Characterization of Phospholipase C�� Enzymes with Gain-of-Function Mutations

Phospholipase Cγ isozymes (PLCγ1 and PLCγ2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLCγ2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLCγ1 and PLCγ2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLCγ2 without increasing Rac binding. Importantly, the activation of the ALI14-PLCγ2 and corresponding PLCγ1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.Phosphoinositide-specific phospholipase C (PLC)² enzymes, comprising several families (PLCβ, γ, δ, ϵ, η, and ζ), have been established as crucial signaling molecules involved in regulation of a variety of cellular functions (1–4). PLC-catalyzed formation of the second messengers, inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, from phosphatidylinositol 4,5-bisphosphate (PIP₂), constitutes one of the major cell signaling responses. These second messengers provide a common link from highly specific receptors for hormones, neurotransmitters, antigens, and growth factors to downstream, intracellular targets; thus, they contribute to regulation of biological functions as diverse as cell motility, fertilization, and sensory transduction. Despite this central role for PLC enzymes in signaling networks, the molecular details of their regulation and possible subversion of these regulatory mechanisms in disease remain poorly understood.Of two PLCγ enzymes, PLCγ1 is ubiquitously expressed and appears to regulate a multitude of cellular functions in many tissues. Plcg1-null mice die by embryonic day 9, highlighting the widespread importance of this enzyme (5). PLCγ1 is activated in response to growth factor stimulation; in addition, its function in T-cell responses has been extensively documented (1). PLCγ2, in contrast, is most highly expressed in cells of the hematopoietic system and plays a key role in regulation of the immune response. Consistent with this, Plcg2-null mice display defects in the functioning of B cells, platelets, mast cells, and natural killer cells (6).Both PLCγ enzymes have also been implicated in signaling events underlying aberrant cellular responses. PLCγ1 is critically involved in the regulation of cancer cell motility (7–11) while PLCγ2 has been implicated in deregulation of the immune responses resembling Btk-dependent X-linked agammaglobulinaemia and SLE disease in humans (12–14). It has been suggested that, in cancer cells, PLCγ1 could function as a key, rate-limiting, common component involved in cell motility triggered by several growth factors and integrins (7). In some cancer cells, this increased motility could result from deregulation i.e. higher levels of expression of PLCγ1 (15, 16). The possibility that the activity of PLCγ could be up-regulated due to mutation has not yet been fully investigated in cancer. Previous studies of PLCγ2, however, have demonstrated the first gain-of-function mutation in a PLC molecule in the context of an organism, and shown that, in principle, PLC activity can be greatly enhanced by point mutations (13). Furthermore, this work has demonstrated that such a mutation is linked to a dramatic phenotypic disorder. By using a large scale ENU mutagenesis to discover new immune regulators, several mouse strains were generated with spontaneous autoimmune and inflammatory symptoms; two of these strains harbor a mutation in PLCγ2. In addition to the previously described ALI5 mutation (13) the ALI14 mutation has been identified very recently.³ Strikingly, the well-characterized ALI5 phenotype has shown that the mutation affects many cellular functions deregulated in Plcg2-null mice. Notably, while in Plcg2-null mice such responses are lacking, the ALI5 mutation resulted in their enhancement. In particular, further analyses of the ALI5 mutation in the context of signaling in B-cells have demonstrated that calcium responses to the crosslinking of the B-cell receptor were enhanced and prolonged resulting in enhanced deletion of B cells and autoreactivity (13).The domain organization of PLCγ enzymes is characterized by the insertion of a highly structured region (PLCγ-specific array, γSA) between the two halves of the TIM-barrel catalytic domain common to all PLCs. The γSA comprises a split PH (spPH) domain flanking two tandem SH2 domains and a SH3 domain (1). A distinct regulatory feature of PLCγ enzymes is that their activation is linked to an increase in phosphorylation of specific tyrosine residues (most notably within the γSA) by receptor and non-receptor tyrosine kinases (17, 18). Furthermore, multiple protein-protein interactions (mainly mediated by SH2 domains) also contribute to activation and have an important role in localizing PLCγ into protein complexes with different binding partners, depending on cell type and specific cellular compartments. One mode of activation that is specific for the PLCγ2 isozyme is direct binding to and activation by Rac. The interaction involves the spPH domain, and this activation mechanism does not require tyrosine phosphorylation (19, 20). In molecular terms, changes that lead to PLC activation in response to different input signals, or due to point mutations, are not well understood and require further studies.Here we describe further analysis of the two gain-of-function mutations, ALI5 and ALI14, obtained using ENU mutagenesis. These mutations map to different regions in PLCγ2, and we performed detailed analysis of these regions in both PLCγ isozymes. To characterize the molecular mechanism of gain-of-function, we combined studies in vitro and in different cellular signaling contexts. We have found that ALI5- and ALI14-type point mutations lead, by distinct mechanisms, to an enhancement of responses to a variety of input signals while their combination results in a constitutively active PLC enzyme.     

