Changes of Wnt/β-catenin signalling,BMP2, and BMP4 in the jejunum during ageing in rats

Background and study aimsThe renewal of intestinal epithelium is maintained by intestinal stem cells (ISCs). Studies have found an age-dependent increase of Esg⁺/Dl⁺ progenitor cells in the midgut of Drosophila. However, changes of ISCs and the molecular regulation in mammalian animals with age are yet unknown. The aim of this study was to find out the changes of ISCs and molecular regulation in mammalian animals during the process of ageing.Material and methodsThirty Sprague–Dawley rats were divided into three groups: young (3 months old), adult (6 months old), and ageing (24 months old). Levels of PCNA, Bmi1, β-catenin and BMP4 were examined by Immunohistochemistry staining. Levels of Bmi1, GSK-3β, Dkk1 and BMP2 were determined by Western Blot.ResultsOur results showed that the proliferation of ISCs was decreased and the number of intestinal stem cells declined in ageing rats. The niches of ISCs, including Wnt signalling pathway and some proteins of Bone morphogenetic protein (BMP) signalling pathway, were downregulated in the jejunum of ageing rats.ConclusionOur study indicated that age-related decreased proliferation of intestinal stem cells in the jejunum could be associated with the alleviation of niches, including Wnt signalling pathway and some proteins of BMP signalling pathway.     

Modulation of glucose transport in hamster adipocytes by insulin and byβ- andα 2-adrenoceptor agonists

Summary Glucose transport in hamster adipocytes and its modulation by insulin and isoprenaline was characterized with the aid of the non-metabolizable hexose 3-0-methylglucose. Insulin stimulated the initial uptake rates by an increase in V_max of the transport without any detectable change in K_m. The hormone concentration producing half maximal stimulation was identical to that required in rat adipocytes. However, hamster adipocytes were much less responsive to insulin (3-fold stimulation as compared to a 12-fold stimulation in rat fat cells), and maximal transport rates were 10-fold lower than that observed in rat adipocytes. Accordingly, the number of glucose transporters, as assessed by glucose-inhibitable cytochalasin-B binding, was considerably lower in plasma membranes of hamster adipocytes. Moreover, no transporters were detected in the low-density microsomes which in insulin-sensitive cell types represent the intracellular pool of recruitable glucose transporters. The relative insulin resistance of the hamster fat cells may therefore be due to a depleted pool of intracellular glucose transporters. In the presence of adenosine, the-adrenoceptor agonist isoprenaline produced a moderate stimulation of the basal transport rate which was antagonized by the ₂-agonist clonidine. If adenosine deaminase was added in order to remove endogenous adenosine, isoprenaline inhibited the insulin-stimulated transport by 50%. In contrast to the stimulatory effects of insulin and isoproterenol, the inhibitory effect of the catecholamine was reversed by cooling the cells to 22°. Glucagon produced a comparable inhibition, suggesting that the inhibitory effect was mediated by adenylate cyclase or its regulatory subunits. The ₂-adrenoceptor agonist clonidine fully antagonized the inhibitory effect of isoproterenol. The effect of glucagon was reversed by adrenaline, if propranolol was added in order to unmask the ₂-agonist action of the catecholamine. The data demonstrate that ₂-adrenoceptors participate in the regulation of glucose transport by antagonizing both the-receptor mediated stimulation and inhibition produced by catecholamines.     

Neuron-like phenotypic changes in pancreatic β-cells induced by NGF, FGF, and dbcAMP

We studied the effects of nerve growth factor (NGF), fibroblast growth factor (FGF), and dibutyryl-cAMP (dbcAMP) on rat pancreatic β-cell morphology and of NGF and dbcAMP on insulin secretion. After 2 wk in culture, nearly 3% of β-cells extended neurite-like processes spontaneously; when cells were treated with NGF, almost 30% of them extended processes. In the presence of dbcAMP, almost all β-cells flattened, and the extension of neurite-like processes was more pronounced in fetal than in adult cells. The most prominent effect, regardless of age, was observed in cells treated with NGF and dbcAMP together, since the percentage of neurite-like bearing β-cells increased to 50%. β-cells cultured under these conditions maintained their immunoreactivity to insulin and nearly all β-cells and their neurite-like processes were also positive to GABA, tubulin, tau protein, and N-CAM. FGF increased the percentage of adult β-cells bearing neurite-like processes to 13%, and FGF and dbcAMP applied together to 40%. β-cells treated with NGF and dbcAMP for 5 to 7 d preserved their capability to secrete the hormone in response to different extracellular glucose concentrations. Insulin secretion of dbcAMP-treated β-cells was 2.5-fold higher than in control cells. NGF-treated cells were able to discriminate between different glucose concentrations, a property lost in control cells with time in culture.     

