Preparation and in vitro cytocompatibility of chitosan-siloxane hybrid hydrogels.

Injectable systems can be used in minimally invasive surgical applications. Although chitosan-glycerophosphate hydrogel systems are biodegradable and biocompatible, the long periods of time required for their effective gelation have severely limited their clinical application. The challenges currently facing researchers in this field are therefore focused on shortening the gelation time and biocompatibility of these materials to develop hydrogels suitable for clinical application. Chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) hybrids have recently demonstrated good cytocompatibility with respect to human osteoblastic cells (MG63) and human bone marrow cells. Although these precursor sols could form gels under physiological conditions, they required neutralization with a sodium hydroxide solution. In this study, the chitosan-GPTMS hybrid systems were neutralized with glycerophosphate to prepare injectable hydrogels. The results revealed that the gelation time of the hydrogels could be controlled by the amount of GPTMS in the precursor sols. The in vitro cytocompatibility of the hydrogels were evaluated in terms of the proliferation of MG63 cells cultured either directly onto the hydrogels or indirectly onto the cell culture plate under a hydrogel insert. In the former case, the cells showed good attachment and proliferated for up to 7 days. Similar results were observed in the in direct culture. These results suggest that this new chitosan-GPTMS hydrogel could potentially be used as an injectable biomaterial in clinical applications.

Construction of a chromosome-assigned, sequence-tagged linkage map for the radish, Raphanus sativus L. and QTL analysis of morphological traits.

The radish displays great morphological variation but the genetic factors underlying this variability are mostly unknown. To identify quantitative trait loci (QTLs) controlling radish morphological traits, we cultivated 94 F4 and F5 recombinant inbred lines derived from a cross between the rat-tail radish and the Japanese radish cultivar 'Harufuku' inbred lines. Eight morphological traits (ovule and seed numbers per silique, plant shape, pubescence and root formation) were measured for investigation. We constructed a map composed of 322 markers with a total length of 673.6 cM. The linkage groups were assigned to the radish chromosomes using disomic rape-radish chromosome-addition lines. On the map, eight and 10 QTLs were identified in 2008 and 2009, respectively. The chromosome-linkage group correspondence, the sequence-specific markers and the QTLs detected here will provide useful information for further genetic studies and for selection during radish breeding programs.

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.).

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, 'Harufuku'. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3-15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.

QTL mapping of clubroot resistance in radish (Raphanus sativus L.).

A QTL analysis for clubroot resistance (CR) of radish was performed using an F(2) population derived from a crossing of a CR Japanese radish and a clubroot-susceptible (CS) Chinese radish. F(3) plants obtained by selfing of F(2) plants were used for the CR tests. The potted seedlings were inoculated and the symptom was evaluated 6 weeks thereafter. The mean disease indexes of the F(3) plants were used for the phenotype of the F(2). The results of two CR tests were analyzed for the presence of QTL. A linkage map was constructed using AFLP and SSR markers; it spanned 554 cM and contained 18 linkage groups. A CR locus was observed in the top region of linkage group 1 in two tests. Therefore, the present results suggest that a large part of radish CR is controlled by a single gene or closely linked genes in this radish population, although minor effects of other genomic areas cannot be ruled out. The CR locus was named Crs1. Markers linked to Crs1 showed sequence homology to the genomic region of the top of chromosome 3 of Arabidopsis, as in the case of Crr3, a CR locus in Brassica rapa. These markers should be useful for breeding CR cultivars of radish. As Japanese radishes are known to be highly resistant or immune to clubroot, these markers may also be useful in the introgression of this CR gene to Brassica crops.

Neuropathy is associated with depression independently of health-related quality of life in Japanese patients with diabetes.

OBJECTIVES: To identify factors independently associated with depression in Japanese patients with diabetes, after controlling for potential confounding factors. METHODS: Among 197 outpatients with diabetes, 129 (type 1: 24, type 2: 105) completed a questionnaire concerning socio-demographic and health-related variables. Depression screening was done using Zung's Self-Rating Depression Scale test, followed by diagnostic interviews by experienced psychiatrists employing the Diagnostic Statistical Manual of Mental Disorders, 4th edition (DSM-IV). RESULTS: Forty-seven patients (36.4%) had symptomatological depression. A Self-Rating Depression Scale cut-off score of 40 had good sensitivity (100%) and modest specificity (59%) for detecting major depressive episode, in accordance with the DSM-IV. Diabetic patients suffering from depression were more likely to have neuropathy, retinopathy, body pain, a feeling of poor general health, and lack of social support, than the non-depressed patients. However, age, gender, marital status, diabetes type, insulin requirement, duration of diabetes, hemoglobin A1c (HbA1c) and the presence of nephropathy did not differ between the two groups. In multivariate logistic regression analysis, body pain (OR 3.26, 95% CI 1.31-8.08) and the presence of microvascular complications (OR 2.81, 95% CI 1.13-6.98) were independent factors associated with depression. Specifically, diabetic neuropathy (OR 3.10, 95% CI 1.17-8.22) was associated with depression independently of age, gender, marital status, social supports, quality of life, diabetes type, duration of diabetes, HbA1c, and insulin requirement. CONCLUSIONS: A diabetic complication, specifically neuropathy, was independently associated with depression in patients with diabetes. The present findings indicate the need to find a biological base common to both depression in diabetes and diabetic neuropathy.

