Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) by suppression subtractive hybridization

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5- and 3-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17–1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970–20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293–51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.The nucleotide sequences of OsGGT cDNA has been submitted to GenBank DDBJ under accession numbers AB164463.     

Papillomavirus capsid mutation to escape dendritic cell-dependent innate immunity in cervical cancer

Infection with oncogenic human papillomaviruses (HPVs), typified by HPV type 16 (HPV16), is a necessary cause of cervical cancer. Prophylactic vaccination with HPV16 L1 virus-like particles (VLPs) provides immunity. HPV16 VLPs activate dendritic cells and a potent neutralizing immunoglobulin G (IgG) response, yet many cervical cancer patients fail to generate detectable VLP-specific IgG. Therefore, we examined the role of the innate recognition of HPV16 L1 in VLP-induced immune responses and its evasion during carcinogenesis. Nonconservative mutations within HPV16 L1 have been described in isolates from cervical cancer and its precursor, high-grade cervical intraepithelial neoplasia (CIN). We determined the effect of mutations in L1 upon in vitro self-assembly into VLPs and their influence upon the induction of innate and adaptive immune responses in mice. Several nonconservative mutations in HPV16 L1 isolated from high-grade CIN or cervical carcinoma prevent self-assembly of L1 VLPs. Intact VLPs, but not assembly-defective L1, activate dendritic cells to produce proinflammatory factors, such as alpha interferon, that play a critical role in inducing adaptive immunity. Indeed, effective induction of L1-specific IgG1 and IgG2a was dependent upon intact VLP structure. Dendritic cell activation and production of virus-specific neutralizing IgG by VLPs requires MyD88-dependent signaling, although the L1 structure that initiates MyD88-mediated signaling is distinct from the neutralizing epitopes. We conclude that innate recognition of the intact L1 VLP structure via MyD88 is critical in the induction of high-titer neutralizing IgG. Tumor progression is associated with genetic instability and L1 mutants. Selection for assembly-deficient L1 mutations suggests the evasion of MyD88-dependent immune control during cervical carcinogenesis.     

Fixative-Stain Sequences for Selective Demonstration of Juxtaglomerular Cells

The effects of 31 fixatives, containing alcohol, acids, formalin and metallic salts, and representing many of the standard fixatives, were observed for selectivity and intensity of staining of juxtaglomerular granules in mouse kidney. Four staining methods: 1:400,000 aqueous methyl violet 2B; Bowie's ethyl violet-Biebrich scarlet; 1:200,000 aldehyde fuchsin; and periodic acid-Schiff were used. Fixatives containing HgCl₂, trichloroacetic acid or formalin were found to be the most satisfactory for subsequent staining of the granules.     

Functional magnetic resonance imaging of macaque monkeys performing visually guided saccade tasks: comparison of cortical eye fields with humans

The frontal and parietal eye fields serve as functional landmarks of the primate brain, although their correspondences between humans and macaque monkeys remain unclear. We conducted fMRI at 4.7 T in monkeys performing visually-guided saccade tasks and compared brain activations with those in humans using identical paradigms. Among multiple parietal activations, the dorsal lateral intraparietal area in monkeys and an area in the posterior superior parietal lobule in humans exhibited the highest selectivity to saccade directions. In the frontal cortex, the selectivity was highest at the junction of the precentral and superior frontal sulci in humans and in the frontal eye field (FEF) in monkeys. BOLD activation peaks were also found in premotor areas (BA6) in monkeys, which suggests that the apparent discrepancy in location between putative human FEF (BA6, suggested by imaging studies) and monkey FEF (BA8, identified by microstimulation studies) partly arose from methodological differences.     

Establishment and characterization of cultured epithelial cells lacking expression of ZO-1

In well polarized epithelial cells, closely related ZO-1 and ZO-2 are thought to function as scaffold proteins at tight junctions (TJs). In epithelial cells at the initial phase of polarization, these proteins are recruited to cadherin-based spotlike adherens junctions (AJs). As a first step to clarify the function of ZO-1, we successfully generated mouse epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/- cells were highly polarized with well organized AJs/TJs, which were indistinguishable from those in ZO-1+/+ cells by electron microscopy. In good agreement, by immunofluorescence microscopy, most TJ proteins including claudins and occludin appeared to be normally concentrated at TJs of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ scaffold protein, respectively, to TJs. When the polarization of ZO-1-/- cells was initiated by a Ca2+ switch, the initial AJ formation did not appear to be affected; however, the subsequent TJ formation (recruitment of claudins/occludin to junctions and barrier establishment) was markedly retarded. This retardation as well as the disappearance of cingulin were rescued completely by exogenous ZO-1 but not by ZO-2 expression. Quantitative evaluation of ZO-1/ZO-2 expression levels led to the conclusion that ZO-1 and ZO-2 would function redundantly to some extent in junction formation/epithelial polarization but that they are not functionally identical. Finally, we discussed advantageous aspects of the gene knock-out system with cultured epithelial cells in epithelial cell biology.     

Identification and characterization of a cathepsin B-like protease in Physarum sclerotium

In response to dry stress the plasmodium of a true slime mold, Physarum polycephalum, undergoes formation of sclerotium, which is a dormant body resistant to desiccation. The sclerotium can germinate within several hours after addition of water, followed by generation of the plasmodium. In the early phase of the germination many enzymes and other proteins of the sclerotium are required for formation of the plasmodium. As dehydration of proteins often leads to destruction of their structure or reduction in their activity, it is important to elucidate whether the dehydrated enzymes are present as the intact in the sclerotium. In this study three peaks of protease activity were detected with anion exchange column chromatography of the extract from the sclerotia. From among them, an acid protease was purified to homogeneity by gel filtration column chromatography, hydroxyapatite column chromatography, acid treatment, and cation-exchange column chromatography. Treatment of the protease fractions with pH 4.0 resulted in approximately 20-fold activation of the activity. The purified protease was a monomer with a molecular mass of 35 kDa. The optimum pH and temperature were 6.3 and 40 degrees C, respectively. Beta-casein, histone H1, and H2B were degraded by the 35 kDa protease, but human hemoglobin and human serum albumin were very poor substrates. In addition, the enzyme was sensitive to the cysteine protease inhibitors chymostatin, E-64, and leupeptin. These results indicate that, in the sclerotium, a premature form of a cathepsin B-like protease remains non-denatured under dehydrated conditions.     

Inhibitory effect of amines on polar filament extrusion byPlistophora anguillarum spores

In in vitro tests, amines were screened for the inhibition of polar filament extrusion by spores ofPlistophora anguillarum, a microsporidian parasite of the eel. Primary amines having C₈ ⁻ C₁₈ alkyl chains were effective, irrespective of the anions bound. Secondary amines having a branched C₈ chain and tertiary amines having C₁₄−C₁₈ chains were also effective. Asymmetry in side chains seemed to be required for inhibitory action. Quaternary ammonium salts having C₁₂−C₁₆ alkyl chains and some germicides or disinfectants such as cetyl pyridinium chloride and benzethonium chloride were also effective. Diamines and amides were ineffective regardless of the length of their alkyl chins. In in vivo tests, eel larvae-fed for 4 days with a commercial feed supplemented with 5×10⁸ cells of spores and 4g of laurylamine aspartate/100 g feed, and thereafter with the usual commercial feed for 26 days—were not infected.