Depression-like behavior is associated with lower Per2 mRNA expression in the lateral habenula of rats

Circadian rhythm dysfunction is primary symptom of depression and is closely related to depression onset. The role of the lateral habenula (LHb) of the thalamus in the pathogenesis of depression has been a research topic of great interest. The neuronal activity of this structure has circadian characteristics, which are related to the regulation of circadian rhythms. However, in depression model of rats, the role of clock genes in the LHb has not been assessed. To address this gap, we used a clomipramine (CLI) injection-induced depression model in rats to assess the daily expression of rhythmic genes in the LHb and depression-like behavior in rats at multiple time points. In determining the role of the Per2 gene in the development of depression-like behavior in the LHb, we found that the expression of this clock gene differed in a circadian manner. Per2 expression was also significantly decreased in CLI-treated rats in late afternoon (17:00) and in the middle of the night (1:00). Furthermore, silencing Per2 in the LHb of normal rats induced depression-like behavior at night, suggesting that Per2 may play an important role in the pathogenesis of depression. Collectively, these results indicate that decreased Per2 expression in the LHb may be related to increased depression-like behavior at night in depression model of rats.     

Rab1 GTPase promotes expression of β-adrenergic receptors in rat pulmonary microvascular endothelial cells

It is known that Rab1 regulates the expression and function of beta-adrenoceptors (β-ARs) in many cells. However, the effect of these changes in rat pulmonary microvascular endothelial cells (RPMVECs) is not known. In the present study, we investigated the role of Rab1, a Ras-like GTPase that coordinates protein transport from the endoplasmic reticulum (ER) to the Golgi body and regulates the cell-surface targeting and function of endogenous β-ARs in RPMVECs in the presence of lipopolysaccharide (LPS).We found that lentivirus-driven expression of wild-type Rab1 (Rab1WT) in RPMVECs strongly enhanced the amount of β-ARs on the cell surface, whereas the dominant-negative mutant Rab1N124I significantly attenuated β-ARs expression on the cell surface. In addition, LPS stimulation significantly reduced β-ARs expression on the cell surface in RPMVECs; however, this effect was reversed by over-expression of wild-type Rab1WT. Fluorescent microscopy analysis demonstrated that expression of Rab1N124I and Rab1 small interfering RNA (siRNA) significantly induced the accumulation of green fluorescent protein (GFP)-tagged β₂-AR in the ER. Consistent with their effects on β-ARs export, Rab1WT and Rab1N124I differentially modified the β-AR-mediated activation of extracellular signal-regulated kinase1/2 (ERK1/2). Importantly, over-expression of Rab1WT markedly reduced LPS-induced hyper-permeability of RPMVECs by increasing the expression of β₂-AR on the cell surface. These data reveal that β-ARs function in RPMVECs could be modulated by manipulating β-ARs traffic from the ER to the Golgi body. We propose the ER-to-Golgi transport as a regulatory site for control of permeability of RPMVECs.     

Decreased interleukin 27 expression is associated with active uveitis in Beh?et’s disease

Instruction

Interleukin 27 (IL-27) is an important regulator of the proinflammatory T-cell response. In this study, we investigated its role in the pathogenesis of Behçet’s disease (BD).

Methods

IL-27 mRNA in peripheral blood mononuclear cells (PBMCs) was examined by performing RT-PCRs. Cytokine levels in sera or supernatants of PBMCs, naïve CD4⁺ T cells, dendritic cells (DCs) and DC/T cells were determined by enzyme-linked immunosorbent assay. We used RNA interference in naïve CD4⁺ T cells to study the role of interferon regulatory factor 8 (IRF8) in the inhibitory effect of IL-27 on Th17 cell differentiation. Flow cytometry was used to evaluate the frequency of IL-17- and interferon γ–producing T cells.

Results

The expression of IL-27p28 mRNA by PBMCs and IL-27 in the sera and supernatants of cultured PBMCs were markedly decreased in patients with active BD. A higher frequency of IL-17-producing CD4⁺ T (Th17) cells and increased IL-17 production under Th17 polarizing conditions were observed in patients with active BD. IL-27 significantly inhibited Th17 cell differentiation. Downregulation of IRF8 by RNA interference abrogated the suppressive effect of IL-27 on Th17 differentiation. IL-27 inhibited the production of IL-1β, IL-6 and IL-23, but promoted IL-10 production, by DCs. IL-27-treated DCs inhibited both the Th1 and Th17 cell responses.