Transfer of rice mitochondrial ribosomal protein L6 gene to the nucleus: acquisition of the 5'-untranslated region via a transposable element.

BACKGROUND: The mitochondria of contemporary organisms contain fewer genes than the ancestral bacteria are predicted to have contained. Because most of the mitochondrial proteins are encoded in the nucleus, the genes would have been transferred from the mitochondrion to the nucleus at some stage of evolution and they must have acquired cis-regulatory elements compatible with eukaryotic gene expression. However, most of such processes remain unknown. RESULTS: The ribosomal protein L6 gene (rpl6) has been lost in presently-known angiosperm mitochondrial genomes. We found that each of the two rice rpl6 genes (OsRpl6-1 and OsRpl6-2) has an intron in an identical position within the 5'-untranslated region (UTR), which suggests a duplication of the rpl6 gene after its transfer to the nucleus. Each of the predicted RPL6 proteins lacks an N-terminal extension as a mitochondrial targeting signal. Transient assays using green fluorescent protein indicated that their mature N-terminal coding regions contain the mitochondrial targeting information. Reverse transcription-PCR analysis showed that OsRpl6-2 expresses considerably fewer transcripts than OsRpl6-1. This might be the result of differences in promoter regions because the 5'-noncoding regions of the two rpl6 genes differ at a point close to the center of the intron. There are several sequences homologous to the region around the 5'-UTR of OsRpl6-1 in the rice genome. These sequences have characteristics similar to those of the transposable elements (TE) belonging to the PIF/Harbinger superfamily. CONCLUSION: The above evidences suggest a novel mechanism in which the 5'-UTR of the transferred mitochondrial gene was acquired via a TE. Since the 5'-UTRs and introns within the 5'-UTRs often contain transcriptional and posttranscriptional cis-elements, the transferred rice mitochondrial rpl6 gene may have acquired its cis-element from a TE.

Increased hepatic expression of ganglioside-specific sialidase, NEU3, improves insulin sensitivity and glucose tolerance in mice.

Membrane microdomains rich in gangliosides are recognized as being critical for proper compartmentalization of insulin signaling. Plasma membrane-associated sialidase, NEU3, is a key enzyme for ganglioside hydrolysis. We previously reported that mice overexpressing NEU3 mainly in muscles developed severe insulin-resistant diabetes. To examine the possible contributions of NEU3 to in vivo insulin sensitivity and glucose tolerance, NEU3 was expressed by using adenoviral vectors in the livers of C57BL/6 mice on standard and high-fat diets, and insulin-resistant KKAy mice on standard diets. Hepatic NEU3 overexpression paradoxically improved glucose tolerance and insulin sensitivity in the C57BL/6 mice fed standard diets, and glucose tolerance in the C57BL/6 mice fed high-fat diets and in KKAy mice. Hepatic NEU3 overexpression increased hepatic glycogen deposition and triglyceride accumulation, and enhanced the hepatic peroxisome proliferator-activated receptor gamma and fetuin expression in the C57BL/6 mice on standard and high-fat diets, and in KKAy mice. Thin-layer chromatographic analysis demonstrated increased levels of GM1 and markedly reduced GM3 in the livers of mice with hepatic NEU3 overexpression (NEU3 mice). Basal and insulin-stimulated tyrosine phosphorylations of insulin receptor substrate 1 were significantly increased, but tyrosine phosphorylations of the insulin receptor and insulin receptor substrate 2 in the NEU3 liver were unchanged. Insulin-stimulated tyrosine phosphorylations of the insulin receptor were increased in adipose tissues of NEU3 mice. These results suggest that hepatic NEU3 overexpression improves insulin sensitivity and glucose tolerance through modification of ganglioside composition and peroxisome proliferator-activated receptor gamma signaling. Our findings also provide further evidence that NEU3 is an important regulator of insulin sensitivity and glucose tolerance.

