalean fruit in the Early Coniacian–Early Santonian of Japan provides the earliest record of this group in the fossil record. It also establishes a minimum age for the early divergence of the asterid clade, a major group of living angiosperms comprising more than a third of all species of extant flowering plants. Electronic Publication     

Rearing conditions required for behavioral compensation after unilateral cercal ablation in the cricket Gryllus bimaculatus

The rearing condition necessary for behavioral compensation after sensory deprivation was investigated in the cricket Gryllus bimaculatus. The right-cercus-ablated cricket was reared in a glass vial with a slightly larger diameter than the body length of the cricket. After two weeks of rearing in the vial, the air-puff-evoked escape behavior of the cricket was investigated. The response rate (relative occurrence of the escape behavior after a standard air puff) obtained was identical with that of crickets reared in a large cage. On the other hand, unlike crickets reared in a large cage, the distorted escape directional property of the cricket reared in the vial was not compensated at all. Control experiments proved that the restraint in the vial did not affect the motor system, and the air motion from environments was not essential for the compensational recovery of the escape direction. Therefore, the ablated crickets required spontaneous walking in order to compensate the directionality of their escape. A self-generated wind caused by spontaneous walking appears necessary for the crickets to realize the defect of their sensory system and to compensate the related escape behavior. A hypothesis for the compensation mechanism based on the efference copy signal is proposed.     

Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.     

Connexin-30 deletion analysis in connexin-26 heterozygotes

Mutations in the Connexin-26 gene (Cx 26, GJB2) are the most common cause of hereditary nonsyndromic sensorineural hearing loss (SNHL). DNA analysis of the Cx 26 gene in deaf or hard-of-hearing individuals frequently demonstrates heterozygosity despite the fact that most mutations are known to be recessive. A 342-kb deletion in a gene adjacent to Cx 26, the Connexin-30 gene (Cx 30, GJB6), has been reported to cause deafness in the homozygous state or in combination with heterozygous mutations in Cx 26 (digenic inheritance). We have analyzed deaf or hard-of-hearing Cx 26 heterozygotes and individuals with no mutations in Cx 26 for this Cx 30 deletion. We found that 4/20 (20%) of the Cx 26 heterozygotes are heterozygous for this deletion and that no individuals were homozygous for the Cx 30 deletion. Cx 30 deletion analysis is recommended for all individuals with nonsyndromic SNHL following Cx 26 sequencing that does not demonstrate two recessive mutations.     

Atlas of the embryonic brain in the pygmy squid,Idiosepius paradoxus

Gross structural changes and neuropil formation in the brain during development were described in Idiosepius paradoxus, a sepioid that we chose as a model cephalopod. The brain originates in 4 pairs of ectodermal placodes, which occur separately in the embryonic surface undergoing epiboly. In the final period of epiboly, neuroblasts internalize from the placodes and gather into 4 pairs of ganglionic masses. The ganglionic masses assemble into a ring-like cluster encircling the inner yolk and the foregut anlage, gradually integrated into the 4 domains of a massive brain, a subesophageal mass (SBM), a supraesophageal mass (SPM), and a pair of optic lobes. In the early brain, neuropil forms a framework composed of a longitudinal ladder lying in the SBM, and a transverse arch standing on the lateral sides of the SBM and crossing the SPM. Differentiation of brain lobes proceeds from ventral to dorsal along this framework; first the magnocellular lobes and the posterior pedal lobe appear first in the SBM, the other lobes in the SBM and the basal lobes follow in the proximal region of the SPM, and the accessory lobes develop last in the most dorsal zone of the SPM. In the hatchlings, the brain lobes show almost the same arrangement as in the adults, but the accessory lobes, particularly the vertical lobe, are much smaller than those in the adults. Comparison of the present results with those in the teuthoid and the octopod indicates that developmental sequences of the brain are highly conserved in the coleoid cephalopods.     

Des-γ-Carboxyprothrombin (DCP) and NX-DCP Expressions and Their Relationship with Clinicopathological Features in Hepatocellular Carcinoma

Aim

Des-γ-carboxyprothrombin (DCP) has been used as a tumor marker for hepatocellular carcinoma (HCC). Recently the DCP/NX-DCP ratio, calculated by dividing DCP by NX-DCP, has been reported useful in detecting HCC. The purpose of this study is to clarify the significance of DCP and NX-DCP expression in HCC tissues.

