Polymeric Hydrogelator-Based Molecular Gels Containing Polyaniline/Phosphoric Acid Systems.

To expand the range of applications of hydrogels, researchers are interested in developing novel molecular hydrogel materials that have affinities for the living body and the ability to mediate electrical signals. In this study, a simple mixing method for creating a novel composite molecular gel is employed, which combines a hydrophilic conductive polymer, a polyaniline/phosphoric acid complex, and a polymer hydrogelator as a matrix. The composite hydrogel showed an improved gel-forming ability; more effective mechanical properties, with an increased strain value at the sol-gel transition point compared to the single system, which may be sufficient for paintable gel; and a better electrochemical response, due to the electrically conducting polyaniline component. These findings demonstrate the applicability of the new composite hydrogels to new potential paintable electrode materials.

[Chemo-Resistant Breast Carcinoma with Cartilaginous Differentiation Manifestation after Neoadjuvant Chemotherapy-A Case Report].

A 49-year-old woman was admitted to our hospital because of a tumor in her right breast. The tumor was localized to the C area and was approximately 3 cm in size. A right axillary lymphadenopathy was also found. Histopathological examination and needle biopsy of the breast tumor revealed invasive lobular carcinoma, and she was diagnosed with Stage ⅡB triple-negative breast cancer(cT2N1M0). Paclitaxel plus bevacizumab chemotherapy followed by ddAC chemotherapy was administered as neoadjuvant chemotherapy, but the tumor remained stable. Thus, she underwent mastectomy and lymph node dissection. Pathological findings of the resected specimen showed invasive carcinoma with cartilaginous differentiation. She was then treated with capecitabine 15 days after the surgery; however, multiple lung metastases were found on CT after 6 courses. Therefore, she was transferred to another hospital and received other chemotherapies, but died after 5 months.

[A Case of Ascending Colon Cancer Probably Arising from a Diverticulum].

A 70's male was admitted to our hospital with complaints of anorexia and abdominal pain. CT showed thickening of the ascending colon. Colonoscopy revealed multiple diverticula of the ascending colon, but no tumor on the mucosa. The patient was diagnosed as a case of diverticulitis of the ascending colon and was advised laparoscopic ileocecal resection. The resected specimen showed wall thickening; however, no remarkable findings were observed, with the exception of multiple diverticula on the mucosal surface. Histological examination showed well-differentiated tubular adenocarcinoma extending into the serosa probably arising from the diverticulum. Chemotherapy was performed after surgery. The patient died due to peritoneal dissemination from the ascending colon cancer 14 months after surgery.

Acute appendicitis with a neuroendocrine tumor G1 (carcinoid): pitfalls of conservative treatment.

A man in his early thirties presented to our clinic with right lower abdominal pain. Computed tomography (CT) and ultrasonography (US) revealed a swollen appendix and an appendicolith. Abscess formation was not observed but ongoing appendiceal rupture was not ruled out. Three months after successful conservative therapy, the lumen of the apical portion was kept dilated and laparoscopic interval appendectomy was performed. No tumorous findings were observed macroscopically. However, histology revealed many tiny nests infiltrating the submucosa, muscular layer, and subserosa at the root of the appendix. An appendiceal neuroendocrine tumor G1 (NET G1; carcinoid) was diagnosed immunohistologically. Neither CT nor US visualized the tumor because of its non-tumor-forming but infiltrative growth. In conclusion, after successful conservative treatment, interval appendectomy should be considered to uncover a possible appendiceal NET G1 (carcinoid), particularly when dilatation of the distal lumen is kept under observation.

Extramedullary relapse in RARA rearrangement-negative acute promyelocytic leukemia successfully treated in combination with chemotherapy, local radiotherapy, and cord blood transplantation.

RARA rearrangement-negative acute promyelocytic leukemia (APL) is uncommon, and its extramedullary relapse is extremely rare. We report a 5-year-old girl with RARA rearrangement-negative APL, which recurred solely at the external auditory canal and mastoid air cells. She was successfully treated with chemotherapy, local radiotherapy, and unrelated cord blood transplantation. She has maintained complete remission for 24 months after transplantation. The clinical features and our therapeutic strategy in this patient will provide valuable information for extramedullary relapse of RARA rearrangement-negative APL.