Brain Swelling and Loss of Gray and White Matter Differentiation in Human Postmortem Cases by Computed Tomography

The purpose of this study was to evaluate the brain by postmortem computed tomography (PMCT) versus antemortem computed tomography (AMCT) using brains from the same patients. We studied 36 nontraumatic subjects who underwent AMCT, PMCT, and pathological autopsy in our hospital between April 2009 and December 2013. PMCT was performed within 20 h after death, followed by pathological autopsy including the brain. Autopsy confirmed the absence of intracranial disorders that might be related to the cause of death or might affect measurements in our study. Width of the third ventricle, width of the central sulcus, and attenuation in gray matter (GM) and white matter (WM) from the same area of the basal ganglia, centrum semiovale, and high convexity were statistically compared between AMCT and PMCT. Both the width of the third ventricle and the central sulcus were significantly shorter in PMCT than in AMCT (P < 0.0001). GM attenuation increased after death at the level of the centrum semiovale and high convexity, but the differences were not statistically significant considering the differences in attenuation among the different computed tomography scanners. WM attenuation significantly increased after death at all levels (P<0.0001). The differences were larger than the differences in scanners. GM/WM ratio of attenuation was significantly lower by PMCT than by AMCT at all levels (P<0.0001). PMCT showed an increase in WM attenuation, loss of GM–WM differentiation, and brain swelling, evidenced by a decrease in the size of ventricles and sulci.     

Co-localization of polysomes,cytoskeleton, and membranes with protein bodies from corn endosperm

Summary Frozen corn endosperm was homogenized in a cytoskeleton-stabilizing buffer and stained directly (without pelleting) with rhodamine-phalloidin for actin and either thiazole orange to stain RNA or DiOC₆ to stain membranes prior to examination under the fluorescence microscope. Other samples were treated with a non-ionic detergent alone or in conjunction with a ionic detergent prior to staining and fluorescence microscopy. Very gentle homogenization in unsupplemented buffer yielded a massive aggregate containing protein bodies that fluoresced after treatment with the ER stain DiOC₆. This aggregate was capped by an aggregate of unstained starch grains. More vigorous homogenization yielded more disperse patterns showing almost identical co-localization of ER, actin and RNA (polysomes). Homogenization in buffer plus non-ionic detergent removed most of the membrane yet maintained co-localization of actin and polysomes, while homogenization in double detergent removed the last traces of membrane and actin, and released over 70% of the polysomes. We interpret these results to suggest that protein bodies are surrounded by membranes, cytoskeleton and RNA (polysomes) and that the majority of the polysomes are attached more firmly to the cytoskeleton than to the membrane. This provides evidence from fluorescence microscopy to supplement that from biochemical analyses for the existence of cytomatrix-bound polysomes in plants.Abbreviations CBP cytoskeleton-bound polysomes - CMBP cyto-matrix-bound polysomes - CSB cytoskeleton-stabilizing buffer - DOC sodium deoxycholate - DiOC₆ 3,3-dihexyloxacarbocyanine iodide - DTE dithioerythritol - MBP membrane-bound polysomes - FP free polysomes - PMSF phenylmethyl-sulfonyl fluoride - PTE polyoxy-ethylene-10-tridecyl ether - Rh-Ph rhodamine-phalloidin - TO thiazole orange - Tris tris-(hydroxymethyl) aminomethane     

