Effects of low-fat diet and aging on metabolic profiles of Creb3l4 knockout mice.

BACKGROUND/OBJECTIVES: Increased adipose tissue mass closely associates with the development of insulin resistance and type 2 diabetes mellitus. Previously, we reported that CREB3L4 expressed in adipose tissue negatively regulates adipogenesis, and Creb3l4 knockout mice fed a high-fat diet for 16 weeks showed fat cell hyperplasia, with improved glucose tolerance and insulin sensitivity. These mice did not show significant weight gain and fat mass. Because fat diet or aging is known to be associated with the development of obesity, we examined the effects of Creb3l4 gene subjected to low-fat diet (LFD) or aging process on body composition and obesity risk. SUBJECTS/METHODS: We fed Creb3l4 knockout mice a low-fat diet for 16 weeks (LFD group) or chow diet for over 1 year (aged group) and observed various metabolic parameters in the LFD-fed and aged Creb3l4 knockout mice. RESULTS: LFD-fed and aged Creb3l4 knockout mice showed significant weight gain and adiposity, impaired glucose tolerance and decreased insulin sensitivity, compared with wild-type mice. CONCLUSIONS: Creb3l4 has a critical role in metabolic phenotypes and a better understanding of its function may provide improved insight into the etiology of diabetes and other metabolic disorders.

ELAS1-mediated inhibition of the cyclin G1-B'γ interaction promotes cancer cell apoptosis via stabilization and activation of p53.

Radiation therapy (RT) is useful for selectively killing cancer cells. However, because high levels of ionizing radiation (IR) are toxic to normal cells, RT cannot be applied repeatedly to cancer patients. Therefore, novel chemicals that enhance the efficacy of chemoradiotherapy (CRT) would be valuable. Here, we report that ELAS1, a peptide corresponding to the protein phosphatase 2A (PP2A) association domain of cyclin G1 (CycG1), can enhance the efficacy of CRT. ELAS1 interacts with the PP2A B'γ-subunit and competitively inhibits association with CycG1, thereby preventing the PP2A holoenzyme from dephosphorylating target proteins, Mdm2 (pT218) and p53 (pS46), following DNA double-strand break (DSB) insults. Doxycycline (Dox)-induced overexpression of Myc-ELAS1 caused γ-irradiation to induce apoptosis in human osteosarcoma (U2OS) cells, at 1/10th the effective dosage of γ-irradiation required for apoptosis in Myc-vector-expressing cells; ELAS1 peptide incorporation into U2OS cells also showed similar apoptotic effects. Moreover, administration of DSB-inducing chemicals, camptothecin (CPT) or irinotecan, to Myc-ELAS1-expressing U2OS cells also induced efficient apoptosis with only 1/100th (CPT) or 1/5th (irinotecan) of the amounts of drugs required for this effect in Myc-vector-expressing cells. Taken together, ELAS1 may be important for the design of ELAS1-mimetic compounds to improve CRT efficacy.

Identification of Creb3l4 as an essential negative regulator of adipogenesis.

Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPß or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

[Metastatic lung tumor from colon cancer with cavitation: report of a case].

A 55-year-old man underwent rectal amputation for rectal cancer in August 2005. A tiny thin-walled cavity lesion in his left S1+2 was found on computed tomography (CT) of the chest in November 2008. The cavity lesion in the left S1+2 gradually increased in size over 3 months and positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) showed FDG accumulation at the lesion. Videoassisted thoracoscopic (VATS) wedge resection was performed to make a definite diagnosis in March 2009. The pathological findings revealed a metastatic lung tumor from the rectal cancer. It is necessary to consider the possibility of metastatic lung tumors in a case with the cavity lesions especially in patients with a history of colon cancer.

[Simultaneously treated thymoma and lung cancer; report of a case].

A 74-year-old man was admitted to our hospital in order to treat a mediastinal mass and 2 ground-glass attenuations in the right upper lobe detected by chest X-ray and computed tomography (CT). Partial resection of right lung and thymectomy were performed. The mediastinal mass and 2 ground-glass attenuations in the right upper lobe proved to be thymoma and bronchioloalveolar carcinomas, respectively by pathology.

Silencing of the Lats2 tumor suppressor overrides a p53-dependent oncogenic stress checkpoint and enables mutant H-Ras-driven cell transformation.