Characterization of genome-reduced fission yeast strains.

The Schizosaccharomyces pombe genome is one of the smallest among the free-living eukaryotes. We further reduced the S. pombe gene number by large-scale gene deletion to identify a minimal gene set required for growth under laboratory conditions. The genome-reduced strain has four deletion regions: 168.4 kb in the left arm of chromosome I, 155.4 kb in the right arm of chromosome I, 211.7 kb in the left arm of chromosome II and 121.6 kb in the right arm of chromosome II. The deletions corresponded to a loss of 223 genes of the original ~5100. The quadruple-deletion strain, with a total deletion size of 657.3 kb, showed a decreased ability to uptake glucose and some amino acids in comparison with the parental strain. The strain also showed increased gene expression of the mating pheromone M-factor precursor and the nicotinamide adenine dinucleotide phosphate -specific glutamate dehydrogenase. There was also a 2.7-fold increase in the concentration of cellular adenosine triphosphate, and levels of the heterologous proteins, enhanced green fluorescent protein and secreted human growth hormone were increased by 1.7- and 1.8-fold, respectively. The transcriptome data from this study have been submitted to the Gene Expression Omnibus (GEO: http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE38620 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=vjkxjewuywgcovc&acc=GSE38620).

Exome sequencing identifies a new candidate mutation for susceptibility to diabetes in a family with highly aggregated type 2 diabetes.

The aim of this study was to investigate the genetic background of familial clustering of diabetes using genome-wide linkage analysis combined with exome sequencing. We recruited a Japanese family with a 3-generation history of diabetes. The family comprised 16 members, 13 having been diagnosed with diabetes. Nine members had been diagnosed before the age of 40. Linkage analysis was performed assuming an autosomal dominant model. Linkage regions were observed on chromosomes 4q34, 5q11-q13, and 12p11-q22 and the logarithm of odds (LOD) scores were 1.80. To identify the susceptibility variants, we performed exome sequencing of an affected family member. We predicted that the familial clustering of diabetes is caused by a rare non-synonymous variant, and focused our analysis on non-synonymous variants absent in dbSNP131. Exome sequencing identified 10 such variants in the linkage regions, 7 of which were concordant with the affection status in the family. One hundred five normal subjects and 67 lean diabetes subjects were genotyped for the 7 variants; the only variant found to be significantly more frequent in the diabetes subjects than in the normal subjects was the N1072K variant of the early endosome antigen 1 (EEA1) gene (0 in normal subjects and 4 in diabetes subjects, p=0.022). We therefore propose that the N1072K variant of the EEA1 gene is a candidate mutation for susceptibility to diabetes in the Japanese population.

Reduction of reactive oxygen species ameliorates metabolism-secretion coupling in islets of diabetic GK rats by suppressing lactate overproduction.

We previously demonstrated that impaired glucose-induced insulin secretion (IS) and ATP elevation in islets of Goto-Kakizaki (GK) rats, a nonobese model of diabetes, were significantly restored by 30-60-min suppression of endogenous reactive oxygen species (ROS) overproduction. In this study, we investigated the effect of a longer (12 h) suppression of ROS on metabolism-secretion coupling in ß-cells by exposure to tempol, a superoxide (O2(-)) dismutase mimic, plus ebselen, a glutathione peroxidase mimic (TE treatment). In GK islets, both H2O2 and O2(-) were sufficiently reduced and glucose-induced IS and ATP elevation were improved by TE treatment. Glucose oxidation, an indicator of Krebs cycle velocity, also was improved by TE treatment at high glucose, whereas glucokinase activity, which determines glycolytic velocity, was not affected. Lactate production was markedly increased in GK islets, and TE treatment reduced lactate production and protein expression of lactate dehydrogenase and hypoxia-inducible factor 1α (HIF1α). These results indicate that the Warburg-like effect, which is characteristic of aerobic metabolism in cancer cells by which lactate is overproduced with reduced linking to mitochondria metabolism, plays an important role in impaired metabolism-secretion coupling in diabetic ß-cells and suggest that ROS reduction can improve mitochondrial metabolism by suppressing lactate overproduction through the inhibition of HIF1α stabilization.

