Involvement of connexin 43 in meiotic maturation of bovine oocytes.

In ovarian follicles, cumulus cells provide the oocyte with small molecules that permit growth and control maturation. These nutrients reach the germinal cell through gap junction channels, which are present between the cumulus cells and the oocyte, and between the cumulus cells. In this study the involvement of intercellular communication mediated by gap junction channels on oocyte maturation of in vitro cultured bovine cumulus-oocyte complexes (COCs) was investigated. The stages of oocyte maturation were determined by Hoechst 33342 staining, which showed that 90% of COCs placed in the maturation medium for 24 h progress to the metaphase II stage. Bovine COC gap junction communication was disrupted initially using n-alkanols, which inhibit any passage through gap junctions. In the presence of 1-heptanol (3 mmol l(-1)) or octanol (3.0 mmol l(-1) and 0.3 mmol l(-1)), only 29% of the COCs reached metaphase II. Removal of the uncoupling agent was associated with restoration of oocyte maturation, indicating that treatment with n-alkanols was neither cytotoxic nor irreversible. Concentrations of connexin 43 (Cx43), the major gap junction protein expressed in the COCs, were decreased specifically using a recombinant adenovirus expressing the antisense Cx43 cDNA (Ad-asCx43). The efficacy of adenoviral infection was > 95% in cumulus cells evaluated after infection with recombinant adenoviruses expressing the green fluorescence protein. RT-PCR performed on total RNA isolated from Ad-asCx43-infected COCs showed that the rat Cx43 cDNA was transcribed. Western blot analysis revealed a three-fold decrease in Cx43 expression in COCs expressing the antisense RNA for Cx43. Injection of cumulus cells with Lucifer yellow demonstrated further that the resulting lower amount of Cx43 in infected COCs is associated with a two-fold decrease in the extent of coupling between cumulus cells. In addition, oocyte maturation was decreased by 50% in the infected COC cultures. These results indicate that Cx43-mediated communication between cumulus cells plays a crucial role in maturation of bovine oocytes.

Pancreatic-specific expression of the glucose transporter type 2 gene: identification of cis-elements and islet-specific trans-acting factors.

A defect in glucose sensing of the pancreatic beta-cells has been observed in several animal models of type II diabetes and has been correlated with a reduced gene expression of the glucose transporter type 2 (Glut2). In a transgenic mouse model, expression of Glut2 antisense RNA in pancreatic beta-cells has recently been shown to be associated with an impaired glucose-induced insulin secretion and the development of diabetes. To identify factors that may be involved in the specific decrease of Glut2 in the beta-cells of the diabetic animal, an attempt was made to localize the cis-elements and trans-acting factors involved in the control of Glut2 expression in the endocrine pancreas. It was demonstrated by transient transfection studies that only 338 base pairs (bp) of the murine Glut2 proximal promoter are needed for reporter gene expression in pancreatic islet-derived cell lines, whereas no activity was detected in nonpancreatic cells. Three cis-elements, GTI, GTII, and GTIII, have been identified by DNAse I footprinting and gel retardation experiments within these 338 bp. GTI and GTIII bind distinct but ubiquitously expressed trans-acting factors. On the other hand, nuclear proteins specifically expressed in pancreatic cell lines interact with GTII, and their relative abundance correlates with endogenous Glut2 expression. These GTII-binding factors correspond to nuclear proteins of 180 and 90 kilodaltons as defined by Southwestern analysis. The 180-kilodalton factor is present in pancreatic beta-cell lines but not in an alpha-cell line. Mutation of the GTI or GTIII cis-elements decreases transcriptional activity directed by the 338-bp promoter, whereas mutation of GTII increases gene transcription. Thus negative and positive regulatory sequences are identified within the proximal 338 bp of the GLUT2 promoter and may participate in the islet-specific expression of the gene by binding beta-cell specific trans-acting factors.

The scaffold protein IB1/JIP-1 controls the activation of JNK in rat stressed urothelium.

The c-Jun N-terminal kinase (JNK) is critical for cell survival, differentiation, apoptosis and tumorigenesis. This signalling pathway requires the presence of the scaffold protein Islet-Brain1/c-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1). Immunolabeling and in situ hybridisation of bladder sections showed that IB1/JIP-1 is expressed in urothelial cells. The functional role of IB1/JIP-1 in the urothelium was therefore studied in vivo in a model of complete rat bladder outlet obstruction. This parietal stress, which is due to urine retention, reduced the content of IB1/JIP-1 in urothelial cells and consequently induced a drastic increase in JNK activity and AP-1 binding activity. Using a viral gene transfer approach, the stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1. Conversely, the JNK activity was increased in urothelial cells where the IB1/JIP-1 content was experimentally reduced using an antisense RNA strategy. Furthermore, JNK activation was found to be increased in non-stressed urothelial cells of heterozygous mice carrying a selective disruption of the IB1/JIP-1 gene. These data established that mechanical stress in urothelial cells in vivo induces a robust JNK activation as a consequence of regulated expression of the scaffold protein IB1/JIP-1. This result highlights a critical role for that scaffold protein in the homeostasis of the urothelium and unravels a new potential target to regulate the JNK pathway in this tissue.