Genetic Variation in the Catechol-O-Methyl Transferase Val108/158Met Is Linked to the Caudate and Posterior Cingulate Cortex Volume in Healthy Subjects: Voxel-Based Morphometry Analysis of Brain Magnetic Resonance Imaging

The effect of the catechol-O-methyltransferase (COMT) Val158Met polymorphism on brain morphology has been investigated but remains controversial. We hypothesized that a comparison between Val/Val and Val/Met individuals, which may represent the most different combinations concerning the effects of the COMT genotype, may reveal new findings. We investigated the brain morphology using 3-Tesla magnetic resonance imaging in 27 Val/Val and 22 Val/Met individuals. Voxel-based morphometry revealed that the volumes of the bilateral caudate and posterior cingulate cortex were significantly smaller in Val/Val individuals than in Val/Met individuals [right caudate: false discovery rate (FDR)-corrected p = 0.048; left caudate: FDR-corrected p = 0.048; and bilateral posterior cingulate cortex: FDR-corrected p = 0.048]. This study demonstrates that interacting functional variants of COMT affect gray matter regional volumes in healthy subjects.     

Perlecan proteolysis induces an alpha2beta1 integrin- and Src family kinase-dependent anti-apoptotic pathway in fibroblasts in the absence of focal adhesion kinase activation

Dysregulation of apoptosis in endothelial cells (EC) and fibroblasts contributes to fibrosis. We have shown previously that apoptosis of EC triggers the proteolysis of extracellular matrix components and the release of a C-terminal fragment of perlecan, which in turn inhibits apoptosis of fibroblasts. Here we have defined the receptors and pathways implicated in this anti-apoptotic response in fibroblasts. Neutralizing alpha2beta1 integrin activity in fibroblasts exposed to either medium conditioned by apoptotic EC (SSC) or a recombinant perlecan C-terminal fragment (LG3) prevented resistance to apoptosis and is associated with decreased levels of Akt phosphorylation. Co-incubation of fibroblasts for 24 h with SSC or LG3 in the presence of PP2 (AG1879), a biochemical inhibitor of Src family kinases (SFKs) and focal adhesion kinase, showed a significantly decreased anti-apoptotic response. However, focal adhesion kinase gene silencing with RNA interference did not inhibit the anti-apoptotic response in fibroblasts. Src phosphorylation was increased in fibroblasts exposed to SSC, and transfection of fibroblasts with constitutively active Src mutants induced an anti-apoptotic response that was not further increased by SSC. Also, Src(-/-)Fyn(-/-) fibroblasts failed to mount an anti-apoptotic response in presence of SSC for 24 h but developed a complete anti-apoptotic response when exposed to SSC for 7 days. These results suggest that extracellular matrix fragments produced by apoptotic EC initiate a state of resistance to apoptosis in fibroblasts via an alpha2beta1 integrin/SFK (Src and Fyn)/phosphatidylinositol 3-kinase (PI3K)-dependent pathway. In the long term, additional SFK members are recruited for sustaining the anti-apoptotic response, which could play crucial roles in abnormal fibrogenic healing.     

Hyperuricemia is independently associated with coronary heart disease and renal dysfunction in patients with type 2 diabetes mellitus

Aims

To investigate the relationship between hyperuricemia (HUA) and the clinical backgrounds in Japanese patients with type 2 diabetes mellitus.