Self-monitoring of blood glucose (SMBG) improves glycaemic control in oral hypoglycaemic agent (OHA)-treated type 2 diabetes (SMBG-OHA study).

BACKGROUND: We conducted a clinical research study to determine the effect of self-monitoring of blood glucose (SMBG) on glycaemic control and the value of a putatively less painful blood sampling technique on SMBG in oral hypoglycaemic agent-treated type 2 diabetes patients; SMBG has not been broadly applied in non-insulin-treated patients in Japan. METHODS: One hundred thirty-seven subjects were recruited for the 24-week, prospective, comparison study and randomized into three groups: 46, no SMBG group; 46, fingertip group; and 45, palm group. The primary endpoint was change in HbA(1c). The secondary endpoints were SMBG compliance, dropout rate, treatment changes, and patient's and physician's satisfaction. RESULTS: Six subjects in the fingertip group (13.2%) and one subject in the palm group (2.2%) were dropped because of pain. A(1C) level of all subjects at 24-week was decreased more in the fingertip (-0.23%) and palm (-0.16%) groups than that in the no SMBG group (+0.31%) (p < 0.05). SMBG compliance was higher in the fingertip group (2.17 times/day) than that in the palm group (1.65 times/day) (p < 0.05). A(1C) level of treatment-unchanged subjects was decreased more in the fingertip (-0.25%) and palm (-0.21%) groups than that in the no SMBG group (+0.30%) (p < 0.05). SMBG compliance was higher in the fingertip group (2.24 times/day) than that in the palm group (1.65 times/day) (p < 0.05). Patient's questionnaire showed that 84.1% of the fingertip group and 90.2% of the palm group were satisfied with SMBG. Physician's satisfaction was higher in the palm group (94.0%) than that in the fingertip group (80.0%) (p < 0.05). CONCLUSION: SMBG is beneficial for glycaemic control, and palm blood sampling is a useful procedure for oral hypoglycaemic agent-treated type 2 diabetes.

Role of mitochondrial phosphate carrier in metabolism-secretion coupling in rat insulinoma cell line INS-1.

In pancreatic ß-cells, glucose-induced mitochondrial ATP production plays an important role in insulin secretion. The mitochondrial phosphate carrier PiC is a member of the SLC25 (solute carrier family 25) family and transports Pi from the cytosol into the mitochondrial matrix. Since intramitochondrial Pi is an essential substrate for mitochondrial ATP production by complex V (ATP synthase) and affects the activity of the respiratory chain, Pi transport via PiC may be a rate-limiting step for ATP production. We evaluated the role of PiC in metabolism-secretion coupling in pancreatic ß-cells using INS-1 cells manipulated to reduce PiC expression by siRNA (small interfering RNA). Consequent reduction of the PiC protein level decreased glucose (10 mM)-stimulated insulin secretion, the ATP:ADP ratio in the presence of 10 mM glucose and elevation of intracellular calcium concentration in response to 10 mM glucose without affecting the mitochondrial membrane potential (Δψm) in INS-1 cells. In experiments using the mitochondrial fraction of INS-1 cells in the presence of 1 mM succinate, PiC down-regulation decreased ATP production at various Pi concentrations ranging from 0.001 to 10 mM, but did not affect Δψm at 3 mM Pi. In conclusion, the Pi supply to mitochondria via PiC plays a critical role in ATP production and metabolism-secretion coupling in INS-1 cells.

GCKR mutations in Japanese families with clustered type 2 diabetes.