Campylobacter, polyneuropathy, and Guillain-Barré syndrome in Denmark, 1994-2003

We examined the incidence of Campylobacter infections, Guillain-Barré syndrome and unspecified polyneuropathies in an ecological study from 1994 to 2003. The increase in Campylobacter cases in Denmark in the 1990s was comparable to an increase in cases of unspecified polyneuropathies; however, the incidence of Guillain-Barré syndrome cases remained stable during the period.     

The role of α-, δ- and F cells in insulin secretion and action

Nowadays, most of the basic pancreatic islet research is focused on the beta and alpha cells. Nonetheless, the pancreatic islets are complete organs with five, up to now, different cell types and dedicated vasculature and neural supply. Every cell contributes in the integration of the islet function and deserves a closer lookup in the saga of the glucose homeostasis. In this brief report we describe the current concepts about the contribution of the non-beta cells in the overall islet contribution to glucose metabolism.     

Control of transient,resurgent, and persistent current by open-channel block by Na channel β4 in cultured cerebellar granule neurons

Voltage-gated Na channels in several classes of neurons, including cells of the cerebellum, are subject to an open-channel block and unblock by an endogenous protein. The Na_Vβ4 (Scn4b) subunit is a candidate blocking protein because a free peptide from its cytoplasmic tail, the β4 peptide, can block open Na channels and induce resurgent current as channels unblock upon repolarization. In heterologous expression systems, however, Na_Vβ4 fails to produce resurgent current. We therefore tested the necessity of this subunit in generating resurgent current, as well as its influence on Na channel gating and action potential firing, by studying cultured cerebellar granule neurons treated with siRNA targeted against Scn4b. Knockdown of Scn4b, confirmed with quantitative RT-PCR, led to five electrophysiological phenotypes: a loss of resurgent current, a reduction of persistent current, a hyperpolarized half-inactivation voltage of transient current, a higher rheobase, and a decrease in repetitive firing. All disruptions of Na currents and firing were rescued by the β4 peptide. The simplest interpretation is that Na_Vβ4 itself blocks Na channels of granule cells, making this subunit the first blocking protein that is responsible for resurgent current. The results also demonstrate that a known open-channel blocking peptide not only permits a rapid recovery from nonconducting states upon repolarization from positive voltages but also increases Na channel availability at negative potentials by antagonizing fast inactivation. Thus, Na_Vβ4 expression determines multiple aspects of Na channel gating, thereby regulating excitability in cultured cerebellar granule cells.     

Urinary excretion of albumin, α-1-microglobulin,and N-acetyl-B-D-glucosaminidase in relation to smoking habits in diabetic and nondiabetic subjects

Smoking may be a risk factor for the development of diabetic nephropathy. Therefore, the urinary excretion of albumin, α-1-microglobulin, and N-acetyl-BD glucosaminidase was studied in 24 young adult diabetic patients who smoked. None of these patients had urine samples positive for albumin as determined by the Albustix method (i.e., a urinary concentration of albumin of < 0.5 g in 24 hr). Control groups were nonsmoking diabetic patients (matched for age and duration of diabetes) and nondiabetic subjects (smokers and nonsmokers). Explred breath carbon monoxide and the urinary nicotine metabolite continine were measured as objective markers of smoking load. No significant differences in concentrations of urinary proteins were found among any of the four groups. Therefore, smoking is not associated with the development of an increased urinary excretion of albumin within the “microalbuminuria” range. However, further studies are required to determine whether smoking is a risk factor for the progression of established microalbuminuria to Albustix positive proteinuria in diabetic patients.     