Reliability and validity of the Japanese version of the World Health Organization-Five Well-Being Index in the context of detecting depression in diabetic patients.

The present study had two aims. The first was to evaluate the reliability and the validity of the Japanese version of the World Health Organization (WHO)-Five Well-Being Index (WHO-5-J) as a brief well-being scale. The second was to examine the discriminatory validity of this test as a screening tool for current depressive episodes in diabetic patients. A sample of 129 diabetic patients completed the WHO-5-J. Of these, 65 were also interviewed by psychiatrists to assess whether they had any current depressive episodes according to DSM-IV. The internal consistency was evaluated using Cronbach's alpha, the Loevinger coefficient of homogeneity, and factor analysis. The external concurrent validity was evaluated by correlations with the external scales potentially related to subjective well-being. Discriminatory validity was evaluated using receiver operating characteristic (ROC) analysis. Cronbach's alpha and the Loevinger coefficient were estimated to be 0.89 and 0.65, respectively. A factor analysis identified only one factor. The WHO-5-J was significantly correlated with a number of major diabetic complications, depression, anxiety, and subjective quality of life. ROC analysis showed that the WHO-5-J can be used to detect a current depressive episode (area under curve: 0.92; 95% confidence interval: 0.85-0.98). A cut-off of <13 yielded the best sensitivity/specificity trade-off: sensitivity, 100%; specificity, 78%. The WHO-5-J was thus found to have a sufficient reliability and validity, indicating that it is a useful instrument for detecting current depressive episodes in diabetic patients.

Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance.

An SSR-based linkage map was constructed in Brassica rapa. It includes 113 SSR, 87 RFLP, and 62 RAPD markers. It consists of 10 linkage groups with a total distance of 1005.5 cM and an average distance of 3.7 cM. SSRs are distributed throughout the linkage groups at an average of 8.7 cM. Synteny between B. rapa and a model plant, Arabidopsis thaliana, was analyzed. A number of small genomic segments of A. thaliana were scattered throughout an entire B. rapa linkage map. This points out the complex genomic rearrangements during the course of evolution in Cruciferae. A 282.5-cM region in the B. rapa map was in synteny with A. thaliana. Of the three QTL (Crr1, Crr2, and Crr4) for clubroot resistance identified, synteny analysis revealed that two major QTL regions, Crr1 and Crr2, overlapped in a small region of Arabidopsis chromosome 4. This region belongs to one of the disease-resistance gene clusters (MRCs) in the A. thaliana genome. These results suggest that the resistance genes for clubroot originated from a member of the MRCs in a common ancestral genome and subsequently were distributed to the different regions they now inhabit in the process of evolution.

Isolation and characterization of the pea cytochrome c oxidase Vb gene.

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

WRN gene 1367 Arg allele protects against development of type 2 diabetes mellitus.

Werner's syndrome is an autosomal recessive disease caused by mutation of the WRN gene, which may lead to DNA repair failure and acceleration of aging. A polymorphism at amino acid 1367 Cys (TTG)/Arg (CTG) reportedly reduces the risk of myocardial infarction in Japanese. We studied the possible involvement of this polymorphism in type 2 diabetes. When polymorphism of the WRN gene was analyzed in 272 randomly recruited type 2 diabetic subjects (age 64.5+/-11.1), we found those with Cys/Arg to be older than those with Cys/Cys (p=0.021) and that the age at diagnosis of diabetes was greater in Cys/Arg than in Cys/Cys subjects (p=0.011). Diabetes-free survival rate over the age, analyzed by Kaplan-Meier method, differed significantly between these two genotype groups (p=0.0125) and the survival curve was shifted to the right in the Cys/Arg group as compared to the Cys/Cys group. No difference in allele frequency was observed between our diabetic (n=272) and non-diabetic subjects (n=171, age 66.0+/-8.0). These results suggest that the 1367 Arg allele of the WRN gene protects against the development of type 2 diabetes mellitus in Japanese.

Secondary sulfonylurea failure: comparison of period until insulin treatment between diabetic patients treated with gliclazide and glibenclamide.