OBJECTIVE: The aim was to investigate the genetic background of familial clustering of type 2 diabetes. SUBJECTS AND METHODS: We recruited Japanese families with a 3-generation history of diabetes. Genome-wide linkage analysis was performed assuming an autosomal dominant model. Genes in the linkage region were computationally prioritized using Endeavour. We sequenced the candidate genes, and the frequencies of detected nucleotide changes were then examined in normoglycemic controls. RESULTS: To exclude known genetic factors, we sequenced 6 maturity onset diabetes of the young (MODY) genes in 10 familial cases. Because we detected a MODY3 mutation HNF1A R583G in one case, we excluded this case from further investigation. Linkage analysis revealed a significant linkage region on 2p25-22 (LOD score=3.47) for 4 families. The 23.6-Mb linkage region contained 106 genes. Those genes were scored by computational prioritization. Eleven genes, i.e., top 10% of 106 genes, were selected and considered primary candidates. Considering their functions, we eliminated 3 well characterized genes and finally sequenced 8 genes. GCKR ranked highly in the computational prioritization. Mutations (minor allele frequency less than 1%) in exons and the promoter of GCKR were found in index cases of the families (3 of 18 alleles) more frequently than in controls (0 of 36 alleles, P=0.033). In one pedigree with 9 affected members, the mutation GCKR g.6859C>G was concordant with affection status. No mutation in other 7 genes that ranked highly in the prioritization was concordant with affection status in families. CONCLUSIONS: We propose that GCKR is a susceptibility gene in Japanese families with clustered diabetes. The family based approach seems to be complementary with a large population study.

Exendin-4 suppresses SRC activation and reactive oxygen species production in diabetic Goto-Kakizaki rat islets in an Epac-dependent manner.

OBJECTIVE: Reactive oxygen species (ROS) is one of most important factors in impaired metabolism secretion coupling in pancreatic ß-cells. We recently reported that elevated ROS production and impaired ATP production at high glucose in diabetic Goto-Kakizaki (GK) rat islets are effectively ameliorated by Src inhibition, suggesting that Src activity is upregulated. In the present study, we investigated whether the glucagon-like peptide-1 signal regulates Src activity and ameliorates endogenous ROS production and ATP production in GK islets using exendin-4. RESEARCH DESIGN AND METHODS: Isolated islets from GK and control Wistar rats were used for immunoblotting analyses and measurements of ROS production and ATP content. Src activity was examined by immunoprecipitation of islet lysates followed by immunoblotting. ROS production was measured with a fluorescent probe using dispersed islet cells. RESULTS: Exendin-4 significantly decreased phosphorylation of Src Tyr416, which indicates Src activation, in GK islets under 16.7 mmol/l glucose exposure. Glucose-induced ROS production (16.7 mmol/l) in GK islet cells was significantly decreased by coexposure of exendin-4 as well as PP2, a Src inhibitor. The Src kinase-negative mutant expression in GK islets significantly decreased ROS production induced by high glucose. Exendin-4, as well as PP2, significantly increased impaired ATP elevation by high glucose in GK islets. The decrease in ROS production by exendin-4 was not affected by H-89, a PKA inhibitor, and an Epac-specific cAMP analog (8CPT-2Me-cAMP) significantly decreased Src Tyr416 phosphorylation and ROS production. CONCLUSIONS: Exendin-4 decreases endogenous ROS production and increases ATP production in diabetic GK rat islets through suppression of Src activation, dependently on Epac.

Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism.

Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity.

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway.

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Delta, vps22Delta, and vps34Delta, were determined on account of their hGH secretion efficiency. The mutant vps10Delta was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.

Production of heterologous proteins using the fission-yeast (Schizosaccharomyces pombe) expression system.

The fission yeast Schizosaccharomyces pombe is a particularly useful model for studying the function and regulation of genes from higher eukaryotes. The genome of Sc. pombe has been sequenced, and DNA microarray, proteome and transcriptome analyses have been carried out. Among the well-characterized yeast species, Sc. pombe is considered an attractive host for the production of heterologous proteins. Expression vectors for high-level expression in Sc. pombe have been developed and many foreign proteins have been successfully expressed. However, further improvements in the protein-expressing host systems are still required for the production of heterologous proteins involved in post-translational modification, metabolism and intracellular trafficking. This minireview focuses on recent advances in heterologous protein production by use of engineered fission-yeast strains.

