Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death

The homeostatic control of beta-cell mass in normal and pathological conditions is based on the balance of proliferation, differentiation, and death of the insulin-secreting cells. A considerable body of evidence, accumulated during the last decade, has emphasized the significance of the disregulation of the mechanisms regulating the apoptosis of beta-cells in the sequence of events that lead to the development of diabetes. The identification of agents capable of interfering with this process needs to be based on a better understanding of the beta-cell specific pathways that are activated during apoptosis. The aim of this article is fivefold: (1) a review of the evidence for beta-cell apoptosis in Type I diabetes, Type II diabetes, and islet transplantation, (2) to review the common stimuli and their mechanisms in pancreatic beta-cell apoptosis, (3) to review the role of caspases and their activation pathway in beta-cell apoptosis, (4) to review the caspase cascade and morphological cellular changes in apoptotic beta-cells, and (5) to highlight the putative strategies for preventing pancreatic beta-cells from apoptosis.     

Sarcoptic mange in wild carnivores and its co-occurrence with parasitic helminths in the Western Italian Alps

Between 2001 and 2004, 229 foxes, 36 stone martens and 48 badgers from the western Italian Alps were examined for sarcoptic mange and for gastrointestinal helminths to investigate their prevalence and geographical distribution and to point out the existence of potential interactions among them. Sarcoptic mange was observed in 25.3±2.8% SE of foxes and in 5.6±3.8% SE of stone martens, while no badger was found infected. Helminths belonged to Cestoidea Cyclophillidea (3.0±1.1% SE), Nematoda Trichurida (Capillaria aerophila and Trichuris vulpis: 6.5±1.6% SE; Trichinella britovi: 3.0±1.1% SE), Ascaridida (12.2±2.2% SE) and Strongylida (6.9±1.7% SE). Sarcoptic mange infection and the presence of helminths proved to be associated, with mangy foxes showing significantly higher prevalence of both cestode and nematode (particularly Ascaridida) worms. Moreover, considering three clusters of parasites (S. scabiei, nematodes and cestodes), more foxes than expected hosted simultaneously 2 and 3 taxa. These evidences suggest the existence of some kind of interaction, whose modalities and implications are discussed in this paper.     

