MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity.

Adipocytes secrete a variety of bioactive molecules that affect the insulin sensitivity of other tissues. We now show that the abundance of monocyte chemoattractant protein-1 (MCP-1) mRNA in adipose tissue and the plasma concentration of MCP-1 were increased both in genetically obese diabetic (db/db) mice and in WT mice with obesity induced by a high-fat diet. Mice engineered to express an MCP-1 transgene in adipose tissue under the control of the aP2 gene promoter exhibited insulin resistance, macrophage infiltration into adipose tissue, and increased hepatic triglyceride content. Furthermore, insulin resistance, hepatic steatosis, and macrophage accumulation in adipose tissue induced by a high-fat diet were reduced extensively in MCP-1 homozygous KO mice compared with WT animals. Finally, acute expression of a dominant-negative mutant of MCP-1 ameliorated insulin resistance in db/db mice and in WT mice fed a high-fat diet. These findings suggest that an increase in MCP-1 expression in adipose tissue contributes to the macrophage infiltration into this tissue, insulin resistance, and hepatic steatosis associated with obesity in mice.

PKClambda regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic beta cells.

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the lambda isoform of PKC in pancreatic beta cells (betaPKClambda(-/-) mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from betaPKClambda(-/-) mice. Neither the beta cell mass nor the islet insulin content of betaPKClambda(-/-) mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3beta was reduced in islets of betaPKClambda(-/-) mice, and the expression of genes regulated by HNF3beta was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3beta expression by infection of islets from betaPKClambda(-/-) mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKClambda plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for beta cell function.

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon.

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.

Osteoblast differentiation and bone formation gene expression in strontium-inducing bone marrow mesenchymal stem cell.

Osteoblastic differentiation from human mesenchymal stem cell (hMSCs) is an important step of bone formation. We studied the in vitro induction of hMSCs by using strontium ranelate, a natural trace amount in water, food and human skeleton. The mRNA synthesis of various osteoblast specific genes was assessed by means of reverse transcription polymerase chain reaction (RT-PCR). In the hMSCs culture, strontium ranelate could enhance the induction of hMSCs to differentiate into osteoblasts. Cbfa1 gene was earlier expressed on day 4 of cell culture (the control group, on day 14) and osteonectin on day 11 (control, on day 21). The early Cbfa1 expression indicates that strontium could enhance osteoblastic differentiation. The detection of osteonectin using strontium induction indicates the role of strontium in enhancing bone remodeling, bone structure stabilization of hydroxyapatite molecule and collagen fibril organization. The cultured hMSCs in the presence of strontium expressed genes of bone extracellular matrix: collagen type I, bone sialoprotein and osteocalcin on the same days as control (same medium with no strontium). Concentration of strontium ranelate has been recommended to be optimized in between 0.2107 - 21.07 microg/ml whereas the high concentration up to 210.7 microg/ml have delayed effect on osteoblastic differentiation with delayed expression on Cbfa1 and osteonectin, and inhibitory effect on bone sialoprotein expression. In addition, strontium could help cell expansion by maintaining cell proliferation rate of hMSCs and osteoblast lineage. We recommend that the strontium is an important factor for inducing mesenchymal stem cells to differentiate into osteoblasts with further enhancement on bone formation. This model might provide a useful cell source for tissue engineering and bone repair.

Methylation adjacent to negatively regulating AP-1 site reactivates TrkA gene expression during cancer progression.

Nerve growth factor and its high-affinity receptor TrkA are thought to be involved in the progression of various cancers. This study investigated the mechanism that regulates aberrant or increased TrkA expression in various cancer cell lines and in the course of pancreatic cancer progression. We found that the negative cis-acting AP-1-like sequence TGAGCGA was located in the 5'-untranslated region of the TrkA gene. Sodium bisulfite mapping revealed that steady-state TrkA expression correlated positively with the accumulation of methylated CpG around the AP-1-like site. Electrophoretic mobility shift assay showed that the AP-1-like site was bound mainly by c-Jun homodimers; the binding was directly blocked by Sss I methylase-induced methylation or by an excess of oligonucleotides containing consensus AP-1 sequences. Consequently, activation of TrkA gene expression by methylation was considered to be caused by the direct interference of c-Jun binding to the negatively regulating AP-1-like site. Furthermore, the accumulation of methylated CpG around the AP-1-like site was also observed with increased TrkA immunohistochemical staining in cases of advanced pancreatic adenocarcinoma with extensive perineural invasion. Unlike global methylation at CpG islands that leads to gene silencing, specific methylation at non-CpG islands would play a crucial epigenetic role in the versatility and plasticity of TrkA expression during cancer progression.

