Increases in Bcl-2 and cleavage of caspase-1 and caspase-3 in human brain after head injury.

The bcl-2 and caspase families are important regulators of programmed cell death in experimental models of ischemic, excitotoxic, and traumatic brain injury. The Bcl-2 family members Bcl-2 and Bcl-xL suppress programmed cell death, whereas Bax promotes programmed cell death. Activated caspase-1 (interleukin-1beta converting enzyme) and caspase-3 (Yama/Apopain/Cpp32) cleave proteins that are important in maintaining cytoskeletal integrity and DNA repair, and activate deoxyribonucleases, producing cell death with morphological features of apoptosis. To address the question of whether these Bcl-2 and caspase family members participate in the process of delayed neuronal death in humans, we examined brain tissue samples removed from adult patients during surgical decompression for intracranial hypertension in the acute phase after traumatic brain injury (n=8) and compared these samples to brain tissue obtained at autopsy from non-trauma patients (n=6). An increase in Bcl-2 but not Bcl-xL or Bax, cleavage of caspase-1, up-regulation and cleavage of caspase-3, and evidence for DNA fragmentation with both apoptotic and necrotic morphologies were found in tissue from traumatic brain injury patients compared with controls. These findings are the first to demonstrate that programmed cell death occurs in human brain after acute injury, and identify potential pharmacological and molecular targets for the treatment of human head injury.     

Disparate molecular evolution of two types of repetitive DNAs in the genome of the grasshopper Eyprepocnemis plorans

Wide arrays of repetitive DNA sequences form an important part of eukaryotic genomes. These repeats appear to evolve as coherent families, where repeats within a family are more similar to each other than to other orthologous representatives in related species. The continuous homogenization of repeats, through selective and non-selective processes, is termed concerted evolution. Ascertaining the level of variation between repeats is crucial to determining which evolutionary model best explains the homogenization observed for these sequences. Here, for the grasshopper Eyprepocnemis plorans, we present the analysis of intragenomic diversity for two repetitive DNA sequences (a satellite DNA (satDNA) and the 45S rDNA) resulting from the independent microdissection of several chromosomes. Our results show different homogenization patterns for these two kinds of paralogous DNA sequences, with a high between-chromosome structure for rDNA but no structure at all for the satDNA. This difference is puzzling, considering the adjacent localization of the two repetitive DNAs on paracentromeric regions in most chromosomes. The disparate homogenization patterns detected for these two repetitive DNA sequences suggest that several processes participate in the concerted evolution in E. plorans, and that these mechanisms might not work as genome-wide processes but rather as sequence-specific ones.     

Treatment of Chronic Viral Hepatitis in Woodchucks by Prolonged Intrahepatic Expression of Interleukin-12

Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10¹⁰ viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-γ) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-γ in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10¹⁰ vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.Hepatitis B virus (HBV) infection is estimated to cause approximately 1 million deaths per year (http://www.who.int/emc). Current therapies against chronic HBV include pegylated alpha interferon (IFN-α) and nucleoside/nucleotide analogs, such as lamivudine, entecavir, adefovir, and tenofovir (16). Sustained antiviral responses are achieved in only one-third of the patients treated with pegylated IFN-α (16, 32). Nucleoside/nucleotide analogs are effective, but treatment must be continued for many years, resulting in high costs, the emergence of drug-resistant variants, and frequent relapses after the discontinuation of therapy (32).Chronic HBV infection is associated with defects in antiviral immunity (3). Patients with an acute self-limiting HBV infection develop neutralizing anti-HBs antibodies and multispecific CD4⁺ and CD8⁺ T-cell responses with a type 1 cytokine profile (3). In contrast, patients with chronic HBV infection show no protective humoral immunity and a weak or undetectable virus-specific T-cell response (5). The precise mechanism responsible for this immunotolerance is still unknown. Recently, several studies have indicated that regulatory T cells (Tregs), immunosuppressive cytokines, and inhibitory receptor-ligand interactions, such as PD1-PDL1, contribute to the impairment of virus-specific T-cell responses in chronic HBV infection (1, 17, 23, 26, 28).Interleukin-12 (IL-12) is a cytokine produced by antigen-presenting cells that is essential for the induction of effective cell-mediated immunity against viruses and other pathogens (30). IL-12 promotes Th1-type responses, enhances cytotoxic-T-cell activity, and stimulates T lymphocytes and NK cells to produce IFN-γ (30). The administration of recombinant IL-12 (rIL-12) to HBV-transgenic mice resulted in the inhibition of HBV replication in the liver (8). In vitro studies demonstrated that IL-12 was able to enhance HBV-specific T-cell responses in chronic HBV carriers (15, 22, 31). Moreover, the upregulation of IL-12 production has been shown to be associated with HBe seroconversion, indicating an important role for this cytokine in the control of HBV infection (22). In two studies, rIL-12 administered to patients with chronic HBV infection once per week as monotherapy (7) or twice weekly in combination with lamivudine (21) increased virus-specific T-cell reactivity and exerted significant antiviral activity. However, in both studies, a rebound of viremia occurred following drug withdrawal. The antiviral effects of rIL-12 were dose dependent, but the therapy was limited by severe toxicity when high doses of the cytokine were used (7, 21).Gene therapy can significantly increase cytokine expression in the target organ without excessively elevating systemic cytokine levels, which leads to an increased efficacy/toxicity ratio. In the present study, we tested the antiviral potential of IL-12-mediated gene therapy using a high-capacity adenovirus (HC-Ad) encoding murine IL-12 (mIL-12) under the control of a liver-specific inducible promoter that is responsive to the progesterone antagonist RU486 (30). HC-Ad is a nonintegrating vector characterized by strong hepatotropism, low toxicity, long-term transgene expression, and high cloning capacity (13). All these properties make HC-Ad a useful tool for therapeutic applications in human liver diseases.As an animal model of chronic HBV infection, we used woodchucks that were chronically infected with woodchuck hepatitis virus (WHV). WHV is a hepadnavirus with a genomic organization, biological properties, and a replicative strategy that are essentially identical to those of HBV. When WHV infects woodchucks in the perinatal period of life, the infection causes chronic hepatitis in most animals. This condition resembles the pathological features and natural history of chronic hepatitis B (18). Here, we demonstrate that prolonged intrahepatic expression of IL-12 overcomes immunological tolerance for WHV antigens and induces sustained antiviral effects in woodchucks with chronic WHV infection and viral loads below 10¹⁰ viral genomes (vg)/ml. These observations indicate that IL-12 gene therapy is an alternative approach for the treatment of chronic HBV infection.     

Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria

Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.     

Mapping of phospholipids by MALDI imaging (MALDI-MSI): realities and expectations

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has emerged as a novel powerful MS methodology that has the ability to generate both molecular and spatial information within a tissue section. Application of this technology as a new type of biochemical lipid microscopy may lead to new discoveries of the lipid metabolism and biomarkers associated with area-specific alterations or damage under stress/disease conditions such as traumatic brain injury or acute lung injury, among others. However there are limitations in the range of what it can detect as compared with liquid chromatography-MS (LC-MS) of a lipid extract from a tissue section. The goal of the current work was to critically consider remarkable new opportunities along with the limitations and approaches for further improvements of MALDI-MSI. Based on our experimental data and assessments, improvements of the spectral and spatial resolution, sensitivity and specificity towards low abundance species of lipids are proposed. This is followed by a review of the current literature, including methodologies that other laboratories have used to overcome these challenges.     

PDL241, a novel humanized monoclonal antibody,reveals CD319 as a therapeutic target for rheumatoid arthritis

Introduction

Targeting the CD20 antigen has been a successful therapeutic intervention in the treatment of rheumatoid arthritis (RA). However, in some patients with an inadequate response to anti-CD20 therapy, a persistence of CD20^- plasmablasts is noted. The strong expression of CD319 on CD20^- plasmablast and plasma cell populations in RA synovium led to the investigation of the potential of CD319 as a therapeutic target.

