Regulation of B-cell survival by BAFF-dependent PKCdelta-mediated nuclear signalling

Approximately 65% of B cells generated in human bone marrow are potentially harmful autoreactive B cells. Most of these cells are clonally deleted in the bone marrow, while those autoreactive B cells that escape to the periphery are anergized or perish before becoming mature B cells. Escape of self-reactive B cells from tolerance permits production of pathogenic auto-antibodies; recent studies suggest that extended B lymphocyte survival is a cause of autoimmune disease in mice and humans. Here we report a mechanism for the regulation of peripheral B-cell survival by serine/threonine protein kinase Cdelta (PKCdelta): spontaneous death of resting B cells is regulated by nuclear localization of PKCdelta that contributes to phosphorylation of histone H2B at serine 14 (S14-H2B). We show that treatment of B cells with the potent B-cell survival factor BAFF ('B-cell-activating factor belonging to the TNF family') prevents nuclear accumulation of PKCdelta. Our data suggest the existence of a previously unknown BAFF-induced and PKCdelta-mediated nuclear signalling pathway which regulates B-cell survival.     

Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK) raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP). Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.     

Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.     

Regulation of calcium signalling in T lymphocytes by the second messenger cyclic ADP-ribose

Cyclic ADP-ribose (cADPR) is a natural compound that mobilizes calcium ions in several eukaryotic cells. Although it can lead to the release of calcium ions in T lymphocytes, it has not been firmly established as a second messenger in these cells. Here, using high-performance liquid chromatography analysis, we show that stimulation of the T-cell receptor/CD3 (TCR/CD3) complex results in activation of a soluble ADP-ribosyl cyclase and a sustained increase in intracellular levels of cADPR. There is a causal relation between increased cADPR concentrations, sustained calcium signalling and activation of T cells, as shown by inhibition of TCR/CD3-stimulated calcium signalling, cell proliferation and expression of the early- and late-activation markers CD25 and HLA-DR by using cADPR antagonists. The molecular target for cADPR, the type-3 ryanodine receptor/calcium channel, is expressed in T cells. Increased cADPR significantly and specifically stimulates the apparent association of [3H]ryanodine with the type-3 ryanodine receptor, indicating a direct modulatory effect of cADPR on channel opening. Thus we show the presence, causal relation and biological significance of the major constituents of the cADPR/calcium-signalling pathway in human T cells.     

Mediators of vascular remodelling co-opted for sequential steps in lung metastasis

Metastasis entails numerous biological functions that collectively enable cancerous cells from a primary site to disseminate and overtake distant organs. Using genetic and pharmacological approaches, we show that the epidermal growth factor receptor ligand epiregulin, the cyclooxygenase COX2, and the matrix metalloproteinases 1 and 2, when expressed in human breast cancer cells, collectively facilitate the assembly of new tumour blood vessels, the release of tumour cells into the circulation, and the breaching of lung capillaries by circulating tumour cells to seed pulmonary metastasis. These findings reveal how aggressive primary tumorigenic functions can be mechanistically coupled to greater lung metastatic potential, and how such biological activities may be therapeutically targeted with specific drug combinations.     

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.     

Regulation by angiotensin II of its receptors in resistance blood vessels

The sensitivity of blood vessels to the vasoconstrictor effects of the hormone angiotensin II appears to be modulated by the activity of the renin-angiotensin system. Elevation of circulating angiotensin II levels by sodium depletion or renal artery stenosis is associated with a diminished pressor response to infused angiotensin II (refs 1-3). Conversely, the vasocontrictor response to the hormone is enhanced when endogenous angiotensin II levels are reduced by sodium loading or nephrectomy. The mechanisms of these varying effects are not known, but physiological and pharmacological experiments suggest involvement of the vascular smooth receptor for angiotensin II (refs 5-8). Modification of the interaction between angiotensin II and its vascular receptor, resulting in altered responsiveness to the hormone, could occur either via 'prior occupancy' of receptors by elevated levels of endogenous angiotensin II resulting in fewer free receptors available to respond to circulating angiotensin II (ref. 5), or, elevated levels of angiotensin II could result in a decrease in receptor affinity for the hormone or a decrease in total receptor number in the vascular smooth muscle cell. We now report the first direct evidence, by radioligand binding assay, that angiotensin II regulates the number of its own receptors in resistance vasculature.     