Conclusions

The results of the present study suggest that a decreased IL-27 expression is associated with disease activity in BD patients. Low IL-27 expression may result in a higher Th1 and Th17 cell response and thereby promote the autoinflammatory reaction observed in BD. Manipulation of IL-27 may offer a new treatment modality for this disease.     

Inhibition of associative long-term depression by activation of β-adrenergic receptors in rat hippocampal CA1 synapses

The aim of this study was to investigate the role of β-adrenergic receptors in modulating associative long-term depression (LTD) at CA1 synapses in rat hippocampal slices. Standard extracellular electrophysiological techniques were employed to record field excitatory post-synaptic potential (fEPSP) activity and to induce associative LTD. Two independent Schaffer collateral pathways were elicited in hippocampal CA1 areas. In one (weak) pathway, the stimulating intensity was adjusted to elicit small fEPSP activity (20–30% of the maximum response). In contrast, 80–90% of the maximum response was evoked in the other (strong) pathway. Associative LTD of weak pathway could be induced by paired stimulation of weak and the strong pathways, repeated 100 times at 0.167 Hz. The associative LTD of weak pathway was NMDA receptor- and phophatase 2B dependent, because bath application of 50 μM D, L-AP5 or 10 μM cypermethrin blocked its induction. Bath application of 1 μM isoproterenol inhibited associative LTD, and this effect was blocked by timolol, suggesting the involvement of β-adrenergic receptors. The inhibitory effect of β-adrenergic receptors on LTD induction was blocked in slices pretreated with inhibitors of protein kinase A and mitogen-activated protein kinase, suggesting that these signal cascades are downstream effectors following activation of β-adrenergic receptors. Nevertheless, bath application of timolol or cypermethrin alone did not have significant effect on associative LTD induction, suggesting neither endogenous function of β-adrenergic receptor nor endogenous PKA activity does have a role in associative LTD induction.     

Thyroid dependent maturation of β-adrenergic receptors in the rat lung

β-Adrenergic receptors were identified in membranes of fetal and postnatal rat lung with (?)-[³H]dihydroalprenolol, [³H]DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to adult levels by day 28 of postnatal life. The increase of β-adrenergic receptors occurring between postnatal days 15 and 28 was dependent on thyroxine (T4) in propylthiouracil treated pups. β-Adrenergic receptors on day 28 were identical in euthyroid (PTU + T4) as compared to normal control pups (489±31 and 491±30 femtomoles·mg^?1) however receptors were markedly reduced in 28 day hypothyroid pups (PTU alone), Bmax = 294±21.5, m±S.E. p<0.01. Treatment of the hypothyroid pups with T4 for three days on postnatal day 25 increased β-adrenergic receptors approximately two-fold by day 28. This thyroid hormone dependent increase in lung β-adrenergic receptors occurs between postnatal days 15 and 28 coincident with the known increase in thyroid gland activity in the rat pup.     

Ectopic calcification in β-thalassemia patients is associated with increased oxidative stress and lower MGP carboxylation

A number of beta-thalassemia (β-thal) patients in the course of the disease exhibit ectopic calcification affecting skin, eyes and the cardiovascular system. Clinical and histopathological features have been described similar to those in pseudoxanthoma elasticum (PXE), although different genes are affected in the two diseases. Cultured dermal fibroblasts from β-thal patients with and without PXE-like clinical manifestations have been compared for parameters of redox balance and for the expression of proteins, which have been already associated with the pathologic mineralisation of soft connective tissues. Even though oxidative stress is a well-known condition of β-thal patients, our results indicate that the occurrence of mineralized elastin is associated with a more pronounced redox disequilibrium, as demonstrated by the intracellular increase of anion superoxide and of oxidized proteins and lipids. Moreover, fibroblasts from β-thal PXE-like patients are characterized by decreased availability of carboxylated matrix Gla protein (MGP), as well as by altered expression of proteins involved in the vitamin K-dependent carboxylation process. Results demonstrate that elastic fibre calcification is promoted when redox balance threshold levels are exceeded and the vitamin K-dependent carboxylation process is affected decreasing the activity of MGP, a well-known inhibitor of ectopic calcification. Furthermore, independently from the primary gene defect, these pathways are similarly involved in fibroblasts from PXE and from β-thal PXE-like patients as well as in other diseases leading to ectopic calcification, thus suggesting that can be used as markers of pathologic mineralisation.     

