Function of endoplasmic reticulum calcium ATPase in innate immunity‐mediated programmed cell death

Programmed cell death (PCD) initiated at the pathogen‐infected sites during the plant innate immune response is thought to prevent the development of disease. Here, we describe the identification and characterization of an ER‐localized type IIB Ca²⁺‐ATPase (NbCA1) that function as a regulator of PCD. Silencing of NbCA1 accelerates viral immune receptor N‐ and fungal‐immune receptor Cf9‐mediated PCD, as well as non‐host pathogen Pseudomonas syringae pv. tomato DC3000 and the general elicitor cryptogein‐induced cell death. The accelerated PCD rescues loss‐of‐resistance phenotype of Rar1, HSP90‐silenced plants, but not SGT1‐silenced plants. Using a genetically encoded calcium sensor, we show that downregulation of NbCA1 results in the modulation of intracellular calcium signalling in response to cryptogein elicitor. We further show that NbCAM1 and NbrbohB function as downstream calcium decoders in N‐immune receptor‐mediated PCD. Our results indicate that ER‐Ca²⁺‐ATPase is a component of the calcium efflux pathway that controls PCD during an innate immune response.     

The Tomato spotted wilt virus cell‐to‐cell movement protein (NSM) triggers a hypersensitive response in Sw‐5‐containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw‐5b resistance gene copy

Although the Sw‐5 gene cluster has been cloned, and Sw‐5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum ‘SR1‘ plants transformed with a copy of the Sw‐5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw‐5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non‐structural TSWV proteins tested, the TSWV cell‐to‐cell movement protein (NS_M) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non‐replicative pEAQ‐HT expression vector system. HR was induced in Sw‐5b‐transgenic N. benthamiana as well as in resistant near‐isogenic tomato lines after agroinfiltration with a functional cell‐to‐cell movement protein (NS_M) from a resistance‐inducing (RI) TSWV strain (BR‐01), but not with NS_M from a Sw‐5 resistance‐breaking (RB) strain (GRAU). This is the first biological demonstration that Sw‐5‐mediated resistance is triggered by the TSWV NS_M cell‐to‐cell movement protein.     

Metabotropic glutamate receptor 5 responses dictate differentiation of neural progenitors to NMDA‐responsive cells in fragile X syndrome

Disrupted metabotropic glutamate receptor 5 (mGluR5) signaling is implicated in many neuropsychiatric disorders, including autism spectrum disorder, found in fragile X syndrome (FXS). Here we report that intracellular calcium responses to the group I mGluR agonist (S)−3,5‐dihydroxyphenylglycine (DHPG) are augmented, and calcium‐dependent mGluR5‐mediated mechanisms alter the differentiation of neural progenitors in neurospheres derived from human induced pluripotent FXS stem cells and the brains of mouse model of FXS. Treatment with the mGluR5 antagonist 2‐methyl‐6‐(phenylethynyl)‐pyridine (MPEP) prevents an abnormal clustering of DHPG‐responsive cells that are responsive to activation of ionotropic receptors in mouse FXS neurospheres. MPEP also corrects morphological defects of differentiated cells and enhanced migration of neuron‐like cells in mouse FXS neurospheres. Unlike in mouse neurospheres, MPEP increases the differentiation of DHPG‐responsive radial glial cells as well as the subpopulation of cells responsive to both DHPG and activation of ionotropic receptors in human neurospheres. However, MPEP normalizes the FXS‐specific increase in the differentiation of cells responsive only to N ‐methyl‐d ‐aspartate (NMDA) present in human neurospheres. Exposure to MPEP prevents the accumulation of intermediate basal progenitors in embryonic FXS mouse brain suggesting that rescue effects of GluR5 antagonist are progenitor type‐dependent and species‐specific differences of basal progenitors may modify effects of MPEP on the cortical development. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Biochemical composition of haemagglutinin‐based influenza virus‐like particle vaccine produced by transient expression in tobacco plants

Influenza virus‐like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant‐based VLP manufacturing platform allowing the large‐scale production of GMP‐grade influenza VLPs. In this article, we report on the biochemical compositions of these plant‐based influenza candidate vaccines, more particularly the characterization of the N‐glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco‐derived lipid content and residual impurities. Mass spectrometry analyses showed that all N‐glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant‐specific complex‐type N‐glycans having core α(1,3)‐fucose, core β(1,2)‐xylose epitopes and Lewis^a extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant‐made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme‐assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals.     