Bradykinin, but not muscarinic, inhibition of M-current in rat sympathetic ganglion neurons involves phospholipase C-β4

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I-K(M)), which can be inhibited by activation of M-1 muscarinic receptors (M-1 mAChR) and bradykinin (BK) B-2 receptors. Inhibition by the M1 mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein G alpha (q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves G alpha (q) and/or G alpha (11) (Jones et al., 1995). G alpha (q) and G alpha (11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I-K(M) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, - beta2, - beta3, and - beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I-K(M) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M-1 mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M-1 mAChR, inhibition of I-K(M) involves PLC and extends this finding by indicating that PLC-beta4 is involved.

Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery.

The purpose of this study was to design microspheres combining sustained delivery and enhanced intracellular penetration for ocular administration of antisense oligonucleotides. Nanosized complexes of antisense TGF-beta2 phosphorothioate oligonucleotides (PS-ODN) with polyethylenimine (PEI), and naked PS-ODN were encapsulated into poly(lactide-co-glycolide) microspheres prepared by the double-emulsion solvent evaporation method. The PS-ODN was introduced either naked or complexed in the inner aqueous phase of the first emulsion. We observed a marked influence of microsphere composition on porosity, size distribution and PS-ODN encapsulation efficiency. Mainly, the presence of PEI induced the formation of large pores observed onto microsphere surface. Introduction of NaCl in the outer aqueous phase increased the encapsulation efficiency and reduced microsphere porosity. In vitro release kinetic of PS-ODN was also investigated. Clearly, the higher the porosity, the faster was the release and the higher was the burst effect. Using an analytical solution of Fick's second law of diffusion, it was shown that the early phase of PS-ODN and PS-ODN-PEI complex release was primarily controlled by pure diffusion, irrespectively of the type of microsphere. Finally, microspheres containing antisense TGF-beta2 nanosized complexes were shown, after subconjunctival administration to rabbit, to significantly increase intracellular penetration of ODN in conjunctival cells and subsequently to improve bleb survival in a rabbit experimental model of filtering surgery. These results open up interesting prospective for the local controlled delivery of genetic material into the eye.

SV40-induced expression of calretinin protects mesothelial cells from asbestos cytotoxicity and may be a key factor contributing to mesothelioma pathogenesis.

The calcium-binding protein calretinin has emerged as a useful marker for the identification of mesotheliomas of the epithelioid and mixed types, but its putative role in tumor development has not been addressed previously. Although exposure to asbestos fibers is considered the main cause of mesothelioma, undoubtedly, not all mesothelioma patients have a history of asbestos exposure. The question as to whether the SV40 virus is involved as a possible co-factor is still highly debated. Here we show that increased expression of SV40 early gene products in the mesothelial cell line MeT-5A induces the expression of calretinin and that elevated calretinin levels strongly correlate with increased resistance to asbestos cytotoxicity. Calretinin alone mediates a significant part of this protective effect because cells stably transfected with calretinin cDNA were clearly more resistant to the toxic effects of crocidolite than mock-transfected control cells. Down-regulation of calretinin by antisense methods restored the sensitivity to asbestos toxicity to a large degree. The protective effect observed in clones with higher calretinin expression levels could be eliminated by phosphatidylinositol 3-kinase (PI3K) inhibitors, implying an important role for the PI3K/AKT signaling (survival) pathway in mediating the protective effect. Up-regulation of calretinin, resulting from either asbestos exposure or SV40 oncoproteins, may be a common denominator that leads to increased resistance to asbestos cytotoxicity and thereby contributes to mesothelioma carcinogenesis.

Isolation from mouse fibroblasts of a cDNA encoding a new form of the fibroblast growth factor receptor (flg).

Structural definition of the receptors for neurotropic and angiogenic modulators such as fibroblast growth factors and related polypeptides will yield insight into the mechanisms that control early development, embryogenesis, organogenesis, wound repair and neovessel formation. We isolated 3 murine cDNAs encoding different binding domains of these receptors (flg). Comparison of these ectoplasmic portions showed that two of the forms corresponded to previously described murine molecules whereas the third one had a different ectoplasmic portion generated by specific changes in two regions. Interestingly, expression of this third form seems to be restricted in its tissue distribution. Such modifications could influence the ligand specificity of the different receptors and/or their binding affinity.