Viral Entry Inhibitors Targeted to the Membrane Site of Action

The fusion of enveloped viruses with the host cell is driven by specialized fusion proteins to initiate infection. The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains, which are central to the complex conformational changes leading to fusion: the first heptad repeat (HRN) is adjacent to the fusion peptide, while the second (HRC) immediately precedes the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one HR peptide, T20 (enfuvirtide), is in clinical use for HIV-1. For paramyxoviruses, the activities of two membrane proteins, the receptor-binding protein (hemagglutinin-neuraminidase [HN] or G) and the fusion protein (F), initiate viral entry. The binding of HN or G to its receptor on a target cell triggers the activation of F, which then inserts into the target cell and mediates the membrane fusion that initiates infection. We have shown that for paramyxoviruses, the inhibitory efficacy of HR peptides is inversely proportional to the rate of F activation. For HIV-1, the antiviral potency of an HRC-derived peptide can be dramatically increased by targeting it to the membrane microdomains where fusion occurs, via the addition of a cholesterol group. We report here that for three paramyxoviruses—human parainfluenza virus type 3 (HPIV3), a major cause of lower respiratory tract diseases in infants, and the emerging zoonotic viruses Hendra virus (HeV) and Nipah virus (NiV), which cause lethal central nervous system diseases—the addition of cholesterol to a paramyxovirus HRC-derived peptide increased antiviral potency by 2 log units. Our data suggest that this enhanced activity is indeed the result of the targeting of the peptide to the plasma membrane, where fusion occurs. The cholesterol-tagged peptides on the cell surface create a protective antiviral shield, target the F protein directly at its site of action, and expand the potential utility of inhibitory peptides for paramyxoviruses.Fusion of enveloped viruses with the host cell is a key step in viral infectivity, and interference with this process can lead to highly effective antivirals. Viral fusion is driven by specialized proteins that undergo an ordered series of conformational changes. These changes facilitate the initial, close apposition of the viral and host membranes, and they ultimately result in the formation of a fusion pore (reviewed in reference 12). The “class I” fusion proteins harbor two regions, typically two heptad repeat (HR) domains: the first one (HRN) adjacent to the fusion peptide and the second one (HRC) immediately preceding the transmembrane domain. Peptides derived from the HR regions can inhibit fusion, and one of them, T20 (enfuvirtide), is in clinical use for HIV-1 (19). Peptides derived from the HRN and HRC regions of paramyxovirus fusion (F) proteins can interact with fusion intermediates of F (3, 20, 22, 37, 46, 49) and provide a promising antiviral strategy.The current model for class I-driven fusion postulates the existence of a so-called prehairpin intermediate, a high-energy structure that bridges the viral and cell membranes, where the HRN and the HRC are separated. The prehairpin intermediate spontaneously collapses into the postfusion structure—a six-helical bundle (6HB), with an inner trimeric coiled-coil formed by the HRN onto which the HRC folds (12, 14, 30, 40). The key to these events is the initial activation step, whereby HN triggers F to initiate the process. Structural and biophysical analyses of the paramyxovirus 6HB (30, 50, 51) suggest that inhibitors bind to the prehairpin intermediate and prevent its transition to the 6HB, thus inhibiting viral entry. The peptides bind to their complementary HR region and thereby prevent HRN and HRC from refolding into the stable 6HB structure required for fusion (3, 10, 40). The efficiency of F triggering by HN critically influences the degree of fusion mediated by F and thus the extent of viral entry (35). In addition, differences in the efficiency of triggering of the fusion process impact the efficacy of potential antiviral molecules that target intermediate states of the fusion protein (36).Paramyxoviruses cause important human illnesses, significantly contributing to global disease and mortality, ranging from lower-respiratory-tract diseases in infants caused by human parainfluenza virus types 1, 2, and 3 (HPIV1, -2, and -3) (9, 48), to highly lethal central nervous system diseases caused by the emerging paramyxoviruses HeV and NiV. No antiviral therapies or vaccines yet exist for these paramyxoviruses, and vaccines would be unlikely to protect the youngest infants. Antiviral agents, therefore, would be particularly beneficial. All paramyxoviruses possess two envelope glycoproteins directly involved in viral entry and pathogenesis: a fusion protein (F) and a receptor-binding protein (HN, H, or G). The paramyxovirus F proteins belong to the group of “class I” fusion proteins (44, 45), which also include the influenza virus hemagglutinin protein and the HIV-1 fusion protein gp120. The F protein is synthesized as a precursor protein (F0) that is proteolytically processed posttranslationally to form a trimer of disulfide-linked heterodimers (F1-F2). This cleavage event places the fusion peptide at the F1 terminus in the mature F protein and is essential for membrane fusion activity. The exact triggers that initiate a series of conformational changes in F leading to membrane fusion differ depending on the pathway the virus uses to enter the cell. In the case of HPIV, HeV, and NiV, the receptor-binding protein, hemagglutinin-neuraminidase (HN) (in HPIV3) or G (in HeV and NiV), binds to cellular surface receptors, brings the viral envelope into proximity with the plasma membrane, and activates the viral F protein. This receptor-ligand interaction is required for the F protein to mediate the fusion of the viral envelope with the host cell membrane (23, 33, 35).The HRC peptide regions of a number of paramyxoviruses, including Sendai virus, measles virus, Newcastle disease virus (NDV), respiratory syncytial virus (RSV), simian virus 5 (SV5), Hendra virus (HeV), and Nipah virus (NiV), can inhibit the infectivity of the homologous virus (17, 20, 31, 37, 47, 49, 52, 53). Recently, we showed that peptides derived from the HRC region of the F protein of HPIV3 are effective inhibitors of both HPIV and HeV/NiV fusion (31) and that, for HeV, the strength of HRC peptide binding to the corresponding HRN region correlates with the potency of fusion and infection inhibition (30). However, peptides derived from the HPIV3 F protein HRC region are more effective at inhibiting HeV/NiV fusion than HPIV3 fusion, despite a stronger homotypic HRN-HRC interaction for HPIV3 (30, 31). We showed (36) that the kinetics of fusion (kinetics of F activation) impacts sensitivity to inhibition by peptides, as is the case for HIV (39). Alterations in HPIV3 HN′s property of F activation affect the kinetics of F''s progression through its conformational changes, thus altering inhibitor efficacy. Once the extended intermediate stage of F has passed, and fusion proceeds, peptide inhibitors are ineffective. We have proposed that the design of effective inhibitors may require either targeting an earlier stage of F activation or increasing the concentration of inhibitor at the location of receptor binding, in order to enhance the access and association of the inhibitor with the intermediate-stage fusion protein (36).A substantial body of evidence supports the notion that viral fusion occurs in confined areas of the interacting viral and host membranes (26). For HIV-1, the lipid composition of the viral membrane is strikingly different from that of the host cell membrane; the former is particularly enriched in cholesterol and sphingomyelin (4, 5, 7, 8). Cholesterol and sphingolipids are often laterally segregated in membrane microdomains or “lipid rafts” (7, 11). In fact, the antiviral potency of the HIV-inhibitory HRC peptide C34 is dramatically increased by targeting it to the “lipid rafts” via the addition of a cholesterol group (16).We applied the targeting strategy based on cholesterol derivatization to paramyxoviruses, and we show here that by adding a cholesterol tag to HPIV3-derived HRC E459V (30) inhibitory peptides, we increased antiviral potency by 2 log units (50% inhibitory concentrations [IC₅₀], <2 nM). We chose to use the HPIV3-derived peptides for HeV/NiV, because we have previously shown that they are far more effective inhibitors of HeV and NiV than the homotypic peptides (30, 31). We propose that the enhanced activity resulting from the addition of a cholesterol tag is a result of the targeting of the peptide to the plasma membrane, where fusion occurs.     

