CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

Structure and function of γ-secretase

The γ-secretase complex is a prime target for pharmacological intervention in Alzheimer’s disease and so far drug discovery efforts have yielded a large variety of potent and rather specific inhibitors of this enzymatic activity. However, as γ-secretase is able to cleave a wide variety of physiological important substrates, the real challenge is to develop substrate-specific compounds. Therefore, obtaining structural information about γ-secretase is indispensable. As crystal structures of the complex will be difficult to achieve, applied biochemical approaches need to be integrated with structural information obtained from other intramembrane-cleaving proteases. Here we review current knowledge about the structure and function of γ-secretase and discuss the value of these findings for the mechanistic understanding of this unusual protease.     

Growth and mortality of individual plants as a function of “available area”

Summary We looked at the relationship between available area, as defined by Thiessen polygons around individual plants, and plant size and mortality in even-aged green-house populations of Lapsana communis L. Polygon area was a good predictor of plant weight in these populations. After nine weeks growth, just prior to the onset of self-thinning, the dry weight of plants was directly proportional to the square root of polygon area. After the onset of selfthinning, plant weight appeared to be directly related to polygon area to the 3/2 power. Plants in small polygons were much more likely to die than those in larger areas. Thinning changed the frequency distribution of polygon sizes from highly skewed and unequal to normal and more equal, while inequality in surviving plant sizes did not appear to be affected by thinning.     

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.     

A model of mate searching and territorial behaviour for “flier” type dragonflies

First, the mating system of a “flier” odonate species that continues patrolling without perching,Cordulia aenea amurensis Selys, is briefly reviewed. Adult males of this species search for mates for a long distance along the shore of a pond if male density is very low, but localize their patrol within a short range and defend the ranges if the density is high (Ubukata 1975, 1979). Secondly, a model of mate searching by vision and territoriality for flier dragonflies is proposed. The main assumptions of the model are: (1) a male with a fixed length of visual range (D) patrols with a constant velocity (V) on a straight line; (2) he turns in the opposite direction at 2 fixed points (distance=L); (3) female arrival probability is uniform in both spatial and temporal dimensions; (4) the females stay at the arrival point for a fixed time (C); (5) if a male discovers a female he copulates with her without fail and leaves the line. From the model the following are predicted: if , the species (type 1) has an optimal length of patrol range even if the female arrival rate is uniform; ifD is smaller than the right hand side of the inequality (type 2), the mating probability becomes higher as the patrol range increases, if there is no other male. On the basis of the above model the concept of an “unusurpable territory” is defined to exist at a given time after the start of patrol and the probability of unusurpability (P) is calculated as a function ofD, V, L, M (male density-1) andT (duration of patrol).P drops abruptly if the length of patrol range (L) exceeds that of unusurpable territory at high male densities. Therefore, there appears to be an optimal length of patrol range when there are other males, agreeing with the data fromC. a. amurensis. Finally, the model is discussed in comparison with some other models. Ecological studies ofCordulia aenea amurensis Selys, VIII.     

“Glial inhibition” of memory in Alzheimer's disease

<正>Two recent studies on Nature Medicine and Nature Communications reported the unusual roles of astrocytes in the brain,that is,release gamma-aminobutyric acid(GABA)and impair hippocampal memory in Alzheimer’s disease(AD).AD represents one common form of neurodegenerative diseases,and is characterized by progressive dementia.The pathological feature of an AD brain includes the appearance     

In vitro evaluation of single- and multi-strain probiotics: Inter-species inhibition between probiotic strains, and inhibition of pathogens

Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit Clostridium difficile, Escherichia coli and S. typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p < 0.05 or less) greater inhibition of pathogens in 12 out of 24 cases. Despite evidence that probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain.     

Selective inhibition of leukotriene receptor BLT-2 reduces vascular oxidative stress and improves endothelial function in ApoE?/? mice

Atherosclerosis is a chronic inflammatory disease and represents the main cause of death in the industrialized world. Metabolites of the arachidonic acid derived from the 5-lipoxygenase pathway are known as leukotrienes that mediate various inflammatory processes during atherogenesis. Leukotriene B4 elicits the overexpression of several proinflammatory proteins, promotes chemotaxis and foam cell formation via BLT receptors. Currently, little is known about the implications of the BLT2 receptor in atherogenesis. Here, we tested whether selective inhibition of this receptor influences the progression of atherosclerosis in mice. Apolipoprotein-E deficient mice were fed a high-fat, cholesterol-rich diet to create atherosclerotic conditions (each group n?=?9). Simultaneously, mice received the pharmacologic BLT2 inhibition (Ly) by intraperitoneal injection every second day 5?mg/kg bw or vehicle. After 8 weeks, mice were killed and experiments were performed. Vascular superoxide release was diminished in mice treated with Ly compared with the control group (68?±?15 vs 131?±?20 RLU, P?=?0.01), as measured by L012 assay. Next, endothelial function was assessed by organ chamber experiments. Endothelial-dependent relaxation was improved in mice treated with the BLT2 receptor antagonist. To determine whether selective inhibition of the BLT2 receptor affects the atherosclerotic plaque growth, immunohistochemical stainings of the aortic root were performed. Oil red O staining revealed no significant differences between both groups (36?±?3% vs 38?±?3%). Monocyte infiltration into the vessel wall was analyzed using Moma-2 staining. No significant differences were observed between both groups (31?±?3% vs 34?±?2%). Selective inhibition of the BLT2 receptor in mice reduces the release of vascular reactive oxygen species and improves endothelial function in mice. Further experiments are necessary in order to obtain tissue-specific and mechanistical insights.     