Development of mammalian production cell lines expressing CNTO736, a glucagon like peptide‐1‐MIMETIBODYTM: Factors that influence productivity and product quality

In an attempt to develop a high producing mammalian cell line expressing CNTO736, a Glucagon like peptide‐1‐antibody fusion protein (also known as a Glucagon like peptide‐1 MIMETIBODY^TM), we have noted that the N‐terminal GLP‐1 portion of the MIMETIBODY^TM was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. Therefore, a number of parameters that had an effect on productivity as well as product quality were examined. Results suggest that the choice of the host cell line had a significant effect on the overall product quality. Product expressed in mouse myeloma host cell lines had a lesser degree of proteolytic degradation and variability in O‐linked glycosylation as compared to that expressed in CHO host cell lines. The choice of a specific CHOK1SV derived clone also had an effect on the product quality. In general, molecules that exhibited minimal N‐terminal clipping had increased level of O‐linked glycosylation in the linker region, giving credence to the hypothesis that O‐linked glycosylation acts to protect against proteolytic degradation. Moreover, products with reduced potential for N‐terminal clipping had longer in vivo serum half‐life. These findings suggest that early monitoring of product quality should be an essential part of production cell line development and therefore, has been incorporated in our process of cell line development for this class of molecules. Biotechnol. Bioeng. 2009;103: 162–176. © 2008 Wiley Periodicals, Inc.     

Involvement of HAb18G/CD147 in T cell activation and immunological synapse formation

HAb18G/CD147, a glycoprotein of the immunoglobulin super‐family (IgSF), is a T cell activation‐associated molecule. In this report, we demonstrated that HAb18G/CD147 expression on both activated CD4⁺ and CD8⁺ T cells was up‐regulated. In vitro cross‐linking of T cells with an anti‐HAb18G/CD147 monoclonal antibody (mAb) 5A12 inhibited T cells proliferation upon T cell receptor stimulation. Such co‐stimulation inhibited T cell proliferation by down‐regulating the expression of CD25 and interleukin‐2 (IL‐2), decreased production of IL‐4 but not interferon‐γ. Laser confocal imaging analysis indicated that HAb18G/CD147 was recruited to the immunological synapse (IS) during T cell activation; triggering HAb18G/CD147 on activated T cells by anti‐HAb18G/CD147 mAb 5A12 strongly dispersed the formation of the IS. Further functional studies showed that the ligation of HAb18G/CD147 with mAb 5A12 decreased the tyrosine phosphorylation and intracellular calcium mobilization levels of T cells. Through docking antibody–antigen interactions, we demonstrated that the function of mAb 5A12 is tightly dependent on its specificity of binding to N‐terminal domain I, which plays pivotal role in the oligomerization of HAb18G/CD147. Taken together, we provide evidence that HAb18G/CD147 could act as a co‐stimulatory receptor to negatively regulate T cell activation and is functionally linked to the formation of the IS.     

Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

Immobilization of 5‐Fluorouridine on Chitosan

The 2′,3′‐O‐levulinic acid derivative 2b of the cancerostatic 5‐fluorouridine as well as its N(3)‐farnesylated nucleolipid 2d were synthesized and coupled to H₂O‐soluble chitosanes of different molecular weight and at various pH values (3.5–5.5) leading to 6 and 7 . In addition, the coumarine fluorophore ATTO‐488 N(9)‐butanoate was bound to the biopolymer by a sequential‐coupling technique to afford 9 and 10 . Moreover, chitosan foils were prepared, to which 2b was coupled. Their degradation by chitosanase (from Streptomyces sp. N174) was studied UV‐spectrophotometrically in a Franz diffusion cell.     

Stirred tank bioreactor culture combined with serum‐/xenogeneic‐free culture medium enables an efficient expansion of umbilical cord‐derived mesenchymal stem/stromal cells

Mesenchymal stem/stromal cells (MSC) are being widely explored as promising candidates for cell‐based therapies. Among the different human MSC origins exploited, umbilical cord represents an attractive and readily available source of MSC that involves a non‐invasive collection procedure. In order to achieve relevant cell numbers of human MSC for clinical applications, it is crucial to develop scalable culture systems that allow bioprocess control and monitoring, combined with the use of serum/xenogeneic (xeno)‐free culture media. In the present study, we firstly established a spinner flask culture system combining gelatin‐based Cultispher^®S microcarriers and xeno‐free culture medium for the expansion of umbilical cord matrix (UCM)‐derived MSC. This system enabled the production of 2.4 (±1.1) x10⁵ cells/mL (n = 4) after 5 days of culture, corresponding to a 5.3 (±1.6)‐fold increase in cell number. The established protocol was then implemented in a stirred‐tank bioreactor (800 mL working volume) (n = 3) yielding 115 million cells after 4 days. Upon expansion under stirred conditions, cells retained their differentiation ability and immunomodulatory potential. The development of a scalable microcarrier‐based stirred culture system, using xeno‐free culture medium that suits the intrinsic features of UCM‐derived MSC represents an important step towards a GMP compliant large‐scale production platform for these promising cell therapy candidates.     