The Lats2 tumor suppressor protein has been implicated earlier in promoting p53 activation in response to mitotic apparatus stress, by preventing Mdm2-driven p53 degradation. We now report that Lats2 also has a role in an ATR-Chk1-mediated stress check point in response to oncogenic H-Ras. Activated mutant H-Ras triggers the translocation of Lats2 from centrosomes into the nucleus, coupled with an increase in Lats2 protein levels. This leads to the induction of p53 activity, upregulation of proapoptotic genes, downregulation of antiapoptotic genes and eventually apoptotic cell death. Many of the cells that survive apoptosis undergo senescence. However, a fraction of the cells escape this checkpoint mechanism, despite maintaining a high mutant H-Ras expression. These escapers display increased genome instability, as evidenced by a substantial fraction of cells with micronuclei and cells with polyploid genomes. Interestingly, such cells show markedly reduced levels of Lats2, in conjunction with enhanced hypermethylation of the Lats2 gene promoter. Our findings suggest that Lats2 might have an important role in quenching H-Ras-induced transformation, whereas silencing of Lats2 expression might serve as a mechanism to enable tumor progression.

Manganese superoxide dismutase Ala16Val polymorphism is associated with the development of type 2 diabetes in Japanese-Americans.

AIMS: Recent evidence indicates that oxidative stress may play an important role in the pathogenesis of insulin resistance and that gene polymorphism (Ala16Val) of manganese superoxide dismutase (MnSOD) may protect against reactive oxygen species (ROS) function. We aimed to test the hypothesis that the Ala16Val variant could be associated with the development of type 2 diabetes. METHODS: We examined 523 nondiabetic Japanese-Americans who underwent a 75g oral glucose tolerance test (OGTT) and were followed for an average of 9.9 years. Cox proportional hazard analysis, stratified by category of OGTT, was used to determine whether the Ala16Val polymorphism was a risk factor in the development of type 2 diabetes. RESULTS: During the follow-up period, 65 subjects developed type 2 diabetes. Compared with Ala allele carriers, subjects with a Val homozygote showed significantly higher risk for developing diabetes (stratified hazard ratio=2.05 [95% confidence interval 1.03-4.08]; P=0.041) after adjustment for age, gender, systolic blood pressure, total cholesterol, body mass index, and homeostasis model assessment. CONCLUSIONS: We demonstrated that the MnSOD Ala16Val polymorphism might be associated with development of type 2 diabetes among Japanese-Americans. These results suggest that insufficient ROS scavenging might be associated with a susceptibility to glucose intolerance.

Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection.

Although many reports have described laparoscopic minor liver resections, major hepatic resection, including right or left lobectomy, has not been widely developed because of technical difficulties. This article describes a new technique for performing laparoscopy-assisted right or left hepatic lobectomy using hilar Glissonean pedicle transection. Laparoscopic mobilization of the right or left hepatic lobe is performed, including dissection of the round, faliciform, triangular, and coronary ligaments. The right or left Glissonean pedicle is encircled and divided laparoscopically. A parenchymal dissection is then performed though the upper median or right subcostal incision, through which the resected liver is removed. We successfully performed this procedure in 6 patients without blood transfusion or serious complications. Laparoscopy-assisted hepatic lobectomy using hilar Glissonean pedicle transection can be feasible and safe in highly selected patients.

Protein kinases that regulate chromosome stability and their downstream targets.

Upon genotoxic stress, checkpoint machinery in eukaryotic cells induces cell-cycle arrest, thus allowing the cells to repair damaged DNA or stalled replication forks. The checkpoint machinery is mediated by phosphorylation cascades involving protein kinases and their target proteins. Since the genome is under constant threat from DNA damage due to radiation, chemicals and replication errors, checkpoint dysregulation can cause catastrophic DNA damage, resulting in chromosome instability, aneuploidy, and even tumorigenesis. Two parallel pathways that respond to DNA-damage stress have been extensively studied. The first is the ATM pathway, which responds to double-stranded DNA breaks, while the second is the ATR pathway, which primarily responds to agents that interfere with normal DNA replication. The ATM and ATR kinases activate their downstream target proteins by phosphorylating specific serine or threonine residues. Dephosphorylation by protein phosphatase (PP2A) also participates in the regulation of these phosphorylation signals. Of the target proteins, the two effector kinases CHK1 and CHK2 are particularly important because they phosphorylate additional substrates to maintain chromosome stability after various DNA damaging insults. Recent observations indicate that other protein kinases that control centrosome duplication and chromosome segregation during the cell cycle also play essential roles in maintaining genomic stability.

Increased expression of a nucleolar Nop5/Sik family member in metastatic melanoma cells: evidence for its role in nucleolar sizing and function.

F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.

Inhibition of itch-scratch response by fruits of Cnidium monnieri in mice.

