Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8⁺ T-cells and the use of an in vitro model of naïve CD8⁺ T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8⁺ T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8⁺ T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8⁺ and CD4⁺ T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.     

G-protein beta subunit of Cochliobolus heterostrophus involved in virulence, asexual and sexual reproductive ability, and morphogenesis

Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein alpha (Galpha) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gbeta subunit, are reported here. CGB1 is the sole Gbeta subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gbeta subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gbeta heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gbeta subunit in filamentous fungi.     

New N-arylsulfonyl-N-alkoxyaminoacetohydroxamic acids as selective inhibitors of gelatinase A (MMP-2)

New N-arylsulfonyl-substituted alkoxyaminoaceto hydroxamic acid derivatives of types 8 and 10 designed as oxa-analogues of known sulfonamide-based MMPi of types 2 and 7 were synthesized and tested for their inhibitory activities on some matrix metalloproteinases. The combination of a biphenylsulfonamide group with oxyamino oxygen in the pharmacophoric central skeleton of sulfonamide-based MMPi obtained in the new sulfonamides 10 seems to be able to give selectivity for MMP-2 over MMP-1. The most potent derivative of this type, 10a, shows similar anti-invasive properties to the analogue reference drug CGS27023A, 2, in an in vitro model of invasion on matrigel, carried out on cellular lines of fibrosarcoma HT1080 (tumoural cells over-expressing MMP-2 and MMP-9).     

Biological anthropology of aging--past,present and future

Biological anthropologists have a strong tradition of studying growth and development and research on aging has been limited. This paper explores the past and current contribution of biological anthropologists to the field of aging through an examination of the American Journal of Physical Anthropology (AJPA) and the American Journal of Human Biology (AJHB). It is clear from this survey that biological anthropologists and human biologists have predominantly studied growth and developmental processes relative to aging. However, there is a trend of increasing interest in aging over time. In the AJHB, papers discussing chronic disease were predominant, followed by reproductive aging (19%), bone aging (15%) and body composition (10%). Within the AJPA, the majority of articles were in the field of human biology (43%) and bioarchaelogy (42%) with a lesser contribution from primatology (14%) and dermatogliphics (1%). Biological anthropologists still have great potential to make contributions to gerontology with our evolutionary and holistic perspectives and focus on cross-cultural research.     

Synthesis of 1-D- and 1-L-myo-inosityl 2-N-acetamido-2-deoxy-alpha-D-glucopyranoside establishes substrate specificity of the Mycobacterium tuberculosis enzyme AcGI deacetylase

Mycothiol (MSH, 1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside) is the principal low molecular weight thiol in actinomycetes. The enzyme 1-D-myo-inosityl 2-N-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (AcGI deacetylase) is involved in the biosynthesis of MSH and forms the free amine 1-D-myo-inosityl 2-amino-2-deoxy-alpha-D-glucopyranoside, which is used in the third of four steps of MSH biosynthesis. Here, we report the synthesis of two isomers of AcGI, which contain either 1-L-myo-inositol or 1-D-myo-inositol. These synthetic products were used to investigate substrate specificity of the Mycobacterium tuberculosis enzyme AcGI deacetylase.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.     

Changes in the human mitochondrial genome after treatment of malignant disease

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage.     

Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.