PKC pathway and ERK/MAPK pathway are required for induction of cyclin D1 and p21Waf1 during 12-o-tetradecanoylphorbol 13-acetate-induced differentiation of myeloleukemia cells.

Treatment of human promyelocytic leukemia cell HL60 with 12-o-tetradecanoylphorbol 13-acetate (TPA) induces growth arrest, differentiation towards the monocyte/macrophage lineage, and expression of cell cycle-regulating genes cyclin D1 and p21Waf1. First, we demonstrated that p21Waf1 expression was increased by TPA in other leukemia cell lines also, including THP-1, U937, and KG-1, which differentiate into monocytes/macrophages by TPA. Secondly, we demonstrated the signal transduction pathways of cyclin D1 and p21Waf1 expressions in TPA-treated HL60 cells. Induction of cyclin D1 expression in TPA-treated HL60 cells was inhibited with protein kinase C (PKC) inhibitor bisindolylmaleimide I and mitogen activated protein kinase kinase (MEK) inhibitor PD98059. Induction of p21Waf1 expression in TPA-treated HL60 cells was inhibited with PKC inhibitor bisindolylmaleimide I and Gö6976, MEK inhibitor PD98059, and p38 mitogen-actibated protein kinase (MAPK) inhibitor SB202190. Thus, cyclin D1 and p21Waf1 expressions are considered to be induced via PKC and extracellular signal-regulated kinase/mitogen-activated protein kinase (MAPK/ERK) pathways in TPA-treated HL60 cells. The upregulation of p21Waf1 seems to play a critical role in TPA-induced cell differentiation by suppressing cyclin dependent kinase activity , while the upregulation of cyclin D1 seems to be compensated by p21Waf1.

Crucial role of phospholipase Cepsilon in chemical carcinogen-induced skin tumor development.

Mutational activation of the ras proto-oncogenes is frequently found in skin cancers. However, the nature of downstream signaling pathways from Ras involved in skin carcinogenesis remains poorly understood. Recently, we and others identified phospholipase C (PLC) epsilon as an effector of Ras. Here we have examined the role of PLCepsilon in de novo skin chemical carcinogenesis by using mice whose PLCepsilon is genetically inactivated. PLCepsilon(-/-) mice exhibit delayed onset and markedly reduced incidence of skin squamous tumors induced by initiation with 7,12-dimethylbenz(a)anthracene followed by promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, the papillomas formed in PLCepsilon(-/-) mice fail to undergo malignant progression into carcinomas, in contrast to a malignant conversion rate of approximately 20% observed with papillomas in PLCepsilon(+/+) mice. In all of the tumors analyzed, the Ha-ras gene is mutationally activated irrespective of the PLCepsilon background. The skin of PLCepsilon(-/-) mice fails to exhibit basal layer cell proliferation and epidermal hyperplasia in response to TPA treatment. These results indicate a crucial role of PLCepsilon in ras oncogene-induced de novo carcinogenesis and downstream signaling from TPA, introducing PLCepsilon as a candidate molecular target for the development of anticancer drugs.

How to help Acehnese helping themselves?--a note after a visit with Kobe University medical team.

On December 26, Aceh Province in Indonesia was hit by the worst earthquake and tsunami. A medical team from Kobe University Graduate School of Medicine visited two of the affected areas on the eastern coast of Aceh: Sigli and Lhokseumawe. This article provides a simple description of experience and assessment derived from the visit. The disaster has left Indonesia with complex problems, which will take a long time to overcome. A continuity of the aid and assistance from various resources is crucial to help Indonesia rebuilt the affected areas. These continuous efforts will provide great contribution for Acehnese people to recover and rebuild their life after tsunami.

Ray tracing analysis of overlapping objects in refraction contrast imaging.

We simulated refraction contrast imaging in overlapping objects using the ray tracing method. The easiest case, in which two columnar objects (blood vessels) with a density of 1.0 [g/cm3], run at right angles in air, was calculated. For absorption, we performed simulation using the Snell law adapted to the object's boundary. A pair of bright and dark spot results from the interference of refracted X-rays where the blood vessels crossed. This has the possibility of increasing the visibility of the image.