The gap-filling sequence on the left arm of chromosome 2 in fission yeast Schizosaccharomyces pombe.

We report a gap-filling sequence between SPBPB21E7.09 (in contig c1348) and SPBPB10D8.01 (in contig pB10D8) on the left arm of chromosome 2 in the fission yeast, Schizosaccharomyces pombe. The sequence was determined from a BAC clone overlapping SPBPB21E7.01c (eno102) (in contig c1348) and SPBC1683.07 (mal1) (in contig pB10D8). The gap-filling sequence is 17,881 bp in length and contains five putative open reading frames, which were systematically named as SPBC460.01c, SPBC460.02c, SPBC460.03, SPBC460.04c and SPBC460.05. Their deduced amino acid sequences respectively include protein motifs corresponding to amino acid permease, glutathione S-transferase C-terminal domain, taurine catabolism dioxygenase TauD TfdA family and major facilitator superfamily, whereas their functions are unknown.

Intraperitoneal bleeding due to rupture of the left gastric vein (LGV) in a patient with liver cirrhosis: a case report.

Intraperitoneal bleeding from ruptured ectopic varices is a rare and fatal complication in patients with portal hypertension. Although laparotomy with high mortality is performed, it is difficult to detect correct bleeding site and save the patient. This is probably the first case report of rupture from left gastric vein revealed by transjugular intrahepatic portosystemic shunt (TIPS). We propose the use of TIPS for diagnosing and treating intraperitoneal bleeding from ectopic varices.

ABCA3-mediated choline-phospholipids uptake into intracellular vesicles in A549 cells.

ABCA3 is proposed to function as a lung surfactant lipid transporter. Here we report ABCA3-dependent lipid uptake into intracellular vesicles in lung adenocarcinoma A549 cells. A549 cells stably expressing GFP-tagged wild-type ABCA3 (A549/ABCA3(WT)) had larger LAMP3-positive vesicles than their parental cells as well as A549 cells expressing a Walker A motif mutant (A549/ABCA3(N568D)). The choline-phospholipids level in A549/ABCA3(WT) was increased 1.25-fold compared to that in A549 and A549/ABCA3(N568D) cells, while the cholesterol levels were similar. Sucrose gradient fractionation analysis in A549/ABCA3(WT) cells revealed that choline-phospholipids were enriched in low-density and nile red-positive vesicles. Electronmicroscopic analysis showed multilamellar vesicles in A549/ABCA3(WT) cells. These results indicate that ABCA3 mediates ATP-dependent choline-phospholipids uptake into intracellular vesicles.

ABCA2 deficiency results in abnormal sphingolipid metabolism in mouse brain.

ABCA2, a member of the ATP-binding cassette (ABC) transporter family, is localized mainly to late endosome/lysosomes of oligodendrocytes in brain, but the physiological role and function of ABCA2 are unknown. In this study, we generated mutant mice (ABCA2-null) by targeting the abca2 gene. ABCA2-null mice exhibited a phenotype including lower pregnancy rate and body weight, shorter latency period on the balance beam, and sensitization to environmental stress compared with wild type mice but no abnormality in the cytoarchitectonic and compact myelin structure or oligodendroglial differentiation. Lipid analysis of brain from 11 days to 64 weeks of age revealed significant accumulation of gangliosides along with reduced sphingomyelin (SM) from 4 weeks to 64 weeks of age and accumulation of cerebrosides and sulfatides at 64 weeks of age in ABCA2-null mice compared with wild type mice. In addition, a significant accumulation of the major ganglioside GM1 and reduced SM was detected in the myelin fraction of ABCA2-null brain. Comparison of ABCA2-null and wild type mice revealed weak ABCA2 immunoreactivity in some large pyramidal cells of wild type brain. These results suggest that ABCA2 is involved in the intracellular metabolism of sphingolipids in the brain, particularly SM and gangliosides in oligodendrocytes and certain neurons.