Distribution of melatonin receptors in murine pancreatic islets

Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the β-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function.     

Homozygous deletion of glycogen synthase kinase 3beta bypasses senescence allowing Ras transformation of primary murine fibroblasts

In primary mammalian cells, expression of oncogenes such as activated Ras induces premature senescence rather than transformation. We show that homozygous deletion of glycogen synthase kinase (GSK) 3beta (GSK3beta-/-) bypasses senescence induced by mutant Ras(V12) allowing primary mouse embryo fibroblasts (MEFs) as well as immortalized MEFs to exhibit a transformed phenotype in vitro and in vivo. Both catalytic activity and Axin-binding of GSK3beta are required to optimally suppress Ras transformation. The expression of Ras(V12) in GSK3beta-/-, but not in GSK3beta+/+ MEFs results in translocation of beta-catenin to the nucleus with concomitant up-regulation of cyclin D1. siRNA-mediated knockdown of beta-catenin decreases both cyclin D1 expression and anchorage-independent growth of transformed cells indicating a causal role for beta-catenin. Thus Ras(V12) and the lack of GSK3beta act in concert to activate the beta-catenin pathway, which may underlie the bypass of senescence and tumorigenic transformation by Ras.     

Carvedilol improves left ventricular function in murine coxsackievirus-induced acute myocarditis association with reduced myocardial interleukin-1beta and MMP-8 expression and a modulated immune response

BACKGROUND: Proinflammatory cytokines induce the expression of matrix metalloproteinases that play a crucial role in myocardial remodeling. Beta-adrenergic receptor stimulation influences the production of cytokines heralding the possibility of modulating cytokine production by beta-adrenergic blockers. METHODS AND RESULTS: In a coxsackievirus B3 murine myocarditis model (BALB/c), effects of carvedilol and metoprolol on myocardial cytokine expression, inflammatory cell infiltration and MMP/TIMP profiles were investigated. In carvedilol-treated mice, a significant improvement in left ventricular function was documented 10 days post infection. In infected mice (n=10), IL-1beta, TNF-alpha, TGF-beta(1) and IL-10 myocardial mRNA abundance were increased significantly (240%, 200%, 161%, and 230%) compared to controls (n=10), while IL-15 mRNA was markedly reduced (70%). Infected mice showed significantly increased infiltrations with CD3-, CD4- and CD8-T-lymphocytes (730%, 1110%, 380%). In the infected mice, myocardial MMP/TIMP profiles presented a significant upregulation of membrane type-1 MMP, MMP-9, MMP-8 and MMP-3 (150%, 160%, 340%, and 270%) and a significant decrease in TIMP-4 levels (75%). Carvedilol attenuated over-expression of myocardial TGF-beta(1), IL-1beta and MMP-8 mRNA expression significantly and induced a relevant IL-10 mRNA expression in the infected mice (n=10). By an unchanged infiltration with CD3-T-lymphocytes, carvedilol showed a representative reduction in CD4-T-lymphocytes. CONCLUSION: Carvedilol treatment in experimental myocarditis leads to reduced expression of proinflammatory cytokines and MMPs, which contributes to reduced matrix degradation and ultimately to improved structural integrity of the heart. Besides the antiadrenergic potential, carvedilol is beneficial due to a wide range of biological activities (antiinflammatory, antifibrotic, antioxidative and immunomodulatory).     

Sickle cell anemia as a syndrome: a review of diagnostic features.

Sickle cell (SS) disease is a complex of various genetic conditions. In some, homozygosity for the beta S gene may be present alone or in combination with the heterozygous or homozygous alpha-thalassemia-2 condition. Such combinations might ameliorate the clinical and hematological condition of the patient. The same may be true for the high levels of Hb F and F-cells observed in many Hb S homozygotes. Howeever, the chemical heterogeneity of Hb F appears not to be related to the clinical status of the Hb S homozygote. Combinations of a Hb S heterozygosity with a heterozygosity for a Hb D-type of variant, for either one of two types of beta-thalassemia, two types of alpha beta- thalassemia, and five types of HPFH are discussed, and data are compared with those obtained for Hb S homozygotes. The use of advanced laboratory procedures and family studies is often necessary for an accurate diagnosis.     

Dietary supplementation with melatonin reduces levels of amyloid beta-peptides in the murine cerebral cortex

Melatonin levels decrease with aging in mice. Dietary supplementation with melatonin has recently been shown to result in a significant rise in levels of endogenous melatonin in the serum and all other tissue samples tested. Herein, the effects of dietary melatonin on brain levels of nitric oxide synthase, synaptic proteins and amyloid beta-peptides (Abeta) were determined in mice. Melatonin supplementation did not significantly change cerebral cortical levels of nitric oxide synthase or synaptic proteins such as synaptophysin and SNAP-25. Increased brain melatonin concentrations however, led to a significant reduction in levels of toxic cortical Abeta of both short and long forms which are involved in amyloid depositions and plaque formation in Alzheimer's diseases. Thus, melatonin supplementation may retard neurodegenerative changes associated with brain aging. Depletion of melatonin in the brain of aging mice may in part account for this adverse change.     

