The polyglycine and polyglutamine repeats in the androgen receptor gene in Japanese and Caucasian populations

Human androgen receptor (AR) gene contains two polymorphic trinucleotide repeats of CAG and GGC, which code for polyglutamine and polyglycine tracts in the N-terminal domain in which the receptor activity resides. Longer repeats induce decrease of transactivation function in the AR receptor, weaken an anti-proliferative effect on various steroid-related tissues, and may promote the carcinogenesis of these cancers, such as breast, endometrial, and ovarian cancers. However, the incidences of these steroid-related cancers are remarkably lower in Japanese than in Caucasians. We hypothesize that the GGC and CAG repeats in AR gene correspond to lower incidence of steroid-related cancers in the Japanese population. To test this hypothesis, these two polymorphic trinucleotide repeats in AR gene were genotyped in 221 Japanese and 177 Caucasians. The results of genotyping in these loci clearly show that the distribution of GGC repeat is significantly different between these populations (P<0.001). Japanese (73.7%) had 16 GGC repeats compared to 53.3% for Caucasians. Japanese (3.8%) also had 17 GGC repeats compared to 36.2% for Caucasians. No Japanese had more than 18 GGC repeats compared to 3.4% for Caucasians. The length of CAG repeats in the Japanese population was not significantly different than that of the Caucasian population, although the CAG repeats varied from 14 to 31 and 15 to 29 repeats in Japanese and German populations, respectively. This study demonstrates that the Japanese population has shorter GGC compared to the Caucasian population, which may explain the incidences of estrogen-related cancers in these populations.     

Ions released from a S-PRG filler induces oxidative stress in <Emphasis Type="Italic">Candida albicans</Emphasis> inhibiting its growth and pathogenicity

Candida albicans causes opportunistic fungal infections usually hidden among more dominant bacteria and does not exhibit high pathogenicity in vivo. Among the elderly, due to reduced host resistance to pathogens attributable to immunoscenesence, oral candidiasis is more likely to develop often leading to systemic candidiasis. Surface pre-reacted glass ionomer filler (S-PRG filler) is an ion-releasing functional bioactive glass that can release and recharge six ions which in turn strengthens tooth structure, inhibits demineralization arising from dental caries, and suppresses dental plaque accumulation. However, its effects on C. albicans have never been elucidated. Here, we evaluated the effects of ion released from S-PRG filler on C. albicans. Results show that extraction liquids containing released ions (ELIS) decreased the amount of hydrogen peroxide and catalase activity in C. albicans. Moreover, ELIS presence was found to affect C. albicans: (1) suppression of fungal growth and biofilm formation, (2) prevent adherence to denture base resin, (3) inhibit dimorphism conversion, and (4) hinder the capability to produce secreted aspartyl proteinase. Taken together, our findings suggest that ELIS induces oxidative stress in C. albicans and suppresses its growth and pathogenicity. In this regard, we propose that ELIS has the potential to be clinically used to help prevent the onset and inhibition of oral candidiasis among the elderly population.     

Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis

Extracellular proteases of Staphylococcus aureus are emerging as potential virulence factors that are relevant to the pathogenicity of staphylococcal infections. These proteases may also be involved in the proteolytic cleavage of other exoproteins released from this organism. To define the target exoproteins and their sites of cleavage by proteases, high-resolution two-dimensional polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing of exoprotein spots was performed. Two to three hundred exoprotein spots were detected at the early-stationary phase of cultures of S. aureus NCTC8325, and then at the late-stationary stage most of these high molecular protein spots became invisible due to further proteolytic degradation. As the result of N-terminal analysis, lipase, triacylglycerol lipase, orf619 protein and orf388 protein were detected as multiple spots at the early-stationary phase. We found that these exoproteins were cleaved at 3, 7, 4 and 4 different sites, respectively, by proteases. According to the M.W. and pI of each peptide spot obtained from the gel and their matches with calculated values in addition to their N-terminal sequences, we showed that the positions of putative peptides resulted from proteolytic cleavage of these proteins.     

Involvement of superoxide generation in salicylic acid-induced stomatal closure in Vicia faba.

Salicylic acid (SA), the known mediator of systemic acquired resistance, induced stomatal closure of Vicia faba L. Application of SA to the epidermal peels evoked an elevation of chemiluminescence of Cripridina lucigenin-derived chemiluminescent reagent (CLA) which is sensitive to superoxide anion (O(2)(.-)). The SA-induced generation of chemiluminescence was suppressed by O(2)(.-)-specific scavengers superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron). These results suggest that O(2)(.-) was generated in epidermal peels by SA-treatment. A peroxidase inhibitor salicylhydroxamic acid (SHAM) inhibited guaiacol peroxidase activity and suppressed the SA-induced CLA chemiluminescence in the epidermal peels, suggesting that O(2)(.-) generation occurred by the peroxidase-catalyzed reaction as proposed for SA-treated tobacco cell suspension culture [Kawano et al. (1998) Plant Cell Physiol. 39: 721]. SOD, Tiron or SHAM suppressed the SA-induced stomatal closure. Moreover, application of superoxide-generating system also induced stomatal closure. These results support the concept of involvement of reactive oxygen species in signal transduction in SA-induced stomatal closure.     