We previously screened the anti-itching activities of 33 herbal medicines in substance P (SP)-induced itching model mice. One of the most potent antipruritogenic extracts, the methanol extract of fruits of Cnidium monnieri (Cnidii Fructus) was studied further. The chloroform-soluble fraction of the methanol extract markedly inhibited SP-induced scratching. Among 10 subfractions of the chloroform-soluble fraction, the CS-3 fraction had the most potent inhibitory effect on scratching. Each of 3 subfractions of CS-3 showed significant anti-scratching activities. However, inhibitory potencies were not different among the three and weaker than that of CS-3 itself at a same dose. These 3 subfractions of CS-3 mainly contained xanthotoxin, isopimpinellin, bergapten, imperatorin and osthol. Single administration of osthol did not inhibit SP-induced scratching, and imperatorin very weakly subsided scratching. These results suggest that the strong antipruritic action was focused on the CS-3 fraction of the C. monnieri methanol extract, and it might result from the combined effects of these coumarin derivatives, or by undetermined minor compounds.

A novel meiosis-specific protein of fission yeast, Meu13p, promotes homologous pairing independently of homologous recombination.

Meiotic homologous pairing is crucial to proper homologous recombination, which secures subsequent reductional chromosome segregation. We have identified a novel meiosis-specific protein of fission yeast Schizosaccharomyces pombe, Meu13p, to be a molecule that is required for proper homologous pairing and recombination. Rec12p (homologue of Saccharomyces cerevisiae Spo11p), which is essential for the initiation of meiotic recombination, is also shown for the first time to participate in the pairing process of S.pombe. Meu13p, however, contributes to pairing through a recombination-independent mechanism, as disruption of the meu13(+) gene reduces pairing whether the rec12(+) gene is deleted or not. We also demonstrate a dynamic nature of homologous pairing in living meiotic cells, which is markedly affected by meu13 deletion. Meu13p is not required for telomere clustering and the nuclear movement process, which are well known requirements for efficient pairing in S.pombe. Based on these results, together with the localization of Meu13p on meiotic chromatin, we propose that Meu13p directly promotes proper homologous pairing and recombination.

Induction by carrageenan inflammation of prepronociceptin mRNA in VR1-immunoreactive neurons in rat dorsal root ganglia.

Nociceptin (orphanin FQ) may act on primary afferents and be involved in the regulation of nociceptive processing. We have shown, using reverse transcription-polymerase chain reaction (RT-PCR), that carrageenan-produced peripheral inflammation induces the expression of prepronociceptin (PPN) mRNA in the dorsal root ganglia (DRG). The present experiments were conducted to determine the localization of PPN mRNA in primary sensory neurons after peripheral inflammation, using in situ hybridization. An intraplantar injection of carrageenan induced the expression of PPN mRNA in small and medium sized neurons in the DRG; the effect peaked 0.5 h after carrageenan and subsided by 6 h. All neurons positive for PPN mRNA were positive for vanilloid receptor subtype 1 (VR1)-like immunoreactivity and some VR1-immunoreactive neurons were negative for PPN mRNA. The results suggest that peripheral inflammation induces the production of nociceptin in a sub-population of VR1-positive primary sensory neurons and support the idea that nociceptin produced there is involved in the regulation of nociceptive processing.

Roles of mast cells and histamine in mosquito bite-induced allergic itch-associated responses in mice.

We investigated itch-associated responses (scratching) to mosquito bites and the role of histamine and mast cells in mosquito-induced itching in mice. Although the first bites of mosquito Aedes albopictus did not increase scratching, repeated bites increased scratching. The response was not diminished even after an interval of 2 months. Similarly, repeated intradermal (i.d.) injections of salivary gland extract (SGE) from Aedes albopictus increased scratching after SGE injection itself and mosquito bites. The scratching peaked within 10 min and almost subsided by 60 min. The opioid antagonist naloxone (1 mg/kg, s.c.) inhibited scratching following SGE injection. Although the non-sedative H1-histamine-receptor antagonist terfenadine (30 mg/kg, p.o.) significantly suppressed scratching induced by histamine (100 nmol/site, i.d.) in either naive or mosquito-sensitized mice, it did not affect mosquito-induced scratching in mosquito-sensitized mice. Repeated injections of SGE increased scratching in mast cell-deficient (WBB6F1-W/Wv) mice as well as in normal (WBB6F1-+/+) littermates. Repeated exposure to mosquito bites roughly doubled serum concentrations of total IgE and IgG1, but not IgG2a. Repeated injections of SGE markedly increased plasma extravasation induced by mosquito bites and such an increase was almost completely suppressed by terfenadine (30 mg/kg, p.o.). The results show the presence of histamine-mediated and histamine-independent mechanisms in cutaneous itching and suggest that histamine probably released from mast cells does not play an important role in itching in immediate allergic reaction. Our murine model of mosquito itching may be useful for studying the mechanisms of immediate allergic itching.

Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery.