WIPF2 promotes Shigella flexneri actin‐based motility and cell‐to‐cell spread

Shigella flexneri is an intracellular pathogen that disseminates in colonic epithelial cells through actin‐based motility and formation of membrane protrusions at cell–cell contacts, that project into adjacent cells and resolve into vacuoles, from which the pathogen escapes, thereby achieving cell‐to‐cell spread. Actin nucleation at the bacterial pole relies on the recruitment of the nucleation‐promoting factor N‐WASP, which activates the actin nucleator ARP2/3. In cells, the vast majority of N‐WASP exists as a complex with WIP. The involvement of WIP in N‐WASP‐dependent actin‐based motility of various pathogens, including vaccinia virus and S. flexneri, has been highly controversial. Here, we show that WIPF2 was the only WIP family member expressed in the human colonic epithelial cell line HT‐29, and its depletion impaired S. flexneri dissemination. WIPF2 depletion increased the number of cytosolic bacteria lacking actin tails (non‐motile) and decreased the velocity of motile bacteria. This correlated with a decrease in the recruitment of N‐WASP to the bacterial pole, and among N‐WASP‐positive bacteria, a decrease in actin tail‐positive bacteria, suggesting that WIPF2 is required for N‐WASP recruitment and activation at the bacterial pole. In addition, when motile bacteria formed protrusions, WIPF2 depletion decreased the number of membrane protrusions that successfully resolved into vacuoles.     

Modulations of high‐voltage activated Ca2+ channels in the central neurones of Spodoptera exigua by chlorantraniliprole

The modulation of voltage‐gated calcium channels by chlorantraniliprole in the central neurones isolated from third‐instar larvae of Spodoptera exigua is studied by the whole‐cell patch‐clamp technique. The current of calcium in the third‐instar larvae of S. exigua is identified as a high‐voltage activated Ca²⁺ current. During the 10‐min recording, the current–voltage relationship curves of whole‐cell calcium channels are shifted in a negative direction by 10 mV compared with the control group. The fact that the gravity rundown of calcium current in the treated group is more apparent than in the control group demonstrates that the open channels are constantly inactivated. In addition, chlorantraniliprole inhibits the recorded calcium currents in a concentration‐dependent manner, which is irreversible on washout.     

Ethylene production in Botrytis cinerea‐ and oligogalacturonide‐induced immunity requires calcium‐dependent protein kinases

Plant immunity against pathogens is achieved through rapid activation of defense responses that occur upon sensing of microbe‐ or damage‐associated molecular patterns, respectively referred to as MAMPs and DAMPs. Oligogalacturonides (OGs), linear fragments derived from homogalacturonan hydrolysis by pathogen‐secreted cell wall‐degrading enzymes, and flg22, a 22‐amino acid peptide derived from the bacterial flagellin, represent prototypical DAMPs and MAMPs, respectively. Both types of molecules induce protection against infections. In plants, like in animals, calcium is a second messenger that mediates responses to biotic stresses by activating calcium‐binding proteins. Here we show that simultaneous loss of calcium‐dependent protein kinases CPK5, CPK6 and CPK11 affects Arabidopsis thaliana basal as well as elicitor‐ induced resistance to the necrotroph Botrytis cinerea, by affecting pathogen‐induced ethylene production and accumulation of the ethylene biosynthetic enzymes 1‐aminocyclopropane‐1‐carboxylic acid (ACC) synthase 2 (ACS2) and 6 (ACS6). Moreover, ethylene signaling contributes to OG‐triggered immunity activation, and lack of CPK5, CPK6 and CPK11 affects the duration of OG‐ and flg22‐induced gene expression, indicating that these kinases are shared elements of both DAMP and MAMP signaling pathways.     