Subcellular distribution and activation of rat submandibular cAMP-dependent protein kinase following β-adrenergic receptor stimulation

The extent of activation of rat submandibular protein kinase A (EC 2.7.1.37) isozymes following β-adrenergic receptor stimulation was determined in vitro using dispersed cells and an 8-N₃-[³²P]cAMP photoprobe. The half-maximal binding of the photoprobe for microsomal and cytosolic type I and cytosolic type II was 9 nM, 27 nM and 92 nM, respectively. ‘Cold trap’ studies indicated that 70% of type I protein kinase A was activated following maximal β-adrenergic receptor stimulation, whereas type II activation was less than 40%. Both cytosolic and microsomal type I activation occurred rapidly following β-adrenergic receptor stimulation and both remain activated throughout the entire secretory period. Type I inactivation occurred rapidly subsequent to β-adrenergic receptor blockade. The dose-response relationship for the isotypes following β-adrenergic receptor activation demonstrated a greater extent of type I activation at submaximal concentrations of agonist. Although protein kinase A may not be the only kinase involved in rat submandibular mucin release, these data add further support to a direct regulatory role for this kinase, with type I having potentially a greater role than type II.     

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of β-adrenergic responses

Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.     

Sex difference in cellular calcium metabolism of rat hepatocytes and in α-adrenergic activation of glycogen phosphorylase

Three topics were the subject of these investigations: (i) the difference between males and females in the basal calcium metabolism of hepatocytes; (ii) the source of the calcium which triggers the phosphorylase a stimulation induced by epinephrine through alpha-adrenergic receptors; (iii) the time relation between the rise in phosphorylase activity and the increase in calcium efflux. We found that there was no difference between males and females in total or exchangeable cell calcium. However, there were significant differences in the mitochondrial calcium pool and fluxes measured by steady-state kinetic analyses: they were smaller and the rate constants of mitochondrial calcium influx and efflux were lower in males than in females. The ⁴⁵Ca content of isolated mitochondria and microsomes was also significantly lower in males than in females. In both males and females, epinephrine stimulated phosphorylase activity and calcium efflux even in the absence of extracellular calcium, indicating that the principal source of calcium which triggers the enzyme stimulation is intracellular. During the first 10 min following stimulation by 10^?6 M epinephrine, the total cell calcium, ⁴⁵Ca and the mitochondrial calcium were significantly depressed in male hepatocytes. After 10 min, these changes were reversed and the cell or mitochondrial calcium content was greater than in controls. In females, on the other hand, changes could only be detected if the cells were transferred to calcium-free media before the stimulation. In both males and females, there was a good temporal relationship between the stimulation of calcium efflux and the rise in phosphorylase a activity when hepatocytes were exposed to increasing concentrations of epinephrine: both rose at least 75% in less than 15 s. We conclude that there are important differences in cellular calcium metabolism between males and females. The rise in cytosolic calcium induced by alpha-adrenergic activation is principally due to a mobilization of calcium from an intracellular pool, probably the mitochondria.     

Hypomethylation of the thymosin β(10) gene is not associated with its overexpression in non-small cell lung cancer

Lung cancer is the leading cause of cancer-related deaths worldwide and is usually associated with a late diagnosis and a poor prognosis. Thymosin β₁₀ (TMSB10) is a monomeric actin sequestering protein that regulates actin cytoskeleton organization. The aberrant TMSB10 expression has been implicated in the pathogenesis of human cancers. However, its role in carcinogenesis is still controversial. To better understand the role of TMSB10 in lung tumorigenesis and its regulatory mechanism, we examined the methylation status and expression of the TMSB10 gene in non-small cell lung cancers (NSCLCs) using methylation-specific PCR (MSP) and immunohistochemistry (IHC), respectively. MSP analysis showed that the TMSB10 promoter was already unmethylated in most tumor tissues and became demethylated in 20 (14.4%) of the 139 NSCLCs. TMSB10 hypomethylation was not significantly correlated with the clinicopathological features. IHC showed that the TMSB10 protein was strongly expressed in the cytoplasm of malignant cells and its overexpression was detected in 50.0% of the tumor tissues compared to normal tissues. TMSB10 overexpression was frequently observed in sqaumous cell carcinomas compared to adenocarcinomas with border line significance (P = 0.072). However, TMSB10 methylation status was not linked to its overexpression. Collectively, these results suggest that TMSB10 hypomethylation may be a frequent event in NSCLCs, but it may not be a common mechanism underlying TMSB10 overexpression. However, further studies with large numbers of patients are needed to confirm our findings.     

