Responses of hypertrophied myocytes to reactive species: implications for glycolysis and electrophile metabolism

During cardiac remodelling, the heart generates higher levels of reactive species; yet an intermediate 'compensatory' stage of hypertrophy is associated with a greater ability to withstand oxidative stress. The mechanisms underlying this protected myocardial phenotype are poorly understood. We examined how a cellular model of hypertrophy deals with electrophilic insults, such as would occur upon ischaemia or in the failing heart. For this, we measured energetics in control and PE (phenylephrine)-treated NRCMs (neonatal rat cardiomyocytes) under basal conditions and when stressed with HNE (4-hydroxynonenal). PE treatment caused hypertrophy as indicated by augmented atrial natriuretic peptide and increased cellular protein content. Hypertrophied myocytes demonstrated a 2.5-fold increase in ATP-linked oxygen consumption and a robust augmentation of oligomycin-stimulated glycolytic flux and lactate production. Hypertrophied myocytes displayed a protected phenotype that was resistant to HNE-induced cell death and a unique bioenergetic response characterized by a delayed and abrogated rate of oxygen consumption and a 2-fold increase in glycolysis upon HNE exposure. This augmentation of glycolytic flux was not due to increased glucose uptake, suggesting that electrophile stress results in utilization of intracellular glycogen stores to support the increased energy demand. Hypertrophied myocytes also had an increased propensity to oxidize HNE to 4-hydroxynonenoic acid and sustained less protein damage due to acute HNE insults. Inhibition of aldehyde dehydrogenase resulted in bioenergetic collapse when myocytes were challenged with HNE. The integration of electrophile metabolism with glycolytic and mitochondrial energy production appears to be important for maintaining myocyte homoeostasis under conditions of increased oxidative stress.     

Clinical outcome of henipavirus infection in hamsters is determined by the route and dose of infection

Nipah virus (NiV) and Hendra virus (HeV) are emerging zoonotic viruses and the causative agents of severe respiratory disease and encephalitis in humans. Little is known about the mechanisms that govern the development of respiratory and neurological disease. Using a hamster model of lethal NiV and HeV infection, we describe the role of the route and dose of infection on the clinical outcome and determine virus tropism and host responses following infection. Infection of hamster with a high dose of NiV or HeV resulted in acute respiratory distress. NiV initially replicated in the upper respiratory tract epithelium, whereas HeV initiated infection primarily in the interstitium. In contrast, infection with a low dose of NiV or HeV resulted in the development of neurological signs and more systemic spread of the virus through involvement of the endothelium. The development of neurological signs coincided with disruption of the blood-brain barrier (BBB) and expression of tumor necrosis alpha (TNF-α) and interleukin 1 β (IL-1β). In addition, interferon-inducible protein 10 (IP-10) was identified as playing an important role in NiV and HeV pathogenesis. These studies reveal novel information on the development and progression of NiV and HeV clinical disease, provide a mechanism for the differences in transmission observed between NiV and HeV outbreaks, and identify specific cytokines and chemokines that serve as important targets for treatment.     

Disulfide bond formation contributes to herpes simplex virus capsid stability and retention of pentons

Disulfide bonds reportedly stabilize the capsids of several viruses, including papillomavirus, polyomavirus, and simian virus 40, and have been detected in herpes simplex virus (HSV) capsids. In this study, we show that in mature HSV-1 virions, capsid proteins VP5, VP23, VP19C, UL17, and UL25 participate in covalent cross-links, and that these are susceptible to dithiothreitol (DTT). In addition, several tegument proteins were found in high-molecular-weight complexes, including VP22, UL36, and UL37. Cross-linked capsid complexes can be detected in virions isolated in the presence and absence of N-ethylmaleimide (NEM), a chemical that reacts irreversibly with free cysteines to block disulfide formation. Intracellular capsids isolated in the absence of NEM contain disulfide cross-linked species; however, intracellular capsids isolated from cells pretreated with NEM did not. Thus, the free cysteines in intracellular capsids appear to be positioned such that disulfide bond formation can occur readily if they are exposed to an oxidizing environment. These results indicate that disulfide cross-links are normally present in extracellular virions but not in intracellular capsids. Interestingly, intracellular capsids isolated in the presence of NEM are unstable; B and C capsids are converted to a novel form that resembles A capsids, indicating that scaffold and DNA are lost. Furthermore, these capsids also have lost pentons and peripentonal triplexes as visualized by cryoelectron microscopy. These data indicate that capsid stability, and especially the retention of pentons, is regulated by the formation of disulfide bonds in the capsid.     

A novel gene inactivation system reveals altered periplasmic flagellar orientation in a Borrelia burgdorferi fliL mutant

Motility and chemotaxis are essential components of pathogenesis for many infectious bacteria, including Borrelia burgdorferi, the causative agent of Lyme disease. Motility and chemotaxis genes comprise 5 to 6% of the genome of B. burgdorferi, yet the functions of most of those genes remain uncharacterized, mainly due to the paucity of a nonpolar gene inactivation system. In this communication, we describe the development of a novel gene inactivation methodology to target B. burgdorferi fliL, a putative periplasmic flagellar gene located in a large motility operon and transcribed by RNA polymerase containing σ(70). Although the morphology of nonpolar fliL mutant cells was indistinguishable from that of wild-type cells, the mutant exhibited a defective-motility phenotype. Cryo-electron tomography (cryo-ET) of intact organisms revealed that the periplasmic flagella in the fliL mutant were frequently tilted toward the cell pole instead of their normal orientation toward the cell body. These defects were corrected when the mutant was complemented in cis. Moreover, a comparative analysis of flagellar motors from the wild type and the mutant provides the first structural evidence that FliL is localized between the stator and rotor. Our results suggest that FliL is likely involved in coordinating or regulating the orientation of periplasmic flagella in B. burgdorferi.     

The design and synthesis of guanosine compounds with in vitro activity against the colon cancer cell line SW480: non-taxane derived mimics of taxol?

In the course of our investigation into the use of taxol as a lead compound to design new molecules with anti-cancer activity, we have synthesized four compounds based on protected guanosine coupled to taxol isoserine side-chain analogues. These analogues show in vitro anti-cancer activity against the colon cancer cell line SW480 that their constituent parts do not.     

Immunization with P10 Peptide Increases Specific Immunity and Protects Immunosuppressed BALB/c Mice Infected with Virulent Yeasts of Paracoccidioides brasiliensis

Paracoccidioidomycosis is a systemic granulomatous disease caused by Paracoccidioides spp. A peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4⁺ helper-1 immune response in mice and protects against intratracheal challenge with virulent P. brasiliensis. Previously, we evaluated the efficacy of the P10 peptide alone or combined with antifungal drugs in mice immunosuppressed and infected with virulent isolate of P. brasiliensis. In the present work, our data suggest that P10 immunization leads to an effective cellular immune response associated with an enhanced T cell proliferative response. P10-stimulated splenocytes increased nitric oxide (NO) production and induced high levels of IFN-γ, IL-1β and IL-12. Furthermore, significantly increased concentrations of pro-inflammatory cytokines were also observed in lung homogenates of immunized mice. P10 immunization was followed by minimal fibrosis in response to infection. Combined with antifungal drugs, P10 immunization most significantly improved survival of anergic infected mice. Administration of either itraconazole or sulfamethoxazole/trimethoprim together with P10 immunization resulted in 100 % survival up to 200 days post-infection, whereas untreated mice died within 80 days. Hence, our data show that P10 immunization promotes a strong specific immune response even in immunocompromised hosts and thus P10 treatment represents a powerful adjuvant therapy to chemotherapy.