Stimulation of cell division in the rat by NaCl, KCl, MgCl2, and CaCl2, and inhibition of the sodium chloride effect on the glandular stomach by ascorbic acid and β-carotene

Three questions associated with the stimulation of cell division by chloride salts have been investigated: (i) whether cations other than sodium show a similar effect, (ii) whether vitamins can have a preventive activity, and (iii) whether subchronic treatment with sodium chloride in the diet is also effective. Male Fischer 344 rats were given solutions of the chloride salts of sodium, potassium, magnesium, and calcium by oral gavage. Water was used for control. After 4 h, a 24-h osmotic minipump containing 5-bromo-2′-deoxyuridine was implanted subcutaneously. The forestomach and glandular stomach, as well as liver and bladder were analyzed immunohistochemically 24 h later for the proportion of cells in S phase as an indicator of the rate of replicative DNA synthesis. For both the forestomach and the glandular stomach, potassium was as potent as sodium, and the divalent cations Mg and Ca were even more potent on a molar basis. Supplementation of the diet with ascorbic acid (2 g/kg food) or β-carotene (12.5 mg/kg food) for 1 week before gavage of the sodium chloride solution resulted in an inhibition of the stimulation of cell division. A putative tumor-chemopreventive activity of the two vitamins might therefore not only rely on their antioxidative properties but may include effects on the cell cycle. A 4-week treatment with a sodium chloride supplement in the diet (2% and 4% supplement) resulted in a significant stimulation of cell division not only in both parts of the stomach and in the bladder (with the 4% supplement) but also in the liver (even with the 2% supplement). Sodium-chloride-stimulated cell turnover therefore is a sustained effect. Received: 16 October 1998 / Accepted: 12 November 1998     

Tumor-derived hydrogen sulfide,produced by cystathionine-β-synthase,stimulates bioenergetics,cell proliferation,and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H₂S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H₂S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H₂S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H₂S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H₂S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H₂S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H₂S as a tumor growth factor and anticancer drug target.The endogenous gasotransmitter hydrogen sulfide (H₂S) is a stimulator of vasorelaxation (1–3), angiogenesis (3–5), and cellular bioenergetics (6, 7). H₂S is generated from l-cysteine by two pyridoxal-5′-phospate–dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) (8–10). H₂S exerts its cellular actions via multiple mechanisms (1–15), including activation of potassium channels (1–3), stimulation of kinase pathways (4, 11, 12), and inhibition of phosphodiesterases (3, 15).Both ATP generation and angiogenesis are vital factors for the growth and proliferation of tumors (16–19). Using human colon cancer tissues and cancer-derived cell lines, we have now conducted a series of in vitro and in vivo studies to explore whether endogenous, tumor cell-derived H₂S plays a role as a tumor-derived survival factor. The results show that CBS is selectively overexpressed in colon cancer, and that H₂S produced by it serves to maintain the tumor''s cellular bioenergetics and to promote tumor angiogenesis.     

IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing

Cross-presentation allows antigen-presenting cells to present exogenous antigens to CD8(+) T cells, playing an essential role in controlling infections and tumor development. IFN-α induces the rapid differentiation of human mono-cytes into dendritic cells, known as IFN-DCs, highly efficient in mediating cross-presentation, as well as the cross-priming of CD8(+) T cells. Here, we have investigated the mechanisms underlying the cross-presentation ability of IFN-DCs by studying the intracellular sorting of soluble ovalbumin and nonstructural-3 protein of hepatitis C virus. Our results demonstrate that, independently from the route and mechanism of antigen entry, IFN-DCs are extraordinarily competent in preserving internalized proteins from early degradation and in routing antigens toward the MHC class-I processing pathway, allowing long-lasting, cross-priming capacity. In IFN-DCs, both early and recycling endosomes function as key compartments for the storage of both antigens and MHC-class I molecules and for proteasome- and transporter-associated with Ag processing-dependent auxiliary cross-presentation pathways. Because IFN-DCs closely resemble human DCs naturally occurring in vivo in response to infections and other danger signals, these findings may have important implications for the design of vaccination strategies in neoplastic or chronic infectious diseases.     

1α,25-dihydroxyvitamin D_3 prevents DNA damage and restores antioxidant enzymes in rat hepatocarcinogenesis induced by diethylnitrosamine and promoted by phenobarbital