Effects of low glucose degradation products peritoneal dialysis fluid on the peritoneal fibrosis and vascularization in a chronic rat model

In the present study, we examined the effects of a new peritoneal dialysis fluid (PDF) with a low level of low glucose degradation products (GDP) on the functional and structural stability of the peritoneal membrane (PM). Male Sprague-Dawley rats were divided into three groups: group C (n = 8), without dialysate infusion; group P (n = 12), infused with low-level GDP solution (4.25% Physioneal, pH 7.0-7.4); and group D (n = 12), infused with conventional solution (4.25% Dianeal, pH 5.2, adjusted to pH 7.0). In groups D and P, animals were infused through a permanent catheter with 25 mL of PDF, twice daily for 8 weeks. Lipopolysaccharide was added into the PDF immediately before infusion on days 8, 9 and 10 in the two dialysis groups. When compared with group P, group D showed a higher glucose mass transfer at weeks 6 and 8, D/P urea at week 8, TGF-beta1 at weeks 4 and 8, and VEGF level at week 8. The submesothelial matrix layer of the parietal peritoneum was significantly thickened in group D and the lectin-stained blood vessels in this layer were well-visualized in group D compared with group P. There were significantly more peritoneal blood vessels in group D than group P. The transforming growth factor-beta induced gene-h3 (betaig-h3) and TGF-beta1 levels in the peritoneal effluent correlated with the submesothelial thickness, which correlated with the dialysate-to-plasma ratio (D/P) of protein and, inversely, with the rate of glucose transport (D/D(0) glucose, where D is glucose concentration in the dialysate and D(0) is glucose concentration in the dialysis solution before it is infused into the peritoneal cavity). The present study showed that low-GDP PDF effectively attenuated the peritoneal vascularization and fibrosis related to conventional solution.     

A murine model for evaluating metastatic potential: Characterization of a 90–110-kDa metastasis-binding protein

Reliable discriminatory tests to predict metastatic disease would clearly facilitate the management of cancer in the elderly. We have recently identified a 90–110-kilodalton (kDa) cell surface glycoprotein that is differentially expressed in benign and malignant murine adrenal carcinoma cells. In view of the proteins highly glycosylated nature, we have tested its ability to bind to a panel of agarose-bound lectins. Wheat germ agglutinin (WGA), a lectin specific for terminal sialic acid and N-acetylglucosamine (GlcNAc), had a strong affinity for the metastasis-related protein but failed to detect such a glycoprotein in nonmetastatic cells. Treatment of cells with sialidase to remove terminal sialic acids did not affect the affinity of the protein for the lectin, indicating the presence of terminal GlcNac. We show by in situ that this metastatic binding protein (MBP) is regionally concentrated on the surface of invasive cells but absent in cells unable to invade. We postulate that MBP plays an active role in cell migration through interactions with β-1,4 galactosytransferase and basement membrane glycoproteines.     

HB Q-Thailand-HB H disease in a Chinese living in Geneva, Switzerland: characterization of the variant and identification of the two alpha-thalassemic chromosomes

Data on a 24-year-old Chinese male with Hb Q-Thailand-Hb H disease are presented. The hemoglobin variant was characterized by fast microprocedures, mainly by reverse-phase high-performance liquid chromatography. Gene mapping analyses identified the alpha-thalassemia-2, which is associated with the alpha-Q chain, as caused by a 4.2-kb deletion involving the alpha 2 globin gene, while the alpha-thalassemia-1 anomaly was the common Southeast Asian type in which part of the psi zeta, the psi alpha, and the alpha 2 and alpha 1 globin genes are deleted.     

Cytokine-induced PGE(2) formation is reduced from iNOS deficient murine islets

Cytokines may be involved in islet destruction during Type 1 diabetes. Exposure to interleukin-1β (IL-1β) or IL-1β plus interferon-γ (IFN-γ) of rodent islets induces expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent formation of nitric oxide (NO) and prostaglandin E₂ (PGE₂) may impair β-cell function. Using iNOS deficient (iNOS −/−) islets, we have further investigated the relation between NO formation and PGE₂ induction. We found that iNOS −/− islets responded with a reduced PGE₂ formation following IL-1β or (IL-1β + IFN-γ) treatment compared to wild-type (wt) islets, while COX-2 mRNA or protein content were unchanged. By the addition of an NO donor together with IL-1β, PGE₂ formation could be stimulated from iNOS −/− islets. We conclude that the lowered capacity of PGE₂ formation observed from cytokine exposed iNOS −/− islets is due to a decreased stimulation of PGE₂ formation by the COX-2 enzyme in the absence of NO, rather then differences in expressed COX-2 protein.     