Urotensin Ⅱ increases endothelin production by vascular smooth muscle cells in rats

The aim of this study was to observe the effects of urotensin Ⅱ (UII) on production of endothelin (ET) in rat aortic vascular smooth muscle cells (VSMC). Cultured VSMCs incubated with various concentrations of UII were used to measure the VSMC ³H-TdR incorporation, the amount of ET mRNA and ET production in VSMCs. In this work we found that UII (10^-10—10^-8 mol/L) promoted VSMC ³H-TdR incorporation (47%—83%, P < 0.01) and increased the amount of ET mRNA by 17.1% (P < 0.05) to 112.8% (P < 0.01), respectively, in a concentration dependent manner compared with control. After 4 and 8 h incubation, 10^-10—10^-8 mol/L of UII elevated the ET synthesis and release in a concentration dependent manner. After 4 h incubation, the content of ET in medium was 4.9, 5.36 and 7.12 pg/mL (P < 0.01). After 8 h incubation, the ET content released from VSMCs was 12.6, 12.07 and 17.17 pg/mL (P < 0.01). In addition, it was found that BQ₁₂₃, a specific ET_A receptor antagonist, obviously decreased the VSMC DNA synthesis induced by UII. The results of this study showed that UII could stimulate the ET mRNA expression and ET production in VSMC. The effects of UII on VSMC DNA synthesis were partly mediated by ET autocrine pathway. It suggests that the interaction between UII and ET plays an important biological regulating role as endogenous active peptides.     

Cortical remodelling induced by activity of ventral tegmental dopamine neurons.

Representations of sensory stimuli in the cerebral cortex can undergo progressive remodelling according to the behavioural importance of the stimuli. The cortex receives widespread projections from dopamine neurons in the ventral tegmental area (VTA), which are activated by new stimuli or unpredicted rewards, and are believed to provide a reinforcement signal for such learning-related cortical reorganization. In the primary auditory cortex (AI) dopamine release has been observed during auditory learning that remodels the sound-frequency representations. Furthermore, dopamine modulates long-term potentiation, a putative cellular mechanism underlying plasticity. Here we show that stimulating the VTA together with an auditory stimulus of a particular tone increases the cortical area and selectivity of the neural responses to that sound stimulus in AI. Conversely, the AI representations of nearby sound frequencies are selectively decreased. Strong, sharply tuned responses to the paired tones also emerge in a second cortical area, whereas the same stimuli evoke only poor or non-selective responses in this second cortical field in naive animals. In addition, we found that strong long-range coherence of neuronal discharge emerges between AI and this secondary auditory cortical area.     

SRD的PWM调压调速

根据开关磁阻电动机调速系统 ( SRD)的 PWM调压调速理论 ,论述了采用集成电路TL494实现 SRD的 PWM调压调速的原理。实验证明 ,该方案控制方便 ,可靠性高 ,调速范围较宽 ,简单易行 ,特别适用于小功率开关磁阻电动机的调速系统。     

Chromatin remodelling and epigenetic features of germ cells

Germ cells have the unique capacity to start a new life upon fertilization. They are generated during a sex-specific differentiation programme called gametogenesis. Maturation of germ cells is characterized by an impressive degree of cellular restructuring and gene regulation that involves remarkable genomic reorganization. These events are finely tuned, but are also susceptible to the introduction of various types of error. Because stable genetic transmission to future generations is essential for life, understanding the control of these processes has far-reaching implications for human health and reproduction.     