AIM:To investigate the chemopreventive effects of 1α,25-dihydroxyvitamin D_3 in diethylnitrosamine induced,phenobarbital promoted rat hepatocarcinogenesis.METHODS:The rats were randomly divided into 6 differentgroups (A-F).Groups A,C and E rats received a singleintraperitoneal (i.p) injection of diethylnitrosamine (DEN)at a dose of 200mg/kg body mass in 9g/L NaCl solution at4 wk of age,while group B served as normal vehicle controlreceived normal saline once.After a brief recovery of 2 wk,all the DEN treated rats were given phenobarbital (PB) at0.5g/L daily in the basal diet till wk 20.Group A was DENcontrol.Treatment of 1α,25-(OH)_2D_3 in group C was started4 wk prior to DEN injection and continued thereafter till wk20 at a dose of 0.3μg/100μL propylene glycol per onesingle dose (os) twice a week.Group E received thetreatment of 1α,25-(OH)_2D_3 at the same dose mentionedas above for 15 wk.The rats in group D and F received 1α,25-(OH)_2D_3 alone as in group C without DEN injection.RESULTS:The comet assay showed statistically highermean values for length to width ratios (L:W) of DNA massand tailed cells (P<0.01;P<0.01 respectively) in DEN treatedrats as compared to their normal controls.Continuoussupplementation of 1α,25-dihydroxyvitaminD_3 showed asignificant (P<0.01) decrease in L:W ratio of DNA masstailed cells.Furthermore,1α,25-(OH)_2D_3 supplementationselevated the super oxide dismutase (SOD) activity,hepaticmalondialdehyde (MDA) level,reduced glutathione (GSH)and glutathione S-transferase (GST) activity (P<0.01,P<0.05,P<0.05 and P<0.05 respectively).As an endpointmarker histological changes were observed to establishthe chemopreventive effects of 1α,25-dihydroxyvitaminD_3.CONCLUSION:Supplementations of 1α,25-(OH)_2D_3 has a marked protection against hepatic nodulogenesis,antioxidant enzymes and DNA damages in DEN induced rathepatocarcinogenesis promoted by phenobarbital.     

1α,25-dihydroxyvitamin D3 prevents DNA demage and restores antioxidant enzymes in rat hepatocarcinogenesis induced by diethylnitrosamine and pronoted by phenobarbital

AIM:To investigate the chemopreventive iffects of 1α,25-dihydroxyvitamin D3 in diethylnitrosamine induced,phenobarbital promoted rat hepatocarcinogenesis.METHODS:The rats were randomly divided into 6 different groups(A-F).Group A,C and E rats received a single intraperitoneal (i.p) injection of diethylnitrosamine (DEN) at a dose of 200 mg/kg body mass in 9 g/L NaCl solution at 4 wk of age ,while group B served as normal vehicle control received normal saline once.After a brief recovery of 2 WK, all the DEN treated rats were given phenobarbital (PB) at 0.5g/L daily in the basal diet till WK 20.Group C was started 4 wk prior to DEN injection and continued thereafter till wk 20 at a dose (os) twice a week.Group E received the treatment of 1α,25-(OH)2D3 at the same dose mentioned as above for 15 wk.The rats in group D and F received 1α,25-(OH)2D3 alone as in group C without DEN injection.RESULTS:The comet assay showed statistically higher mean values for length to width ratios (L:W) of DNA mass and tailed cells (p&lt;0.01;p&lt;0.01 respectively) in DEN treated rats as compared to their normal controls.Continuous supplementation of 1α,25-dihydroxyvitaminD3 showed a significant (p&lt;0.01) decrease in L:W ratio of DNA mass tailed cells.Furthermore,1α,25-(OH)2D3 supplementations elevated the super oxide dismutase (SOD) activety,hepatic malondialdehyde(MDA) levle,reduced glutathione (GSH) and glutathione S-transferase (GST) activety (p&lt;0.01,p&lt;0.01,p&lt;0.05 and p&lt;0.05 respectively).As an endpoint marker histological changes were observed to establish the chemopreventive effects of 1α,25-dihydroxyvitaminD3.CONCLUSION:Supplementations of 1α,25-(OH)2D3 has a marked protection against hepatic nodelogenesis,antioxidant enzymes and DNA damages in DEN induced rat hepatocarcinogenesis promoted by phenobarbital.     