MiR‐129‐5p promotes docetaxel resistance in prostate cancer by down‐regulating CAMK2N1 expression

This study focuses on the effect of miR‐129‐5p on docetaxel‐resistant (DR) prostate cancer (PCa) cells invasion, migration and apoptosis. In our study, the expression of CAMK2N1 was assessed by qRT‐PCR in PCa patient tissues and cell lines including PC‐3 and PC‐3‐DR. Cells transfected with miR‐129‐5p mimics, inhibitor, CAMK2N1 or negative controls (NC) were used to interrogate their effects on DR cell invasions, migrations and apoptosis during docetaxel (DTX) treatments. The apoptosis rate of the PCa cells was validated by flow cytometry. Relationships between miR‐129‐5p and CAMK2N1 levels were identified by qRT‐PCR and dual‐luciferase reporter assay. CAMK2N1 was found to be down‐expressed in DR PCa tissue sample, and low levels of CAMK2N1 were correlated with high docetaxel resistance and clinical prediction of poor survival. CAMK2N1 levels were decreased in DR PCa cells treated with DXT. We further explored that up‐regulation of miR‐129‐5p could promote DR PCa cells viability, invasion and migration but demote apoptosis. Involved molecular mechanism studies revealed that miR‐129‐5p reduced downstream CAMK2N1 expression to further impact on chemoresistance to docetaxel of PCa cells, indicating its vital role in PCa docetaxel resistance. Our findings revealed that miR‐129‐5p contributed to the resistance of PC‐3‐DR cells to docetaxel through suppressing CAMK2N1 expression, and thus targeting miR‐129‐5p may provide a novel therapeutic approach in sensitizing PCa to future docetaxel treatment.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

Proteomic analysis provides new insights into the adaptive response of a dinoflagellate Prorocentrum donghaiense to changing ambient nitrogen

Nitrogen (N) is the major nutrient limiting phytoplankton growth and productivity over large ocean areas. Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the ocean. However, very little is known about their adaptive response to changing ambient N. Here, we compared the protein profiles of a marine dinoflagellate Prorocentrum donghaiense grown in inorganic N‐replete, N‐deplete and N‐resupplied conditions using 2‐D fluorescence differential gel electrophoresis. The results showed that cell density, chlorophyll a and particulate organic N contents presented low levels in N‐deplete cells, while particulate organic carbon content and glutamine synthetase (GS) activity maintained high levels. Comparison of the protein profiles of N‐replete, N‐deplete and N‐resupplied cells indicated that proteins involved in photosynthesis, carbon fixation, protein and lipid synthesis were down‐regulated, while proteins participating in N reallocation and transport activity were up‐regulated in N‐deplete cells. High expressions of GS and 60 kDa chaperonin as well as high GS activity in N‐deplete cells indicated their central role in N stress adaptation. Overall, in contrast with other photosynthetic eukaryotic algae, P. donghaiense possessed a specific ability to regulate intracellular carbon and N metabolism in response to extreme ambient N deficiency.     

Induction of systemic disease resistance in Nicotiana benthamiana by the cyclodipeptides cyclo (l‐Pro‐l‐Pro) and cyclo (d‐Pro‐d‐Pro)

Cyclodipeptides, formed from two amino acids by cyclodehydration, are produced naturally by many organisms, and are known to possess a large number of biological activities. In this study, we found that cyclo (l ‐Pro‐l ‐Pro) and cyclo (d ‐Pro‐d ‐Pro) (where Pro is proline) could induce defence responses and systemic resistance in Nicotiana benthamiana. Treatment with the two cyclodipeptides led to a reduction in disease severity by Phytophthora nicotianae and Tobacco mosaic virus (TMV) infections compared with controls. Both cyclopeptides triggered stomatal closure, induced reactive oxygen species production and stimulated cytosolic calcium ion and nitric oxide production in guard cells. In addition, the application of cyclodipeptides significantly up‐regulated the expression of the plant defence gene PR‐1a and the PR‐1a protein, and increased cellular salicylic acid (SA) levels. These results suggest that the SA‐dependent defence pathway is involved in cyclodipeptide‐mediated pathogen resistance in N. benthamiana. We report the systemic resistance induced by cyclodipeptides, which sheds light on the potential of cyclodipeptides for the control of plant diseases.     