Estimation of contrast of refraction contrast imaging compared with absorption imaging-basic approach.

PURPOSE: We discuss the usefulness of the refraction contrast method using highly parallel X-rays as a new approach to minute lung cancer detection. The advantages of refraction contrast images are discussed in terms of contrast, and a comparison is made with absorption images. MATERIALS AND METHODS: We simulated refraction contrast imaging using globules with the density of water in air as models for minute lung cancer detection. The contrast intensified by bright and dark lines was compared on a globule with the contrast of absorption images. We adopted the Monte Carlo simulation to determine the strength of the profile curve of the photon counts at the detector. RESULTS: The obtained contrasts were more intense by two to three digits than those obtainable with the absorption contrast imaging method. CONCLUSION: The contrast in refraction contrast imaging was more intense than that obtainable with absorption contrast imaging. A two to three digit improvement in contrast means that it is possible to greatly reduce the exposure dose necessary for imaging. Therefore, it is expected to become possible to detect the interfaces of soft tissues, which are difficult to capture with conventional absorption imaging, at low dosages and high resolution.

1 alpha,25 dihydroxyvitamin D3 rapidly regulates the mouse osteoprotegerin gene through dual pathways.

UNLABELLED: 1 alpha,25(OH)(2)D(3) rapidly and transiently suppressed OPG gene expression both by accelerating the degradation of mRNA and by suppressing promoter activity. The latter process was mediated through the AP-1 binding site by a reduction in the proportion of phospho-c-Jun in a JNK-independent manner. INTRODUCTION: Osteoclastogenesis is regulated by an integrated network of numerous bone metabolic factors, among which 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)] promotes osteoclastogenesis by reciprocally upregulating the expression of RANKL and downregulating that of osteoprotegerin (OPG). MATERIALS AND METHODS: To analyze the mechanism by which 1 alpha,25(OH)(2)D(3) suppresses OPG, we characterized cis-acting elements of the mouse OPG gene and assessed the post-transcriptional modifications by actinomycin D assays. RESULTS: 1 alpha,25(OH)(2)D(3) rapidly and transiently suppressed OPG expression and shortened the half-life of OPG mRNA; additionally, the c-Jun homodimer bound to the AP-1 binding site (TGACTGA, -293/-287) and maintained steady-state transcription of the OPG gene. Furthermore, mutation of the AP-1 site negated 1 alpha,25(OH)(2)D(3)-driven OPG suppression. Moreover, 1 alpha,25(OH)(2)D(3) treatment of ST2 cells decreased the amount of phosphorylated c-Jun protein (phospho-c-Jun), while the total amount of c-Jun remained constant; however, the amount of phosphorylated Jun N-terminal kinase (JNK) was nearly unchanged by 1 alpha,25(OH)(2)D(3) treatment. CONCLUSION: Taken together with the observation that the OPG promoter has no consensus negative vitamin D-responsive elements, these data suggest that 1 alpha,25(OH)(2)D(3) transrepresses mouse OPG by reducing the proportion of phospho-c-Jun in a JNK-independent manner. Our data indicated that short-term treatment with 1 alpha,25(OH)(2)D(3) effectively downregulated OPG expression both by accelerating the degradation of OPG mRNA and by transrepressing the OPG gene through its AP-1 binding site in the catabolic phase. The OPG gene became insensitive to 1 alpha,25(OH)(2)D(3) treatment, however, and reverted to its steady-state expression level over time, leading to the anabolic phase of the effect of 1 alpha,25(OH)(2)D(3) on bone.

7,12-DMBA-induced rat leukemia: a review with insights into future research.

7,12-Dimethylbenz[a]anthracene (DMBA) elicits leukemia in Long-Evans rats (LE). This leukemia is mostly erythroblastic and 30% of leukemias have total and partial trisomy of #2 chromosome and the rest have diploid karyotype. The common duplication site is in 2q26-q34 and N-ras gene is located in 2q34. 7,8,12-Trimethylbenz[a]anthracene (TMBA) also induces similar leukemias. These leukemias reveal a highly specific mutation of N-ras gene as in human leukemias. N-ras mutation is induced 48h after DMBA treatment. Wild type N-ras allele is frequently lost in diploid leukemias but not in trisomy type. Therefore, a gene dosage problem related to the mutant N-ras gene is involved in development of leukemia. Some secondary genetic rearrangements involving abl and H-ras are also observed in cultured leukemia cells. DMBA-induced chromosome aberrations as well as leukemia are enhanced by erythropoietin and blocked by Sudan III given prior to DMBA treatment. This leukemia will provide an important tool for chemical carcinogenesis and leukemia studies.