Adrenomedullin inhibits the endothelin production induced by urotensin II in rat vascular smooth muscle cells

The aim of this study was to observe the effects of adrenomedullin (ADM) on endothelin (ET) production induced by urotensin Ⅱ (UⅡ) in rat vascular smooth muscle cells (VSMCs). Cultured VSMCs which were incubated with UⅡ (10^-8 mol/L) and with various concentrations of ADM were used to measure the VSMCs ³H-TdR incorpora- tion, the activity of extracellular signal-regulated kinase (ERK), the amount of ET mRNA and ET production in VSMCs. In this work we found that incubation with UⅡ(10^-8 mol/L) increased obviously the amount of ET mRNA in VSMCs and ET production in medium, however, coincubation with ADM (10^-10—10^-8 mol/L) and UⅡ(10^-8 mol/L) reduced the amount of ET mRNA by 15%, 24% and 45% (P< 0.01) respectively, compared with UⅡ alone. The content of ET in medium was 14.13, 11.38 and 11.00 pg/mL. ADM alone (10^-8 mol/L) had no effect on ET production in VSMCs. UⅡ (10^-8 mol/L) promoted the 3H-TdR incorpo- ration and activity of ERK in VSMCs. ADM inhibited VSMCs 3H-TdR incorporation and activation of ERK in a concentration-dependent manner. Compared with UⅡ group, after coincubation with ADM (10^-10—10^-8 mol/L) and UⅡ (10^-8 mol/L) the VSMCs 3H-TdR incorporation was decreased by 7% (P > 0.05), 32% (P < 0.05) and 41% (P < 0.01), respectively, and the activity of ERK was decreased by 24% (P > 0.05), 32% (P < 0.05) and 36% (P < 0.05), re- spectively, in a concentration-dependent manner. The results show that in cultured VSMCs ADM inhibits ET mRNA expression, ET production and proliferation stimulated by UⅡ, and that inhibitory effect of ADM on UⅡ bioaction could be mediated through inhibiting MAPK pathway.     

Silencing of TGF-beta signalling by the pseudoreceptor BAMBI.

Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.     

Initiation of neural induction by FGF signalling before gastrulation

During neural induction, the 'organizer' of the vertebrate embryo instructs neighbouring ectodermal cells to become nervous system rather than epidermis. This process is generally thought to occur around the mid-gastrula stage of embryogenesis. Here we report the isolation of ERNI, an early response gene to signals from the organizer (Hensen's node). Using ERNI as a marker, we present evidence that neural induction begins before gastrulation--much earlier in development than previously thought. We show that the organizer and some of its precursor cells produce a fibroblast growth factor signal, which can initiate, and is required for, neural induction.     

Membrane curvature and mechanisms of dynamic cell membrane remodelling

Membrane curvature is no longer seen as a passive consequence of cellular activity but an active means to create membrane domains and to organize centres for membrane trafficking. Curvature can be dynamically modulated by changes in lipid composition, the oligomerization of curvature scaffolding proteins and the reversible insertion of protein regions that act like wedges in membranes. There is an interplay between curvature-generating and curvature-sensing proteins during vesicle budding. This is seen during vesicle budding and in the formation of microenvironments. On a larger scale, membrane curvature is a prime player in growth, division and movement.     

Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex

Microbial rhodopsins, which constitute a family of seven-helix membrane proteins with retinal as a prosthetic group, are distributed throughout the Bacteria, Archaea and Eukaryota. This family of photoactive proteins uses a common structural design for two distinct functions: light-driven ion transport and phototaxis. The sensors activate a signal transduction chain similar to that of the two-component system of eubacterial chemotaxis. The link between the photoreceptor and the following cytoplasmic signal cascade is formed by a transducer molecule that binds tightly and specifically to its cognate receptor by means of two transmembrane helices (TM1 and TM2). It is thought that light excitation of sensory rhodopsin II from Natronobacterium pharaonis (SRII) in complex with its transducer (HtrII) induces an outward movement of its helix F (ref. 6), which in turn triggers a rotation of TM2 (ref. 7). It is unclear how this TM2 transition is converted into a cellular signal. Here we present the X-ray structure of the complex between N. pharaonis SRII and the receptor-binding domain of HtrII at 1.94 A resolution, which provides an atomic picture of the first signal transduction step. Our results provide evidence for a common mechanism for this process in phototaxis and chemotaxis.