Repression of TXNIP–NLRP3 axis restores intestinal barrier function via inhibition of myeloperoxidase activity and oxidative stress in nonalcoholic steatohepatitis

Dysfunction of the intestinal barrier function occurs in hepatic injury, but the specific mechanisms responsible are largely unknown. Recently, NOD-like receptor 3 (NLRP3) inflammasome functions in impairing endothelial barrier function. In this study, we test the hypothesis that TXNIP–NLRP3 axis repression prevents against intestinal barrier function disruption in nonalcoholic steatohepatitis (NASH). First, lipopolysaccharide (LPS)-induced alterations in expression of ZO-1 and occludin, myeloperoxidase (MPO) activity, reactive oxygen species (ROS) level, and transepithelial electric resistance (TEER) in intestinal epithelial cells (IECs) isolated from C57BL/6 wild-type (WT) and TXNIP^−/− mice were evaluated. The underlying regulatory mechanisms of TXNIP knockout in vivo were investigated with the detection of expressions of TXNIP, NLRP3 and ZO-1, and occludin, the interaction of TXNIP–NLRP3, MPO activity, ROS level, permeability of intestinal mucosa, levels of inflammatory factors in serum, and LPS concentration. We identified that TXNIP knockout promoted ZO-1 and occludin expression, yet reduced MPO activity, ROS level, and cell permeability in IECs, indicating restored the intestinal barrier function. However, LPS upregulated TXNIP and NLRP3 expression, as well as contributed to the interaction between TXNIP and NLRP3 in vitro. Furthermore, TXNIP was significantly upregulated in the intestinal mucosa of NASH mice and its knockout repaired the intestinal barrier disrupt, inhibited expression of inflammatory factors, and reduced LPS concentration as well as hepatic injury in vivo. Taken together, our findings demonstrated that inhibited the activation of the TXNIP–NLRP3 axis reduced MPO activity and oxidative stress and thus restoring the intestinal barrier function in NASH. TXNIP–NLRP3 axis may be a promising therapeutic strategy for the NASH treatment.     

Understanding of basic mechanisms of β-cell function and survival

Type 1 and type 2 diabetes are both diseases of insulin insufficiency, although they develop by distinct pathways. The recent surge in the incidence of type 2 diabetes and the chronic ailments confronted by patients with either form of the disease highlight the need for better understanding of β-cell biology. In this review, we present recent work focused on this goal. Our hope is that basic research being conducted in this and other laboratories will ultimately contribute to the development of methods for enhancing β-cell function and survival in the context of both major forms of diabetes. Our strategy for understanding the β-cell involves a multidisciplinary approach in which tools from the traditional fields of biochemistry, enzymology, and physiology are teamed with newer technologies from the fields of molecular biology, gene discovery, cell and developmental biology, and biophysical chemistry. We have focused on two important aspects of β-cell biology in our studies: β-cell function, specifically the metabolic regulatory mechanisms involved in glucose-stimulated insulin secretion, and β-cell resistance to immune attack, with emphasis on resistance to inflammatory cytokines and reactive oxygen species.     

Synthesis and α-glucosidase inhibition activity of dihydroxy pyrrolidines

A new series of Deacetylsarmentamide A and B derivatives, amides and sulfonamides of 3,4-dihydroxypyrrolidines as α-glucosidase inhibitors were designed and synthesized. The biological screening test against α-glucosidase showed that some of these compounds have the positive inhibitory activity against α-glucosidase. Saturated aliphatic amides were more potent than the olefinic amides. Among all the compounds, 5o/6o having polar –NH₂ group, 10f/11f having polar –OH group on phenyl ring displayed 3–4-fold more potent than the standard drugs. Acarbose, Voglibose and Miglitol were used as standard references. The promising compounds 6i, 5o, 6o, 10a, 11a, 10f and 11f have been identified. Molecular docking simulations were done for compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.     