Prognostic significance of S100A4 expression in gallbladder cancer.

The calcium-binding protein S100A4 has been characterized as a metastasis-inducing molecule, and regulates cell motility and invasiveness of cancer cells. In order to clarify the significance of the expression of S100A4 as a prognostic factor in gallbladder cancer, S100A4 expression in resected gallbladder cancers were examined using an immunohistochemical staining technique. The relationship between S100A4 expression and clinicopathological factors including prognosis were evaluated. Twenty-five of 60 cases (42%) demonstrated positive staining for S100A4. There was no statistically significant association between S100A4 and histological grade, T, N, M factor, presence of stone, or stage. Kaplan-Meier method showed the 5-year survival rate of the group staining positive for S100A4 (31.5%) to be statistically poorer than that of the group staining negative for S100A4 (78.2%). Also in T2 cases, the 5-year survival rate of the group staining positive for S100A4 (57.1%) was statistically poorer than that of the group staining negative for S100A4 (83.3%). On univariate analysis, positive staining for S100A4 was a significant prognostic factor, and the hazard ratio was 4.05. On multivariate analysis, positive staining for S100A4 is also a significant predictor of prognosis second to T factor. These results indicate that positive staining for S100A4 is useful in assessing the prognosis of patients with gallbladder cancer as well as TNM factors.

Decrease in vitamin D receptor and calcium-sensing receptor in highly proliferative parathyroid adenomas.

OBJECTIVE: A significant decrease in vitamin D receptor (VDR) and calcium-sensing receptor (CaSR) protein expression has been demonstrated recently in parathyroid (PT) adenomas. In this study, we investigated the relationships between the proliferative activity of parathyroid glands (PTGs) and the expression of VDR as well as CaSR, and compared it with the clinical severity in patients with primary hyperparathyroidism (1 degrees HPT). DESIGN: Seven patients with 1 degrees HPT were included in this study. Four patients with thyroid carcinoma served as controls. METHODS: Immunohistochemical staining was performed on serial sections of PTGs with specific antibodies against CaSR, VDR, and Ki67. Areas examined in each section were selected at random in relation to the gland size. The number of Ki67-positive cells was expressed as a labeling index (LI; positive cells per 1000 PT cells). The expression of CaSR and VDR was semi-quantitatively analyzed based on the intensity of staining. After averages of the scores from all areas were calculated, CaSR and VDR scores, and Ki67 LI were assigned to each gland for use in statistical analyses. RESULTS: In PT adenomas, scores of VDR and CaSR were markedly lower than in normal PTGs (P<0.01), while the proportion of Ki67-positive cells in PT adenomas was significantly higher than in normal PTGs (P<0.01). Single regression analyses revealed that Ki67 LI was positively correlated with serum levels of intact parathyroid hormone and Ca, and PTG weight (R=0.70, P<0.05, R=0.78, P<0.01 and R=0.84, P<0.05 respectively). Ki67 LI was negatively correlated with CaSR and VDR scores (R=-0.78, P<0.01 and R=-0.72, P<0.05 respectively). Moreover, there was a strong positive relationship between CaSR and VDR expression (R=0.95, P<0.001). CONCLUSIONS: Marked decreases in VDR and CaSR expression could, at least in part, be responsible for the high proliferation of PT cells and the pathological progression of 1 degree HPT.

Adenocarcinoma arising in a tubular duplication of the jejunum.