Effects of protease activated receptor (PAR)2 blocking peptide on endothelin-1 levels in kidney tissues in endotoxemic rat mode

Aims

Septic shock, the severe form of sepsis, is associated with development of progressive damage in multiple organs. Kidney can be injured and its functions altered by activation of coagulation, vasoactive-peptide and inflammatory processes in sepsis. Endothelin (ET)-1, a potent vasoconstrictor, is implicated in the pathogenesis of sepsis and its complications. Protease-activated receptors (PARs) are shown to play an important role in the interplay between inflammation and coagulation. We examined the time-dependent alterations of ET-1 and inflammatory cytokine, such as tumor necrosis factor (TNF)-α in kidney tissue in lipopolysaccharide (LPS)-induced septic rat model and the effects of PAR2 blocking peptide on the LPS-induced elevations of renal ET-1 and TNF-α levels.

Main methods

Male Wistar rats at 8 weeks of age were administered with either saline solution or LPS at different time points (1, 3, 6 and 10 h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide for 3 h to assess whether blockade of PAR2 has a regulatory role on the ET-1 level in septic kidney.

Key findings

An increase in ET-1 peptide level was observed in kidney tissue after LPS administration time-dependently. Levels of renal TNF-α peaked (around 12-fold) at 1 h of sepsis. Interestingly, PAR2 blocking peptide normalized the LPS-induced elevations of renal ET-1 and TNF-α levels.

Significance

The present study reveals a distinct chronological expression of ET-1 and TNF-α in LPS-administered renal tissues and that blockade of PAR2 may play a crucial role in treating renal injury, via normalization of inflammation, coagulation and vaso-active peptide.     

In vivo conformation of mitochondrial DNA revealed by pulsed-field gel electrophoresis in the true slime mold, Physarum polycephalum.

Pulsed-field gel electrophoresis (PFGE) was used to examine the in vivo and in vitro conformations of Physarum polycephalum mitochondrial DNA (mtDNA). We used plugs containing isolated mitochondria, isolated mitochondrial nucleoids (mt-nuclei), and isolated mtDNA, in addition to whole cells. The mtDNA contained in the myxamoebae, plasmodia, isolated mitochondria, and isolated mt-nuclei was circular, but most of the isolated mtDNA had been site-specifically fragmented and linearized during DNA preparation and storage under low ionic strength conditions. Restriction mapping of Physarum mtDNA by the direct digestion of the isolated mt-nuclei from two different strains, DP89 x AI16 and KM88 x AI16, resulted in the circular form. A linear mitochondrial plasmid, mF, is known to promote mitochondrial fusion and integration of itself into the mtDNA in Physarum. Linearization of mtDNA by the integration of the mF plasmid was demonstrated when we used PFGE to analyze isolated mitochondria from the plasmodial strain DP89 x NG7 carrying the mF plasmid (mF+). The PFGE system can be used not only to determine whether the form of mtDNA is linear or circular but also to analyze the dynamic conformational changes of mtDNA.     

Glaucomatous-Type Optic Discs in High Myopia

Purpose

To assess the prevalence of glaucoma in patients with high myopia defined as myopic refractive error of >-8 diopters or axial length ≥26.5 mm.

Methods

The hospital-based observational study included 172 patients (336 eyes) with a mean age of 61.9±12.3 years and mean axial length of 30.1±2.3 mm (range: 24.7–39.1mm). Glaucomatous-type optic discs were defined by glaucomatous optic disc appearance. Glaucoma was defined by glaucomatous optic disc appearance and glaucomatous Goldmann visual field defects not corresponding with myopic macular changes.

Results

Larger disc area (mean: 3.18±1.94 mm²) was associated with longer axial length (P<0.001; standardized correlation coefficient: 0.45). Glaucoma was detected in 94 (28%; 95% Confidence intervals: 23%, 33%) eyes. In multivariate analysis, glaucoma prevalence was 3.2 times higher (P<0.001) in megalodiscs (>3.79 mm²) than in normal-sized discs or small discs (<1.51 mm²) after adjusting for older age. Axial length was not significantly (P = 0.38) associated with glaucoma prevalence in that model. Glaucoma prevalence increased by a factor of 1.39 for each increase in optic disc area by one mm². Again, axial length was not significantly (P = 0.38) associated with glaucoma prevalence when added to this multivariate model.

Conclusion

Within highly myopic individuals, glaucoma prevalence increased with larger optic disc size beyond a disc area of 3.8 mm². Highly myopic megalodiscs as compared to normal sized discs or small discs had a 3.2 times higher risk for glaucomatous optic nerve neuropathy. The increased glaucoma prevalence in axial high myopia was primarily associated with axial myopia associated disc enlargement and not with axial elongation itself.     

Amplification of a plasmid bearing a mammalian replication initiation region in chromosomal and extrachromosomal contexts

Amplified genes in cancer cells reside on extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). We used a plasmid bearing a mammalian replication initiation region to model gene amplification. Recombination junctions in the amplified region were comprehensively identified and sequenced. The junctions consisted of truncated direct repeats (type 1) or inverted repeats (type 2) with or without spacing. All of these junctions were frequently detected in HSRs, whereas there were few type 1 or a unique type 2 flanked by a short inverted repeat in DMs. The junction sequences suggested a model in which the inverted repeats were generated by sister chromatid fusion. We were consistently able to detect anaphase chromatin bridges connected by the plasmid repeat, which were severed in the middle during mitosis. De novo HSR generation was observed in live cells, and each HSR was lengthened more rapidly than expected from the classical breakage/fusion/bridge model. Importantly, we found massive DNA synthesis at the broken anaphase bridge during the G1 to S phase, which could explain the rapid lengthening of the HSR. This mechanism may not operate in acentric DMs, where most of the junctions are eliminated and only those junctions produced through stable intermediates remain.     

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.     

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.