Interspecific incompatibility in Populus: inhibition of tube growth and behaviour of the male germ unit inP. deltoides×P. alba cross

Summary In order to better understand the cellular events controlling interspecific incompatibility in the genus Populus, the incompatible cross betweenP. deltoides andP. alba has been investigated both at the light and electron microscopic levels. Stained in decolourized aniline blue and observed by epifluorescence microscopy, most incompatible pollen grains are seen to germinate at the stigma surface. Numerous incompatible pollen tubes reach the base of the style where they are arrested 19 h after pollination. Ultrastructural observations on in vivo growing incompatible pollen tubes confirm these data. Very few cytoplasmic modifications are seen within living pollen tubes reaching the lower end of the style or within arrested ones, except the presence of polymorphic plastids. In this predominantly tricellular system, the male germ unit (MGU) is apparently initiated at pollen maturity as an association between the vegetative nucleus and sperm cells. It is maintained during pollen tube growth within the style and persists within arrested incompatible pollen tubes. The unique observation of an association between a dividing generative cell at metaphase and the vegetative nucleus is also reported. Arrested pollen tubes are characterized by apical deformations and accumulation of callose within their thickened cell walls. These cytological data provide additional information on the cellular events associated with interspecific incompatibility in Populus.Abbreviations DAPI 4,6-diamino-2-phenylindole - FCR fluorochromatic reaction - MGU male germ unit     

PDK1 is important lipid kinase for RANKL-induced osteoclast formation and function via the regulation of the Akt-GSK3β-NFATc1 signaling cascade

Perturbations in the balanced process of osteoblast-mediated bone formation and osteoclast-mediated bone resorption leading to excessive osteoclast formation and/or activity is the cause of many pathological bone conditions such as osteoporosis. The osteoclast is the only cell in the body capable of resorbing and degrading the mineralized bone matrix. Osteoclast formation from monocytic precursors is governed by the actions of two key cytokines macrophage-colony-stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL). Binding of RANKL binding to receptor RANK initiates a series of downstream signaling responses leading to monocytic cell differentiation and fusion, and subsequent mature osteoclast bone resorption and survival. The phosphoinositide-3-kinase (PI3K)-protein kinase B (Akt) signaling cascade is one such pathway activated in response to RANKL. The 3-phosphoinositide-dependent protein kinase 1 (PDK1), is considered the master upstream lipid kinase of the PI3K-Akt cascade. PDK1 functions to phosphorylate and partially activate Akt, triggering the activation of downstream effectors. However, the role of PDK1 in osteoclasts has yet to be clearly defined. In this study, we specifically deleted the PDK1 gene in osteoclasts using the cathepsin-K promoter driven Cre-LoxP system. We found that the specific genetic ablation of PDK1 in osteoclasts leads to an osteoclast-poor osteopetrotic phenotype in mice. In vitro cellular assays further confirmed the impairment of osteoclast formation in response to RANKL by PDK1-deficient bone marrow macrophage (BMM) precursor cells. PDK1-deficient BMMs exhibited reduced ability to reorganize actin cytoskeleton to form a podosomal actin belt as a result of diminished capacity to fuse into giant multinucleated osteoclasts. Notably, biochemical analyses showed that PDK1 deficiency attenuated the phosphorylation of Akt and downstream effector GSK3β, and reduced induction of NFATc1. GSK3β is a reported negative regulator of NFATc1. GSK3β activity is inhibited by Akt-dependent phosphorylation. Thus, our data provide clear genetic and mechanistic insights into the important role for PDK1 in osteoclasts.     

Prostaglandin inhibition of cartilage chondromucoprotein synthesis: Concept of “intrinsic activity”

We have recently reported that cartilage has two sites for prostaglandin (PG) action. One site (S₁) is stimulated by PGA₁, PGE₁ and PGF_1α and elevates tissue cyclic 3′5′adenosine monophosphate (cAMP). A second site (S₂) is activated by PGA₁ (but not PGE₁ or PGF_1α) and inhibits the synthesis of cartilage macromolecules. The present study is an investigation of the effects of PGB₁ on embryonic chicken cartilage chondromucoprotein synthesis in vitro. PGB₁ was found to inhibit chondromucoprotein synthesis with an apparent affinity for S₂ which was similar to that of PGA₁. The maximal inhibition produced by PGB₁ was, however, approximately one-half the maximal inhibition caused by PGA₁. Studies of the combined effects of PGB₁ and PGA₁ were consistent with the hypothesis that both classes of prostaglandins act at a common site (S₂) with about equal affinity but that PGB₁ has a lower intrinsic activity than PGA₁. Similar studies of the combined effects of PGE₁ or PGF_1α with PGA₁ indicate that neither PGE₁ nor PGF_1α binds significantly to S₂. An independent effect of PGB₁ to activate S₁ and elevate tissue cAMP was also found.