To our knowledge, only five cases of malignant changes in small-bowel duplication have been reported previously in the English-language literature, and these were found only in ileal duplications. We describe a unique case of adenocarcinoma arising in a tubular duplication of the jejunum; this is the oldest patients in whom malignant tumor of alimentary tract duplication has been reported. A 73-year-old man was admitted to our hospital because of lower abdominal pain. Computed tomography (CT) and ultrasonography showed two abdominal masses; one, about 5 cm in size, was observed in the upper abdomen, the superior mesenteric artery (SMA) and the other, which was a well-demarcated heterogeneous mass, was revealed in the lower abdomen. Laparotomy revealed the mass in the upper abdomen involved the SMA and transverse colon, while the mass in the lower abdomen was of white globular form, ping-pong ball size, in the jejunal serosa approximately 40 cm on the anal side from Treiz's ligament. Transverse colostomy and a partial jejunectomy were performed, and the tumor was resected. Histologically, the diagnosis was adenocarcinoma arising in a tubular duplication of the jejunum. The patient died of necrosis and perforation of the intestine due to obstruction of the SMA 7 months after the surgery. Autopsy revealed that the upper abdominal mass consisted of metastatic foci associated with the lymph node of the SMA.

Genetic analysis of xanthogranulomatous cholecystitis: precancerous lesion of gallbladder cancer?

Xanthogranulomatous Cholecystitis is a chronic inflammatory disease of the gallbladder, a variant of the chronic cholecystitis. As xanthogranulomatous cholecystitis is occasionally seen with carcinoma of the gallbladder, the association with cancer is a controversial issue. A focal type of xanthogranulomatous cholecystitis is found simultaneously with gastric cancer diagnosed preoperatively. The resected specimen was genetically studied. Polymerase chain reaction amplification, single-strand conformational polymorphism analysis for mutation of p53 showed no abnormality indicating that less association with cancer in which the mutation of p53 is often seen. Etiopathologic factors of xanthogranulomatous cholecystitis might have relation with cancer, but xanthogranulomatous cholecystitis itself may not be the direct cause for cancer.

T-138C polymorphism of matrix gla protein promoter alters its expression but is not directly associated with atherosclerotic vascular calcification.

Matrix Gla protein (MGP) is a crucial inhibitor of vessel and cartilage calcification. We investigated the association of T-138C MGP promoter polymorphism with the degree of atherosclerosis, vascular calcification and patients' clinical background including calcification of the trachea and costal cartilage. Analysis of 108 autopsy cases was carried out by polymorphism-specific PCR on formalin-fixed paraffin-embedded samples. Statistical correlations among eight risk factors and five markers related to atherosclerosis and extra-bone tissue calcification were multivariantly analyzed. We found very high canonical correlations between the factors and the markers, and Pearson's correlation analysis revealed six significant correlations between age and the Gore index; age and costal cartilage calcification; sex and costal cartilage calcification; hypertension and the Gore index; hypertension and the calcification factor of the Gore index; and hyperlipidemia and costal cartilage calcification. The promoter activity of the -138T allele was significantly higher than that of the -138C allele; treatment with 12-O-tetradecanonylphorbol 13-acetate (TPA) significantly activated the former, but had almost no effect on the latter. The C genotype was significantly common among Japanese subjects, (TT 45.5%, TC 37.6% and CC 16.8%) compared with that reported in the Netherlands, Northern Ireland and France. No significant correlation was observed, however, between T-138C MGP promoter polymorphism and the markers. Although the C genotype (TC+CC) tended to show a higher calcification factor than the TT genotype, no significant difference was observed among the genotypes in the Gore index or in the calcification factor. Although MGP promoter activity and the binding of the AP-1 transcription factor were clearly different between T-138 and C-138 MGP promoter polymorphism in vitro, T-138C polymorphism was, statistically, not an independent factor of atherosclerosis or atherosclerotic vascular calcification in the abdominal aorta.

Double cancer, cholangiocellular and hepatocellular carcinomas, in the cirrhotic liver.

A 70-year-old male patient presented with cirrhotic liver caused by hepatitis C virus and complicated with hepatocellular carcinoma. The carcinoma was effectively treated by transarterial chemoembolization, but cholangiocellular carcinoma occurred in a different segment of the liver and rapidly grew and metastasized systemically. He died 13 months after the first admission. Double cancer of the liver is uncommon, but it must be considered in the differential diagnosis of cases of hepatocellular carcinoma associated with chronic hepatitis or cirrhosis. Failure to note the occurrence of cholangiocellular carcinoma is thought to be a diagnostic pitfall in the follow-up studies of patients with hepatocellular carcinoma.

Imaging of fine structure of bone sample with high coherent X-ray beam and high spatial resolution detector.

In this study, we observed bone specimens of the mouse using a very high coherence beam and high spatial resolution detector (zooming tube: approximately 0.7 micron resolution) and successfully obtained images of the Haversian canal, osteocytes, and osteoclasts.