Seasonal variation in blood pressure is modulated by gender and age but not by BMI in a large Taiwanese population, 1996–2006

Previous research has found that blood pressure tends to be higher in winter and lower in summer. The present study examined seasonal variation in blood pressure by gender, hypertension medication, age group, and body mass index using contemporary Taiwanese data. Over 400,000 health screening records collected biennially between 1996 and 2006 were used to calculate average monthly systolic (SBP) and diastolic blood pressure (DBP) measurements. Generalized estimating equations were used to estimate the difference between the highest and lowest mean monthly blood pressure measurements. Mean monthly blood pressure measurements were higher in winter than in summer for all age groups, regardless of medication for hypertension. The largest difference in mean monthly blood pressure between summer and winter months was 5.3 mm Hg (Standard error = 0.7) for SBP and 3.2 mm Hg (Standard error = 0.7) for DBP. These differences were more pronounced: in SBP than in DBP; in men than in women; and in older than in younger participants. Body mass index was not clearly associated with seasonal variation in blood pressure. Seasonal variation in blood pressure among contemporary Taiwanese populations is modest and may only approach clinical significance for the diagnosis and treatment of hypertension and the prevention of cardiovascular disease amongst older male individuals.     

Layilin, a talin-binding hyaluronan receptor, is expressed in human articular chondrocytes and synoviocytes and is down-regulated by interleukin-1β

Objective

Layilin (LAYN), a 55-kDa transmembrane protein with homology to C-type lectins, has been identified as a receptor of hyaluronan (HA). Interestingly, LAYN does not share any sequence homology with CD44, a primary HA receptor. The primary aim of our study was to examine the expression and potential function of LAYN in human articular chondrocytes and synoviocytes.

Methods

Samples were obtained from patients undergoing joint arthroplasty. Cells were grown in vitro, then stimulated with interleukin (IL)-1β or tumor necrosis factor alpha (TNFα) for 24 h and the expression of LAYN was analyzed. To assess the function of LAYN, we transfected chondrocytes with siRNA against LAYN, treated them with HA and IL-1β, and then analyzed the production of matrix metalloproteinase (MMP)-1 and MMP-13 in the treated chrondrocytes.

Results

The results showed that LAYN was constitutively expressed in human articular chondrocytes and synoviocytes and that IL-1β significantly suppressed the expression of LAYN in these cells. HA repressed IL-1β-induced MMP-1 and MMP-13 production in chondrocytes, but this was significantly abrogated in chondrocytes transfected with siRNA against LAYN.

Conclusions

Our results show that human chondrocytes express LAYN, a novel HA receptor, and that LAYN may contribute to the regulation of HA functions in the arthritic condition. Further investigation of the HA receptor may lead to the development of novel therapeutics to regulate HA signaling in inflammatory arthritis.     

Dynamic changes in the expression of matrix metalloproteinases and their inhibitors,TIMPs,during hepatic fibrosis induced by alcohol in rats

AIM:To determine the dynamic changes in the expressionof matrix metalloproteinases (MMPs) and the endogenoustissue inhibitors of MMPs inhibitors (TIMPs) during hepaticfibrosis induced by alcohol.METHODS:Male Sprague-Dawley rats were randomly dividedinto normal,4 d,2 wk,4 wk,g wk and 11 wk groups,and themodel rats were fed with a mixture of alcohol by gastricinfusion at the designed time,respectively,then decollatedand their livers were harvested for the examination of MMP-2,MMP-3,MMP-9,MMP-13,TIMP-1 and TIMP-2 by immunoh-istochemistry,zymograghy and Western blotting,respectively.RESULTS:Normal rats had moderate expression of MMP-2,which was decreased in the model rats except in the 11 wkgroup,where MMP-2 expression slightly increased.MMP-3had the similar changing pattern to MMP-2 despite weakerexpression.MMP-9 expression decreased in the 4 d and 2 wkgroups,rose in the 4 wk group,decreased again in the 9 wkgroup and returned to normal levels in the 11 wk group.MMP-13 expression decreased in the 4 d and 2 wk groups,and returned to normal levels in the 4 wk,9 wk and 11 wkgroups.TIMP-1 expression decreased in the 4 d and 2 wkgroups,but sharply increased in the 4 wk group and sustainedat a high level even after modeling was stopped for 2 wk.Innormal rats TIMP-2 expression was strong.However,itdecreased as soon as modeling began,and then graduallyrose,but remained to a level lower than that in normal ratseven after modeling was stopped for 2 wk.CONCLUSION:MMP-2 may not always expresses at a highlevel during hepatic fibrosis.MMP-13 and MMP-3 areacutely affected by TIMP-1.In this model TIMP-1 is the mostpowerful factor imposed on capillarization and peri-sinusoidalfibrosis.TIMP-2 is the most effective regulator on the metabolismof type Ⅳ collagen located in the basement of sinus.