Methods

HCC and non-HCC tissue samples were obtained from 157 patients and were immunohistochemically examined for DCP and NX-DCP expression using anti-DCP antibody and anti-NX-DCP antibody. DCP and NX-DCP expression scores were calculated by multiplying staining intensity grade by percentage of stained area. Serum DCP and NX-DCP levels were determined in 89 patients. We evaluated the relationship between tumor expression, serum level, and pathomorphological findings.

Results

Intrahepatic metastasis (im) was significantly more frequent in cases with high DCP expression than in cases with low DCP expression. High NX-DCP expression was associated with significantly lower histological grade, and less frequent im or portal vein invasion (vp) than low NX-DCP expression. Serum DCP was correlated with DCP expression, but serum NX-DCP was not correlated with NX-DCP expression. DCP-positive (≥40 mAU/L), NX-DCP-positive (≥90 mAU/L), and DCP/NX-DCP ratio-positive (≥1.5) cases were associated with significantly larger tumor size and more frequent vp than negative cases. DCP was rarely expressed, but NX-DCP was frequently expressed in non-cancerous liver tissues. Patients with NX-DCP expression-negative tumors showed a lower survival rate than those with NX-DCP expression-positive tumors (p = 0.04), whereas the survival in serum NX-DCP-positive cases was lower than that of serum negative cases (p = 0.02).

Conclusions

DCP and NX-DCP were produced in HCC tissues, but differed in expression level and biological properties. DCP expression, serum DCP or NX-DCP level, and DCP/NX-DCP ratio were closely related to malignant properties of HCC.     

Safety evaluation of probiotic bifidobacteria by analysis of mucin degradation activity and translocation ability

Although probiotic-containing nutrient formulas for infants and toddlers have become very popular, some adverse effects related to translocation of probiotic strains have been reported. We assessed the safety of probiotic bifidobacteria that have been used in clinical investigations and proven to have beneficial effects, by analyzing mucin degradation activity and translocation ability. Mucin degradation activities of three probiotic bifidobacteria strains; Bifidobacterium longum BB536, Bifidobacterium breve M-16V and Bifidobacterium infantis M-63, were evaluated by three in vitro tests comprising growth in liquid medium, SDS-PAGE analysis of degraded mucin residues, and degradation assay in Petri dish. All test strains and control type strains failed to grow in the liquid medium containing mucin as the only carbon source, although good growth was obtained from fecal sample. In the SDS-PAGE analyses of mucin residues and observation of mucinolytic zone in agar plate, the three test strains also showed no mucin degradation activity as the type strains, although fecal sample yielded positive results. In another study, a high dose of B. longum BB536 was administered orally to conventional mice to examine the translocation ability. No translocation into blood, liver, spleen, kidney and mesenteric lymph nodes was observed and no disturbance of epithelial cells and mucosal layer in the ileum, cecum and colon was detected, indicating that the test strain had no translocation ability and induced no damage to intestinal surface. These results resolve the concern about bacterial translocation when using bifidobacteria strains as probiotics, which have been tested in various clinical trials, supporting the continuous use of these probiotic strains without anxiety.     

Expression and function of mouse Sox17 gene in the specification of gallbladder/bile-duct progenitors during early foregut morphogenesis

In early-organogenesis-stage mouse embryos, the posteroventral foregut endoderm adjacent to the heart tube gives rise to liver, ventral pancreas and gallbladder. Hepatic and pancreatic primordia become specified in the posterior segment of the ventral foregut endoderm at early somite stages. The mechanisms for demarcating gallbladder and bile duct primordium, however, are poorly understood. Here, we demonstrate that the gallbladder and bile duct progenitors are specified in the paired lateral endoderm domains outside the heart field at almost the same timing as hepatic and pancreatic induction. In the anterior definitive endoderm, Sox17 reactivation occurs in a certain population within the most lateral domains posterolateral to the anterior intestinal portal (AIP) lip on both the left and right sides. During foregut formation, the paired Sox17-positive domains expand ventromedially to merge in the midline of the AIP lip and become localized between the liver and pancreatic primordia. In Sox17-null embryos, these lateral domains are missing, resulting in a complete loss of the gallbladder/bile-duct structure. Chimera analyses revealed that Sox17-null endoderm cells in the posteroventral foregut do not display any gallbladder/bile-duct molecular characters. Our findings show that Sox17 functions cell-autonomously to specify gallbladder/bile-duct in the mouse embryo.