Methods

After a cross-sectional study evaluating the association of HUA with the clinical characteristics in 1,213 patients with type 2 diabetes mellitus, the estimated glomerular filtration rate (eGFR) and the incidence of diabetic macroangiopathies was investigated in a prospective observational study in 1,073 patients during a 3.5 year period. HUA was defined by serum uric acid levels >327 μmol/L or as patients using allopurinol.

Results

The frequency of HUA was significantly higher in the diabetic patients (32% in men and 15% in women) than in the normal controls (14% in men and 1% in women). In total, HUA was found in 299 (25%) of the patients during the cross-sectional study. Even after adjusting for sex, drinking status, treatment for diabetes mellitus, body mass index, hypertension, use of diuretics, hyperlipidemia, HbA1c and/or the eGFR, the HUA was independently associated with some diabetic complications. The eGFR was significantly reduced in HUA patients compared to those with normouricemia in the 12 months after observation was started. HUA was also an independent risk factor for coronary heart disease even after adjustment in the Cox proportional hazard model.

Conclusions

HUA is a associated with diabetic micro- and macroangiopathies. HUA is a predictor of coronary heart disease and renal dysfunction in patients with type 2 diabetes mellitus. However, the influence of HUA is considered to be limited.     

Low TLR4 expression by liver dendritic cells correlates with reduced capacity to activate allogeneic T cells in response to endotoxin

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8alpha- (myeloid) and CD11c+CD8alpha+ ("lymphoid-related") DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-gamma) and Th2 (IL-4) responses. When higher LPS concentrations (> or =100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.     

Physical restriction of pods causes seed size reduction of a brassinosteroid-deficient faba bean (Vicia faba)

BACKGROUND AND AIMS: A brassinosteroid-deficient mutant faba bean (Vicia faba 'Rinrei') shows dwarfism in many organs including pods and seeds. 'Rinrei' has normal-sized seeds together with dwarf seeds, suggesting that dwarfism in the seed may be indirectly caused by brassinosteroid deficiency. The mechanism of seed size reduction in this mutant was investigated. METHODS: The associations between seed orientation in the pod, seed numbers per pod and pod lengths with seed sizes were analysed in 'Rinrei' and the wild-type plant. KEY RESULTS: 'Rinrei' seeds are tightly arranged in pods containing two or three seeds. Seed size decreased as the number of seeds per pod increased or as the length of the pod decreased. Where no physical restriction occurred between seeds in a pod, the wild-type faba bean seeds had a nearly constant size regardless of seed number per pod or pod length. 'Rinrei' seeds in pods containing single seeds were the same size as wild-type seeds. Brassinolide treatment increased the seed size and the length of pods containing three seeds in 'Rinrei'. CONCLUSION: Seed size of 'Rinrei' is mainly regulated through a reduction of pod length due to brassinosteroid deficiency; physical restriction within pods causes a reduction in seed size. These results suggest a possible mechanism for increasing faba bean yields to optimal levels.     

Inhibition of HIV-1 replication by vesicular stomatitis virus envelope glycoprotein pseudotyped baculovirus vector-transduced ribozyme in mammalian cells

The baculovirus has recently emerged as a promising vector for in vivo gene therapy. To investigate its potential as a delivery vector for an anti-virus ribozyme targeting HIV-1, we constructed recombinant baculovirus vectors bearing a ribozyme-synthesizing cassette driven by the tRNA(i)(Met) promoter with enhanced transduction efficiency by displaying vesicular stomatitis virus glycoprotein (VSV-G) on the viral envelope. Transduction of HeLa CD4(+) cells with a recombinant baculovirus delivering the HIV-1 U5 gene-specific ribozyme dramatically suppressed HIV-1 expression in this cell line. The VSV-G pseudotyped baculovirus vector-transduced ribozyme potently inhibited HIV-1 replication compared to a recombinant baculovirus vector-transduced ribozyme lacking VSV-G. The use of a baculovirus vector might be beneficial for application in gene therapy.