High IFN－α expression is associated with the induction of experimental autoimmune uveitis （EAU） in Fischer 344 rat

INTRODUCTIONThe CD4 T cells can be subdivided intoTh1 and Th2 subsets based on their secreted cy-tokine profile. Th1 cells characteristical1y secreteInterferonry (IFN--ry), whereas Th2 cells maiuly pro--duce IL--4[1]. IL-12 po1arizes the differentiation ofnaitre, CD4 T cells towards Th1 pathWay, in con-trast IL--4 directs T cell differentiation towards Th2pathWay The broken balance between Th1 and Th2immune responses and predominant Th1 responseare crucial factors in initiation…     

Starvation is associated with changes in the elemental composition of the pancreatic β-cell

By using the proton microprobe technique we have investigated the elemental composition of both pancreatic -cells and exocrine pancreas from fed and 24 h or 48 h starved obese hyperglycemic mice. Among the 15 elements measured in the -cells both Ca and Fe increased while Mg and S decreased significantly after 24 h of starvation, the effects being more pronounced after 48 h. When animals were starved for 48 h there was a decrease in the contents of Cl, Rb and Cu, whereas that of Al and Mn increased with 152 and 55%, respectively. There was an initial decrease in Na after 24 h of starvation, which was followed by an increase after 48 h. This is in contrast to Cd, which first increased and then decreased to a value lower than that obtained in the fed animal. The content of K showed a small decrease and that of Pb showed an increase only in the 24 h starved group. In the -cells the contents of Zn and P did not change subsequent to starvation. In the exocrine pancreas Na, Cl and P decreased after 24 h of starvation and except for Na, the decrease was maintained when the starvation period was increased to 48 h. After 24 h there was a significant, though transient, increase in K, Mg and Rb. With regard to the contents of Zn, Cu and S there was a progressive decrease as the starvation continued. In contrast to the endocrine pancreas the content of Al in the exocrine pancreas did not change after 48 h of starvation. There was no change in islet insulin content subsequent to starvation. The extent to which the observed changes in -cell elemental composition is involved in the impaired insulin release associated with starvation, merits further investigations.     

Age-related increase of β1-adrenergic receptor gene expression in rat liver: a potential mechanism contributing to increased β-adrenergic receptor density and responsiveness during aging

In this study we examined whether the levels of gene expressions of the three β- adrenergic receptor (βAR) subtypes, β₁, β₂, and β₃, contribute to age-related increase in βAR density. Liver membranes and total RNA were prepared from young (4- to 6-month-old) and old (24-month-old) male Fischer 344 rats. βAR density (B_max) in liver membranes was measured by a radioligand receptor binding assay using the receptor subtype nonselective βAR antagonist ¹²⁵I-pindolol as the radioligand. Steady-state levels of β₂AR mRNA in rat liver were measured by Northern blot analysis; because of the low abundance of β₁AR and β₃AR mRNA in rat liver, the expressions of these genes were measured by a semiquantitative RT-PCR or an RT-PCR. Scatchard analysis of saturation binding curves of the binding assay confirmed an age-related increase in B_max (young: 7.1?±?0.8?fmol/mg protein vs. old: 18.1?±?4.3?fmol/mg protein). No age-related differences were found in the levels of β₂AR mRNA. However, semiquantitative RT-PCR revealed an approximately twofold increase in β₁AR mRNA level between young and old rats (P?<?0.05). β₁AR mRNA levels were also correlated with B_max values for ¹²⁵I-pindolol binding sites in individual rats (r = 0.67; P?=?0.012). β₃AR mRNA, which was demonstrable in rat white adipose tissue by RT-PCR, was generally not detected in livers from young or old rats, with the exception of two old rats with the highest B_max. These results suggest that an age-related increase of β₁AR gene expression contributes to increased βAR density and β adrenergic responsiveness in rat liver during aging.