Cell death triggering and effector recognition by Sw‐5 SD‐CNL proteins from resistant and susceptible tomato isolines to Tomato spotted wilt virus

Only a limited number of dominant resistance genes acting against plant viruses have been cloned, and further functional studies of these have been almost entirely limited to the resistance genes Rx against Potato virus X (PVX) and N against Tobacco mosaic virus (TMV). Recently, the cell‐to‐cell movement protein (NS_M) of Tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant (Avr) of Sw‐5b‐mediated resistance, a dominant resistance gene which belongs to the class of SD‐CC‐NB‐LRR (Solanaceae domain‐coiled coil‐nucleotide‐binding‐leucine‐rich repeat, SD‐CNL) resistance genes. On transient expression of the NS_M protein in tomato and transgenic Nicotiana benthamiana harbouring the Sw‐5b gene, a hypersensitive cell death response (HR) is triggered. Here, it is shown that high accumulation of the Sw‐5b protein in N. benthamiana leaves, achieved by co‐expression of the Sw‐5b protein with RNA silencing suppressors (RSSs), leads to auto‐activity in the absence of NS_M. In a similar approach, Sw‐5a, the highest conserved paralogue of Sw‐5b from Solanum peruvianum, also triggered HR by auto‐activation, whereas the highest conserved orthologue from susceptible S. lycopersicum, named Sw‐5a^S, did not. However, neither of the last two homologues was able to trigger an NS_M‐dependent HR. Truncated and mutated versions of these Sw‐5 proteins revealed that the NB‐ARC [nucleotide‐binding adaptor shared by Apaf‐1 (from humans), R proteins and CED‐4 (from nematodes)] domain is sufficient for the triggering of HR and seems to be suppressed by the SD‐CC domain. Furthermore, a single mutation was sufficient to restore auto‐activity within the NB‐ARC domain of Sw‐5a^S. When the latter domain was fused to the Sw‐5b LRR domain, NS_M‐dependent HR triggering was regained, but not in the presence of its own Sw‐5a^S LRR domain. Expression analysis in planta revealed a nucleocytoplasmic localization pattern of Sw‐5b, in which the SD‐CC domain seems to be required for nuclear translocation. Although the Sw‐5 N‐terminal CC domain, in contrast with Rx, contains an additional SD, most findings from this study support a conserved role of domains within NB‐LRR (NLR) proteins against plant viruses.     

Functional analysis of the seven in absentia ubiquitin ligase family in tomato

Seven in absentia (SINA) protein is one subgroup of ubiquitin ligases possessing an N‐terminal cysteine‐rich really interesting new gene (RING) domain, two zinc‐finger motifs, and a C‐terminal domain responsible for substrate‐binding and dimerization. In tomato (Solanum lycopersicum), the SINA gene family has six members, and we characterize in this study all tomato SINA (SlSINA) genes and the gene products. Our results show that SlSINA genes are differentially regulated in leaf, bud, stem, flower, and root. All SlSINA proteins possess RING‐dependent E3 ubiquitin ligase activity, exhibiting similar specificity towards the E2 ubiquitin‐conjugating enzyme. SlSINA1/3/4/5/6 are localized in both cytoplasm and nucleus, whereas SlSINA2 is exclusively localized in the nucleus. Moreover, all SlSINAs can interact with each other for homo‐ or hetero‐dimerization. The functionality of SlSINA proteins has been investigated. SlSINA4 plays a positive role in defense signalling, as manifested by elicitation of E3‐dependent hypersensitive response‐like cell death; the other SlSINAs are negative regulator and capable to suppress hypersensitive response cell death. Transgenic tomato plants overexpressing SlSINA2 exhibit pale‐green leaf phenotype, suggesting SlSINA2 regulates chlorophyll level in plant cells, whereas transgenic tomato plants overexpressing SlSINA5 have altered floral structure with exserted stigma, implicating SlSINA5 plays a role in flower development.     

N‐cadherin is dispensable for pancreas development but required for β‐cell granule turnover

The cadherin family of cell adhesion molecules mediates adhesive interactions that are required for the formation and maintenance of tissues. Previously, we demonstrated that N‐cadherin, which is required for numerous morphogenetic processes, is expressed in the pancreatic epithelium at E9.5, but later becomes restricted to endocrine aggregates in mice. To study the role of N‐cadherin during pancreas formation and function we generated a tissue‐specific knockout of N‐cadherin in the early pancreatic epithelium by inter‐crossing N‐cadherin‐floxed mice with Pdx1Cre mice. Analysis of pancreas‐specific ablation of N‐cadherin demonstrates that N‐cadherin is dispensable for pancreatic development, but required for β‐cell granule turnover. The number of insulin secretory granules is significantly reduced in N‐cadherin‐deficient β‐cells, and as a consequence insulin secretion is decreased. genesis 48:374–381, 2010. © 2010 Wiley‐Liss, Inc.