Cyclin G1 is one of the target genes of the transcription factor p53, and is induced in a p53-dependent manner in response to DNA damage. Although cyclin G1 has been implicated in a range of biological phenomena, its precise function remains unclear. Here we present an analysis of the physiological role of cyclin G1 using mice homozygous for a targeted disruption of the cyclin G1 gene. In order to clarify the role of cyclin G1 in the p53 pathway, downstream events such as apoptosis, cell growth and cell cycle checkpoint control were analysed in thymocytes and embryonic fibroblasts derived from cyclin G1-disrupted mice. No difference was detected in induction of apoptosis between mouse embryo fibroblasts (MEFs) derived from cyclin G1+/+ and cyclin G1-/- mice. Following irradiation, cyclin G1-/- MEFs proliferated more slowly and reached lower cell densities in culture dishes than cyclin G1+/+ MEFs. Analysis of cell survival showed that cyclin G1-/- MEFs were about twice as sensitive as cyclin G1+/+ MEFs to gamma radiation or UV radiation. Cyclin G1-/- mice were more sensitive to gamma radiation than wild-type mice. Flow cytometeric analysis revealed that the number of cyclin G1-/- MEFs in G2/M phase after irradiation was reduced by 50% relative to cyclin G1+/+ MEFs. Our results demonstrate that cyclin G1 plays roles in G2/M arrest, damage recovery and growth promotion after cellular stress.

Comprehensive isolation of meiosis-specific genes identifies novel proteins and unusual non-coding transcripts in Schizosaccharomyces pombe.

In order to isolate meiosis-specific genes in Schizosaccharomyces pombe, we have constructed a subtracted cDNA library enriched in clones whose expression is enhanced during meiosis induced by nitrogen starvation. Using northern blot analysis, we isolated 31 kinds of clones whose expression was induced in a meiosis/sporulation-specific manner. We comprehensively named them meu after meiotic expression upregulated. The transcription of 20 meu genes was found to be dependent on the mei4(+) gene, which encodes a transcription factor required for the progression of meiosis. DNA sequencing indicated that most of the meu genes encode novel proteins. Notably, five of the meu genes harbor no apparent protein coding sequences, and the transcripts form stable hairpin structures, suggesting that they may generate non-coding RNAs or antisense RNAS: The results presented here imply that RNAs are also important for the comprehensive characterization of genomic expression.

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells.

We isolated a novel gene, termed MLZE, from a B16-BL6 cDNA library after subtraction of B16-F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1 - 2, which contains the c-myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze-positive cases was significantly larger in Clark levels III, IV and V melanomas (6 / 11 = 55%) than in Clark levels I and II melanomas (2 / 15 = 13%). In two cases of hMlze-positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

Characterization of rec7, an early meiotic recombination gene in Schizosaccharomyces pombe.

rec7 is involved in intra- and intergenic meiotic recombination in all tested regions of the genome of the fission yeast Schizosaccharomyces pombe. Segregational analysis in a rec7 gene disruption mutant revealed frequent occurrence of two-spored asci. Spores giving rise to diploid colonies were shown to derive from skipping of the second meiotic division. Nondisjunction of homologous chromosomes at the first meiotic division was also frequent. The cytological structures and processes, such as formation of linear elements, pairing of homologous chromosomes, and clustering of telomeres and centromeres, are regular in the mutant. Northern blot experiments revealed meiosis-specific expression of rec7. Screening of a meiotic cDNA library also identified transcripts from the opposite strand in the rec7 region. A Rec7-GFP fusion protein was localized in the nucleus of whole cells before karyogamy, during prophase, and after meiosis I. On spreads of prophase nuclei approximately 50 foci of Rec7-GFP were counted. Some of the observed phenotypes of the disruption mutant and the N-terminal sequence homology suggest that Rec7p is a functional homolog of Rec114p of Saccharomyces cerevisiae. The observed phenotypes of the disruption and the appearance of Rec7-GFP in mating haploid cells and after meiosis I are consistent with Rec7p functions before, during, and after meiotic prophase.

Kcc4 associates with septin proteins of Saccharomyces cerevisiae.

Kcc4, a kinase of the budding yeast Saccharomyces cerevisiae, is homologous to the bud neck protein kinases Hsl1/Nik1 and Gin4. We report here that a GFP-Kcc4 fusion protein is localized at the bud neck and that the non-kinase domain is required for this localization. We also demonstrate that Kcc4 associates with septin proteins in vitro and in vivo by two-hybrid analysis, GST pull-down experiments, immunoprecipitation, and analysis of direct association with affinity-purified GST-Kcc4 and MBP-Septin proteins. From the results obtained here, we suggest that Cdc11 is the primary association partner of Kcc4.