FR167653, a potent suppressant of interleukin-1 and tumor necrosis factor-alpha production, ameliorates colonic lesions in experimentally induced acute colitis.

BACKGROUND: Interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are believed to play a significant role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-1 and TNF-alpha possess overlapping and synergetic activities inducing the production in cascade of other cytokines, adhesion molecules, arachidonic acid metabolites, as well as activating immune and non-immune cells. FR167653 (C24H18FN5O2-H2SO4-H2O) is a newly synthesized organic compound with a potent inhibitory effect on IL-1beta and TNF-alpha production. We hypothesized that the suppression of IL-1 and TNF-alpha induced by FR167653 could effectively attenuate experimentally induced colonic damage. METHODS: Colonic lesions were induced in male Sprague-Dawley rats (250-300 g) by intrarectal instillation of 4% acetic acid. The effect of FR167653 administration at 1.0, 1.5, 2.5 mg/kg per 6 h subcutaneously on acetic acid-induced colonic damage was assessed. The lesion area, microscopic findings, colonic and serum levels of TNF-alpha and IL-1beta were also evaluated. RESULTS: Treatment with FR167653 at 1.5 and 2.5 mg/kg per 6 h was able to ameliorate the gross macroscopic appearance of colonic lesions significantly, as well as ameliorate the lesion area induced by acetic acid. Colonic mucosal TNF-alpha and IL-1beta levels of rats treated with FR167653 showed significant decrease in a dose-dependent fashion compared with the control group. In the same manner, serum TNF-alpha of rats treated with FR167653 was significantly lower than that of respective controls. CONCLUSIONS: Subcutaneous administration of FR167653 was able to ameliorate the acute changes induced by acetic acid instillation in a dose-dependent manner. This is the first report to evaluate the dual inhibition of the production of IL-1 and TNF-alpha, offered by FR167653, in acute experimental colitis. Further studies are necessary to evaluate FR167653's efficacy and safety on long-term conditions.     

The effects of PGF2 alpha, PGI2, and TxB2 on human CFU-C in healthy and leukemic patients

This study was undertaken to test the effects of certain arachidonate derivatives, PGF2 alpha, PGI2 and TxB2 on in vitro bone marrow granulocyte colony growth (CFU-C) in leukemia patients receiving maintenance chemotherapy and in normal controls. The addition of PGF2 alpha did not result in increased numbers of colonies, but it did cause a shift in the size of the colonies so that there was a significant increase in larger colonies (P less than 0.001) and significantly fewer small colonies (P less than 0.05) as compared to untreated samples. Of the prostenoids tested in a Tris-buffered system, PGI2 affected the greatest increase in CFU-C (P less than 0.01) followed by PGF2 alpha (P less than 0.05) whereas 6-keto-PGF1 alpha (the stable hydrate of PGI2) did not affect colony growth. Time-response curves revealed a linear growth pattern for PGF2 alpha with a peak at 10 days, whereas there was a 6-day growth lag with PGI2 followed by linear growth with a peak at 13 days. TxB2 added to cultures significantly reduced the number of bone marrow CFU-C at all doses tested. The prostanoid effects on CFU-C derived from leukemic patients on maintenance chemotherapy and from normal individuals were identical in every respect.     

Age-related changes in murine CNS mRNA gene expression are modulated by dietary melatonin

Brain cellular functions decline with normal aging, accompanied by a changing profile of gene expression. Gene array analysis was used to quantitatively estimate messenger RNA (mRNA) expression levels in the cerebral cortex of both young (4-month) and old (27-month) B6C3F1 male mice. A stringent degree of significance was obtained by using multiple gene chips. Out of 12,423 mRNA levels, only 25 changed significantly with age. Nine of these genes coded for inflammatory proteins, all of which were elevated in aged, relative to younger mice. Melatonin (200 p.p.m.) included in the diet of aged animals for 8 wk elevated serum and cortical melatonin and reversed 13 of the 25 genes altered with age. In no case did melatonin potentiate age-related changes in gene expression. The restoration of a more youthful gene profile to brains of aged animals by melatonin, to a large extent, involves reversal of age-induced elevation of basal inflammatory parameters.     

Pioglitazone increases circulating adiponectin levels and subsequently reduces TNF-alpha levels in Type 2 diabetic patients: a randomized study.