Downregulation of IRS-1 expression causes inhibition of corneal angiogenesis.

PURPOSE: The antiangiogenic effect of an antisense oligodeoxynucleotide (ODN) targeting insulin receptor substrate (IRS)-1 was evaluated on rat corneal neovascularization. METHODS: Eyes with neovessels were treated with subconjunctival injections of IRS-1 antisense oligonucleotide (ASODN), IRS-1 sense ODN (SODN), or PBS. At 8 and 24 hours after the first subconjunctival injection, the expression of IRS-1, VEGF, and IL-1beta mRNA was evaluated. IRS-1 protein levels were also measured at 8 hours by Western blot analysis (n = 4/group). On day 10, corneal neovascularization was quantified in flatmount corneas of rats treated daily from days 4 to 9. RESULTS: On day 10, new vessels covered 95.5% +/- 4% of the corneal area in PBS-treated eyes, 92% +/- 7% in SODN-treated eyes and 59% +/- 20% in ASODN-treated eyes (P < 0.001). In the ASODN-treated group, the expression and synthesis of IRS-1 were significantly downregulated when compared with the control groups. ASODN did not significantly affect the expression of VEGF but significantly decreased the expression of IL-1beta at 24 hours (P = 0.04). CONCLUSIONS: Subconjunctival injections of IRS-1 antisense ODN significantly inhibit rat corneal neovascularization. This effect may be mediated by a downregulation of IL-1beta. IRS-1 proteins may be interesting targets for the regulation of angiogenesis mediated by insulin, hypoxia, or inflammation.

Identification and characterization of the members of the pho1 gene family in moss and rice

Inorganic phosphate (Pi) is one of the main nutrients limiting plant growth anddevelopment in many agro-ecosystems. In plants, phosphate is acquired from the soil by theroots, and is then transferred to the shoot via the xylem. In the model plant Arabidopsisthaliana, PHO1 was previously identified as being involved in loading Pi into the xylem ofroots. AtPHO1, belongs to a multigenic family composed of 10 additional members, namelyAtPHO1;H1 to AtPHO1;10. In this study, we aimed at further investigating the role of thePHO1 gene family in Pi homeostasis in plants, and to this end we isolated and characterizedthe PHO1 members of two main model plants, the moss Physcomitrella patens and the riceOryza sativa.In the bryophyte P. patens, bioinformatic analyses revealed the presence of seven AtPHO1homologues, highly similar to AtPHO1. The seven moss PHO1 genes, namely PpPHO1;1 toPpPHO1;7 appeared to be differentially regulated, both at the tissue level and in response toPi status. However only PpPHO1;1 and PpPHO1;7 were specifically up-regulated upon Pistarvation, suggesting a potential role in Pi homeostasis. We also characterized the responseof P. patens to Pi starvation, showing that higher and lower plants share some commonstrategies to adapt to Pi-deficiency.In the second part, focusing on the monocotyledon rice, we showed the existence of threePHO1 homologues OsPHO1;1 to OsPHO1;3, with the unique particularity of each havingNatural Antisense Transcripts (NATs). Molecular analyses revealed that both the sense andthe antisense OsPHO1;2 transcripts were by far the most abundantly expressed transcripts ofthe family, preferentially expressed in the roots. The stable expression of OsPHO1;2 in allconditions tested, in opposition with the highly induced antisense transcript upon Pistarvation, suggest a putative role for the antisense in regulating the sense transcript.Moreover, mutant analyses revealed that OsPHO1;2 plays a key role in Pi homeostasis, intransferring Pi from the root to the shoot. Finally, complementing the pho1 mutant inArabidopsis, characterized by low Pi in the shoot and reduced growth, with the riceOsPHO1;2 gene revealed a new role for PHO1 in Pi signaling. Indeed, the complementedplants showed normal growth, with however low Pi content.

Variations in IB1/JIP1 expression regulate susceptibility of beta-cells to cytokine-induced apoptosis irrespective of C-Jun NH2-terminal kinase signaling.