Methods

PDL241, a novel humanized IgG₁ monoclonal antibody (mAb) to CD319, was generated and examined for its ability to inhibit immunoglobulin production from plasmablasts and plasma cells generated from peripheral blood mononuclear cells (PBMC) in the presence and absence of RA synovial fibroblasts (RA-SF). The in vivo activity of PDL241 was determined in a human PBMC transfer into NOD scid IL-2 gamma chain knockout (NSG) mouse model. Finally, the ability of PDL241 to ameliorate experimental arthritis was evaluated in a collagen-induced arthritis (CIA) model in rhesus monkeys.

Results

PDL241 bound to plasmablasts and plasma cells but not naïve B cells. Consistent with the binding profile, PDL241 inhibited the production of IgM from in vitro PBMC cultures by the depletion of CD319⁺ plasmablasts and plasma cells but not B cells. The activity of PDL241 was dependent on an intact Fc portion of the IgG₁ and mediated predominantly by natural killer cells. Inhibition of IgM production was also observed in the human PBMC transfer to NSG mouse model. Treatment of rhesus monkeys in a CIA model with PDL241 led to a significant inhibition of anti-collagen IgG and IgM antibodies. A beneficial effect on joint related parameters, including bone remodeling, histopathology, and joint swelling was also observed.

Conclusions

The activity of PDL241 in both in vitro and in vivo models highlights the potential of CD319 as a therapeutic target in RA.     

Parental effects modulate seed longevity: exploring parental and offspring phenotypes to elucidate pre-zygotic environmental influences

? Seed longevity, which is essential for germplasm conservation and survival of many land plant species, can vary considerably within species and cultivars. Here, we explore the relationship between parental and offspring phenotypes to elucidate how pre-zygotic environment affects seed longevity. ? Plants of the wild species Plantago cunninghamii were exposed to wet or dry soil within a warm or cool glasshouse until flowering and then moved to a common environment. Seeds subsequently produced were collected at maturity, and longevity was assessed by controlled ageing at 45°C, 60% relative humidity. Multivariate analysis was used to examine relationships between the parental and offspring phenotypes. ? The pre-zygotic environment resulted in a highly plastic parental response which was passed on to offspring seeds and changed their longevity (p(50)) by more than a factor of 2. Seed longevity is a function of the seed population's distribution of deaths in time (σ) and quality (K(i)); σ was associated with plant size, and K(i) with reproductive plant traits. ? The pre-zygotic growth environment modulated seed longevity via a parental effect. Reproductive performance and seed quality (K(i)) were highly correlated with each other and unrelated to the maternal plant phenotype. Hence seed quality may be associated with the paternal plant response to the environment.     

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

The eukaryotic replisome is disassembled in each cell cycle, dependent upon ubiquitylation of the CMG helicase. Studies of Saccharomyces cerevisiae, Caenorhabditis elegans and Xenopus laevis have revealed surprising evolutionary diversity in the ubiquitin ligases that control CMG ubiquitylation, but regulated disassembly of the mammalian replisome has yet to be explored. Here, we describe a model system for studying the ubiquitylation and chromatin extraction of the mammalian CMG replisome, based on mouse embryonic stem cells. We show that the ubiquitin ligase CUL2^LRR1 is required for ubiquitylation of the CMG‐MCM7 subunit during S‐phase, leading to disassembly by the p97 ATPase. Moreover, a second pathway of CMG disassembly is activated during mitosis, dependent upon the TRAIP ubiquitin ligase that is mutated in primordial dwarfism and mis‐regulated in various cancers. These findings indicate that replisome disassembly in diverse metazoa is regulated by a conserved pair of ubiquitin ligases, distinct from those present in other eukaryotes.     

Adsorption of α-amylase and Starch on Porous Zinc Oxide Nanosheet: Biophysical Study

Food Biophysics - Engineered biocatalyst and its desired products using nanotechnology has intensified the research in food industries. Zinc oxide (ZnO) nanosheet is designed and prepared; the...