BACKGROUND: Adipocytokines are involved in the development of insulin resistance and endothelial dysfunction in diabetic patients. However, the relationship between these factors remains unclear. We observed a chronological change in circulating adipocytokines and blood pressure levels with administration of oral hypoglycaemic agents in Type 2 diabetic (T2DM) subjects. METHODS: Thirty poorly controlled T2DM subjects (aged 60.1 +/- 1.5 years, 11 males and 19 females) were randomized into two groups: voglibose (initial dose 0.6 mg/day, increased to 0.9 mg/day) and pioglitazone (initial dose 15 mg/day, increased to 30 mg/day). RESULTS: Both treatment groups showed a similar improvement in glycaemic control. In pioglitazone-treated patients, circulating adiponectin levels were significantly increased from 4 weeks after the start of treatment, and until the end of the study at 12 weeks. Plasma tumour necrosis factor-alpha (TNF-alpha) levels were significantly decreased only at 12 weeks. In contrast, no significant changes in plasma adiponectin or TNF-alpha levels were observed in voglibose-treated patients. Plasma PAI-1 and leptin levels were not significantly changed at 12 weeks in either treatment group. Pioglitazone significantly decreased systolic and diastolic blood pressure levels at 12 weeks, but voglibose had no effect. CONCLUSION: In summary, pioglitazone caused an immediate increase in circulating adiponectin levels, followed by a reduction of TNF-alpha. The observed increase in circulating adiponectin could be related to decreases in both systolic and diastolic blood pressure.     

Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

OBJECTIVES—To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.
METHODS—Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.
RESULTS—HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1.
CONCLUSION—IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

Keywords: synovial lining cells; hyaluronan, interleukin 1β; transforming growth factor β1     

Mechanical stretch-induced hypertrophy of neonatal rat ventricular myocytes is mediated by beta(1)-integrin-microtubule signaling pathways

BACKGROUND: Mechanical stress plays a crucial role in tissue morphogenesis and remodeling. These processes depend in part on force transmission mediated through integrins and the cytoskeleton. METHODS: Ventricular myocytes isolated from neonatal Sprague-Dawley rats (NRVMs) were exposed to persistent centrifugal force stretch for 12 or 24 h. The NRVMs were exposed to colchicine (4 micromol/ml) and anti-integrin beta1 specific antibody (10 microg/ml). Cell viability was assessed by MTT assay and lactate dehydrogenase (LDH) activity. Incorporation of 3H-leucine, and atrial natriuretic peptide (ANP) and angiotensin II (Ang II) levels were assessed. Pixel intensity and distribution of the microtubule were estimated from laser scanning confocal images. RESULTS: Changes in LDH release and the MTT assay showed that 180 rpm. centrifugal force had minimal effect on the viability and number of NRVMs. Mechanical stretch significantly increased 3H-leucine incorporation into cardiomyocytes. Anti-integrin beta1 blocking antibody effectively inhibited the increase in 3H-leucine incorporation and release of ANP (p < 0.05). Following anti-integrin-beta1-blocking antibody, the pixel intensity of the microtubule image was decreased after both12 and 24 h stretch, this was similar to the effect of colchicine. Both treatments also inhibited the secretion of Ang II induced by stretch (p < 0.05). CONCLUSIONS: Anti-integrin-beta1-blocking antibody and colchicine had similar effects, partly inhibiting the stretch-induced increase in microtubule polymerization and the secretion of Ang II in hypertrophic cardiac myocytes.     

Xenogeneic immunosuppression of human umbilical cord mesenchymal stem cells in a major histocompatibility complex-mismatched allogeneic acute graft-versus-host disease murine model

Mesenchymal stem cells (MSCs) hold great promise for treating immune disorders owing to their immunoregulatory capacity, but the mechanism remains controversial. As we show here, the mechanism of human umbilical cord mesenchymal stem cell (HUCMSC)‐mediated immunosuppression involves TGF‐β and indoleamine 2,3‐dioxygenase (IDO). In this study, we investigated the influence of xenogeneic HUCMSCs on acute graft‐versus‐host disease (aGVHD) in murine allogeneic bone marrow transplantation (BMT). In the HUCMSC‐treated group, lethally irradiated DBA/2(H‐2Kd) mice were adoptively transferred with expanded HUCMSCs, bone marrow (BM), and splenocytes (SCs) from C57BL/6 (H‐2Kb) mice. Recipients in the control group were transferred only BM and SCs. The two groups were compared in survival, weight, histopathologic specimens, and aGVHD scoring. In the HUCMSC‐treated group, 60% of the mice survived past day 30 after BMT, but in the control group, all mice died within 18 d. The mice treated with HUCMSCs exhibited light symptoms of aGVHD after day 30. The results suggest that xenogeneic HUCMSCs could alleviate aGVHD symptoms and prolong survival after allogeneic BMT. Our study suggests that in vitro expanded HUCMSCs might be used to inhibit severe aGVHD effectively in allogeneic hematopoietic cell transplantation clinically.