We retrospectively evaluated a possible difference in periods until start of insulin treatment between type 2 diabetic patients treated with gliclazide (GCZ) and glibenclamide (GBC), because GCZ might be protective for beta cells than GBC. Subjects were Japanese patients. GCZ group consisted of patients treated with GCZ alone or with GCZ and GBC in the separate treatment periods in combination with or without other oral hypoglycemic agents (OHAs), while GBC group consisted of patients with GBC alone or in combination with other OHAs except GCZ. The periods until the treatment of insulin commenced were calculated using the Kaplan-Meier method. Proportional hazards models were used to adjust the differing variables between GCZ and GBC groups. The periods until the start of insulin treatment from diabetes onset, diabetes treatment, or GCZ or GBC treatment were significantly longer in the GCZ group than those in GBC group (P<0.001 in each group). Independent variables affecting the period were average HbA1c levels during GCZ or GBC treatment (hazard ratio=2.5 per %), other OHAs combined (hazard ratio=1.9 on combination), and difference between GCZ and GBC groups (hazard ratio=0.5 on GCZ). These results imply that GCZ may be more protective against secondary beta cell failure than GBC.

Genetic variations at urotensin II and urotensin II receptor genes and risk of type 2 diabetes mellitus in Japanese.

Urotensin II is among the most potent vasoactive hormones known and the urotensin II (UTS2) gene is localized to 1p36-p32, one of the regions reported to show possible linkage with type 2 diabetes in Japanese. When we surveyed genetic polymorphisms in the UTS2 and urotensin II receptor (GPR14) gene, we identified two SNPs with amino acid substitutions (designated T21M and S89N and an SNP in the promotor region (-605G>A) of the UTS2 gene, and two SNPs in the non-coding region of the GPR14 gene. We then studied these three SNPs in the UTS2 gene and two SNPs in the GPR14 gene in 152 Japanese subjects with type 2 diabetes mellitus and two control Japanese populations. The allele frequency of 89N was significantly higher in type 2 diabetic patients than in both elderly normal subjects (P = 0.0018) and subjects with normal glucose tolerance (P = 0.0011), whereas the allele frequency of T21M and -605G>A in the UTS2 gene and those of two SNPs in the GPR14 gene were essentially identical in these three groups. Furthermore, in the subjects with normal glucose tolerance, 89N was associated with significantly higher insulin levels on oral glucose tolerance test, suggesting reduced insulin sensitivity in subjects with 89N. These results strongly suggest that subjects with S89N in the UTS2 gene are more insulin-resistant and thus more susceptible to type 2 diabetes mellitus development.

Cultivated tomato has defects in both S-RNase and HT genes required for stylar function of self-incompatibility.

Cultivated tomato (Lycopersicon esculentum), a self-compatible species, evolved from self-incompatible (SI) species in the genus Lycopersicon following a breakdown of the self-incompatibility system. In order to elucidate the molecular basis of this breakdown in L. esculentum, we first analysed the stylar proteins with an in-gel assay for ribonuclease activity and 2D-PAGE. No S-RNase protein or its activity was detected in the style of L. esculentum. We then introduced the S6-RNase gene from an SI relative, L. peruvianum, into L. esculentum. However, the styles of transgenic plants expressing S6-RNase at levels comparable to those found in the L. peruvianum style were unable to reject self-pollen and L. peruvianum pollen in an allele-specific manner. This indicated that defect in the S-RNase expression was not the sole reason for the loss of self-incompatibility in tomato. The asparagine-rich HT protein, originally identified from the style of Nicotiana alata, is the other stylar factor involved in self-incompatibility reaction. We cloned and sequenced two distinct genes encoding HT-A and HT-B proteins from L. peruvianum (LpHT-A and LpHT-B) and L. esculentum (LeHT-A and LeHT-B). A frame shift mutation in the coding sequence of LeHT-A and a stop codon in the ORF of LeHT-B were found, and no LeHT-B transcript was detected in the style of L. esculentum. The results suggest that the breakdown of self-incompatibility in cultivated tomato is associated with loss-of-function mutations in both S-RNase and HT genes.

Correlative polymorphism of NAD(P)H: quinone oxidoreductase (NQO1) with telomere shortening in colorectal cancer.

AIM: The aim of this study was to examine whether and relationships could be found among polymorphism of the NQO1 gene, telomere length and telomerase activity in colorectal cancers. MATERIALS AND METHODS: Fifty-one invasive colorectal cancers were studied. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was undergone to detect mutation of the NQO1 gene. Telomere length was examined by Southern blot analysis. Telomerase activity was assayed by telomeric repeat amplification protocol with minor modifications. RESULTS: Of the 51 tumors, 20 (39.2%) and 9 (17.6%) were heterozygous and homozygous for the mutation, respectively. Most of the cases homozygous for the mutation (88.9%) showed short telomeres and its frequency was significantly higher than in those heterozygous (p = 0.0432). However no relationship was found between the telomerase activity and mutation in the NQO1 gene. CONCLUSION: Our data suggest that oxidative stress by the lack of NQO1 activity could result in telomere shortening through colorectal cancinogenesis.