We previously reported that interleukin-1beta (IL-1beta) alone does not cause apoptosis of beta-cells, whereas when combined with gamma-interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), it exerts a distinct apoptotic effect. Studies in beta-cell lines indicated that IL-1beta reduced expression of islet brain (IB)-1/JNK interacting protein (JIP)-1, a JNK scaffold protein with antiapoptotic action. We examined whether variations in IB1/JIP-1 expression in purified primary beta-cells affect their susceptibility to cytokine-induced apoptosis. Exposure to IL-1beta for 24 h decreased cellular IB1/JIP-1 content by 66 +/- 17%; this IL-1beta effect was maintained in the presence of TNF-alpha + IFN-gamma, which did not influence IB1/JIP-1 levels by themselves. Addition of IL-1beta to TNF-alpha + IFN-gamma increased apoptosis from 20 +/- 2% to 59 +/- 5%. A similar increase in TNF-alpha + IFN-gamma-induced apoptosis was produced by adenoviral expression of antisense IB1/JIP-1 and was not further enhanced by addition of IL-1beta, indicating that IL-1beta-mediated suppression of IB1/JIP-1 in beta-cells increases their susceptibility to cytokine-induced apoptosis. However, adenovirally mediated overexpression of IB1/JIP-1 also potentiated TNF-alpha + IFN-gamma-induced apoptosis, suggesting that the antiapoptotic effect of IB1/JIP-1 depends on well-defined cellular levels. We conclude that the IB1/JIP-1 level in beta-cells can control their susceptibility to apoptosis independent of JNK signaling.

Electrically assisted ocular gene therapy.

Electrotransfer and iontophoresis are being developed as innovative non-viral gene delivery systems for the treatment of eye diseases. These two techniques rely on the use of electric current to allow for higher transfection yield of various ocular cell types in vivo. Short pulses of relatively high-intensity electric fields are used for electrotransfer delivery, whereas the iontophoresis technique is based on the application of low voltage electric current. The basic principles of these techniques and their potential therapeutic application for diseases of the anterior and posterior segments of the eye are reviewed. Iontophoresis has been found most efficient for the delivery of small nucleic acid fragments such as antisense oligonucleotides, siRNA, or ribozymes. Electrotransfer, on the other hand, is being developed for the delivery of oligonucleotides or custom designed plasmids. The wide range of strategies already validated and the potential for targeting specific types of cells confirm the promising early observations made using electrotransfer and iontophoresis. These two nonviral delivery systems are safe and can be used efficiently for targeted gene delivery to ocular tissues in vivo. At the present, their application for the treatment of ocular human diseases is nearing its final stages of adaptation and practical implementation at the bedside.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

New generation vaccine induces effective melanoma-specific CD8+ T cells in the circulation but not in the tumor site.

Although increasing evidence suggests that CTL are important to fight the development of some cancers, the frequency of detectable tumor-specific T cells is low in cancer patients, and these cells have generally poor functional capacities, compared with virus-specific CD8(+) T cells. The generation with a vaccine of potent CTL responses against tumor Ags therefore remains a major challenge. In the present study, ex vivo analyses of Melan-A-specific CD8(+) T cells following vaccination with Melan-A peptide and CpG oligodeoxynucleotides revealed the successful induction in the circulation of effective melanoma-specific T cells, i.e., with phenotypic and functional characteristics similar to those of CTL specific for immunodominant viral Ags. Nonetheless, the eventual impact on tumor development in vaccinated melanoma donors remained limited. The comprehensive study of vaccinated patient metastasis shows that vaccine-driven tumor-infiltrating lymphocytes, although activated, still differed in functional capacities compared with blood counterparts. This coincided with a significant increase of FoxP3(+) regulatory T cell activity within the tumor. The consistent induction of effective tumor-specific CD8(+) T cells in the circulation with a vaccine represents a major achievement; however, clinical benefit may not be achieved unless the tumor environment can be altered to enable CD8(+) T cell efficacy.

State of genomics and epigenomics research in the perspective of HIV cure.

PURPOSE OF REVIEW: One of the seven key scientific priorities identified in the road map on HIV cure research is to 'determine the host mechanisms that control HIV replication in the absence of therapy'. This review summarizes the recent work in genomics and in epigenetic control of viral replication that is relevant for this mission. RECENT FINDINGS: New technologies allow the joint analysis of host and viral transcripts. They identify the patterns of antisense transcription of the viral genome and its role in gene regulation. High-throughput studies facilitate the assessment of integration at the genome scale. Integration site, orientation and host genomic context modulate the transcription and should also be assessed at the level of single cells. The various models of latency in primary cells can be followed using dynamic study designs to acquire transcriptome and proteome data of the process of entry, maintenance and reactivation of latency. Dynamic studies can be applied to the study of transcription factors and chromatin modifications in latency and upon reactivation. SUMMARY: The convergence of primary cell models of latency, new high-throughput quantitative technologies applied to the study of time series and the identification of compounds that reactivate viral transcription bring unprecedented precision to the study of viral latency.

Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3 expression.

Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3' untranslated region (3' UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3' UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.