Stringent requirement for HRD1, SEL1L,and OS-9/XTP3-B for disposal of ERAD-LS substrates

Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L_S substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L_M substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.     

Increased sensitivity to the platelet-derived growth factor (PDGF) receptor inhibitor STI571 in chemoresistant glioma cells is associated with enhanced PDGF-BB-mediated signaling and STI571-induced Akt inactivation

The platelet-derived growth factor receptor (PDGFR) is a tyrosine kinase, implicated in the development and progression of different tumors, including gliomas. Chemoresistance is a common feature of malignant gliomas. Since receptor tyrosine kinases contribute to chemoresistance in tumors, we addressed whether PDGFR signaling might confer selective growth advantage to chemoresistant cells. The effects of the PDGFR inhibitor STI571 on proliferation and PDGFR signaling were compared in chemosensitive and cisplatin-selected, chemoresistant sublines derived from glioma and from two other PDGFR-expressing tumors (ovarian carcinoma and neuroblastoma). The chemoresistant glioma U87/Pt cells were twofold more sensitive to STI571 growth-inhibitory effects than the chemosensitive U87 cells, and two- to threefold more sensitive than five unrelated glioma cell lines. The other two paired cell lines were equally responsive. Sensitization of U87/Pt cells correlated with upregulation of the PDGF-B isoform and with PDGF-BB-induced Akt overactivation, which was prevented by STI571. STI571 specifically inhibited PDGF-BB-, but not PDGF-AA- or stem cell factor-mediated signaling. In serum-containing medium, STI571 decreased phospho-Akt in U87/Pt cells, but not in U87, while activating extracellular signal-regulated kinase (Erk) in both. STI571 antiproliferative effects were partially reverted by constitutively active Akt. Cotreatment with inhibitors of phosphatidylinositol 3'-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) resulted in enhanced growth inhibition in glioma cells. Our results suggest that increased PDGF-BB signaling may sensitize chemoresistant glioma cells to STI571, suggesting a therapeutic potential for STI571 in patients with malignant gliomas refractory to chemotherapy. Simultaneous blockade of PDGFR and PI3K or Erk pathway may enhance therapeutic targeting in gliomas.     

Immunocytochemical study of amelogenin deposition during the early odontogenesis of molars in alendronate-treated newborn rats.

Newborn rats were treated with sodium alendronate to study how enamel is formed and the effect of alendronate during early odontogenesis. Ultrastructural analysis combined with high-resolution immunocytochemistry for amelogenin was carried out. Twelve rats were subjected to daily SC injections of sodium alendronate (2.5 mg/kg/day) for 3 days on their dorsal region, whereas three rats were daily injected with saline solution as a control. Molar tooth germs from 3-day-old rats were fixed under microwave irradiation in 0.1% glutaraldehyde + 4% formaldehyde buffered at pH 7.2 with 0.1 M sodium cacodylate. The specimens were left undecalcified, postfixed with osmium tetroxide, dehydrated, and embedded in LR White resin. Ultrathin sections were incubated with a chicken anti-24-kDa rat amelogenin antibody, a secondary antibody, and finally with a protein A-gold complex. Large patches of amelogenin were present over the unmineralized mantle dentin and at early secretory ameloblasts. At more advanced stages, they were also detected at the enamel matrix, as well as in the mineralized dentin, at the periodontoblastic space of the dentinal tubules, and at the predentin. It is likely that the main effect of alendronate at early stages of odontogenesis is the increase of synthesis/secretion of amelogenin, promoting its deposition within the forming dentin and enamel.     

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.