Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

Purification of rabbit liver phosphofructokinase and its properties under simulatingin vivo conditions

Phosphofructokinase (EC 2.7.1.11) from rabbit liver was purified to homogeneity. Preincubation of enzyme results in nonlinearity of enzyme activity with enzyme concentration. Therefore K_0.5 of enzyme for fructose 6 phosphate in the absence or presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both was determined at physiological concentrations of its various effectors by taking the initial rate obtained by adding the enzyme last. They decrease the K_0.5 value from 4.1 mM to about 0.2mM. The K_0.5 of enzyme for fructose 2,6 bisphosphate was also determined under the above conditions. It is about 4.3ΜM. Transient kinetics of phosphofructokinase at varying concentrations of enzyme in the presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both were studied. It was found that although they decrease t_1/2 i.e. the time to reach half the maximal steady rate by about 5–8 fold, it was about constant at varying concentrations of the enzyme. These results indicate that fructose 2,6 bisphosphate and polyethylene glycol decrease K_0.5 of the enzyme for fructose 6 phosphate not by associating the enzyme to higher aggregates, but by a different mechanism.     

AMP-activated protein kinase (AMPK) regulates autophagy,inflammation and immunity and contributes to osteoclast differentiation and functionabs

Osteoclasts are multinucleated giant cells, responsible for bone resorption. Osteoclast differentiation and function requires a series of cytokines to remove the old bone, which coordinates with the induction of bone remodelling by osteoblast-mediated bone formation. Studies have demonstrated that AMP-activated protein kinase (AMPK) play a negative regulatory role in osteoclast differentiation and function. Research involving AMPK, a nutrient and energy sensor, has primarily focused on osteoclast differentiation and function; thus, its role in autophagy, inflammation and immunity remains poorly understood. Autophagy is a conservative homoeostatic mechanism of eukaryotic cells, and response to osteoclast differentiation and function; however, how it interacts with inflammation remains unclear. Additionally, based on the regulatory function of different AMPK subunits for osteoclast differentiation and function, its activation is regulated by upstream factors to perform bone metabolism. This review summarises the critical role of AMPK-mediated autophagy, inflammation and immunity by upstream and downstream signalling during receptor activator of nuclear factor kappa-B ligand-induced osteoclast differentiation and function. This pathway may provide therapeutic targets for bone-related diseases, as well as function as a biomarker for bone homoeostasis.     

Transfer RNA cleavages by onconase reveal unusual cleavage sites

Onconase, a protein from amphibian eggs and a homologue of pancreatic ribonuclease (RNase) superfamily, is cytotoxic, exhibits antitumor and antiviral activity, and is in phase III clinical trials. It has been shown to predominantly target cellular tRNA on its entry into mammalian cells (Saxena, S. K., Sirdeshmukh, R., Ardelt, W., Mikulski, S. M., Shogen, K., and Youle, R. J. (2002) J. Biol. Chem. 277, 15142-15146). Cleavage site mapping using natural tRNA substrates, in vitro, revealed predominant cleavage sites at UG and GG residues. Cleavages at UG or the less intense cleavages at CG sites are consistent with the known base specificity of onconase. However, predominance of cleavages at selected G-G bonds is unusual for a homologue of pancreatic RNases. Interestingly, in at least three of the four tRNA substrates studied, the predominant cleavages mapped in the triplet UGG located in the context of the variable loop or the D-arm of the tRNA. The cleavage specificity of onconase observed by us thus indicates another special feature of this enzyme, which may be relevant to its cellular actions.     

Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis

Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted rickettsial pathogens that cause human and canine monocytic ehrlichiosis respectively. We tested the hypothesis that these pathogens express unique proteins in response to their growth in vertebrate and tick host cells and that this differential expression is similar in closely related Ehrlichia species. Evaluation of nine E. chaffeensis isolates and one E. canis isolate demonstrated that protein expression was host cell-dependent. The differentially expressed proteins included those from the p28/30-Omp multigene locus. E. chaffeensis and E. canis proteins expressed in infected macrophages were primarily the products of the p28-Omp 19 and 20 genes or their orthologues. In cultured tick cells, E. canis expressed only the p30-10 protein, an orthologue of the E. chaffeensis p28-Omp 14 protein which is the only protein expressed by E. chaffeensis propagated in cultured tick cells. The expressed Omp proteins were post-translationally modified to generate multiple molecular forms. E. chaffeensis gene expression from the p28/30-Omp locus was similar in tick cell lines derived from both vector (Amblyomma americanum) and non-vector (Ixodes scapularis) ticks. Differential expression of proteins within the p28/p30-Omp locus may therefore be vital for adaptation of Ehrlichia species to their dual host life cycle.     

A subset of Cowdria ruminantium genes important for immune recognition and protection

Cowdria ruminantium causes the tick-borne rickettsial disease of heartwater, which is devastating to livestock production in sub-Saharan Africa. Current diagnosis and control methods are inadequate. We have identified and sequenced a subset of genes encoding recombinant antigens recognized by antibody and peripheral blood mononuclear cells from immune ruminants. The identified genes include many with significant similarity to those of Rickettsia prowazekii, genes predicted to encode different outer membrane proteins and lipoproteins and a gene containing an unusual tandem repeat structure. Evidence is presented for immune protection by recombinant antigens in a mouse model of C. ruminantium infection. These data identify new recombinant antigens for evaluation in vaccines and diagnostic tests to control heartwater.     

Fanconi anemia and Bloom's syndrome crosstalk through FANCJ-BLM helicase interaction

Fanconi anemia (FA) and Bloom's syndrome (BS) are rare hereditary chromosomal instability disorders. FA displays bone marrow failure, acute myeloid leukemia, and head and neck cancers, whereas BS is characterized by growth retardation, immunodeficiency, and a wide spectrum of cancers. The BLM gene mutated in BS encodes a DNA helicase that functions in a protein complex to suppress sister-chromatid exchange. Of the 15 FA genetic complementation groups implicated in interstrand crosslink repair, FANCJ encodes a DNA helicase involved in recombinational repair and replication stress response. Based on evidence that BLM and FANCJ interact we suggest that crosstalk between BLM and FA pathways is more complex than previously thought. We propose testable models for how FANCJ and BLM coordinate to help cells deal with stalled replication forks or double-strand breaks (DSB). Understanding how BLM and FANCJ cooperate will help to elucidate an important pathway for maintaining genomic stability.     

DNA repair and replication fork helicases are differentially affected by alkyl phosphotriester lesion

DNA helicases are directly responsible for catalytically unwinding duplex DNA in an ATP-dependent and directionally specific manner and play essential roles in cellular nucleic acid metabolism. It has been conventionally thought that DNA helicases are inhibited by bulky covalent DNA adducts in a strand-specific manner. However, the effects of highly stable alkyl phosphotriester (PTE) lesions that are induced by chemical mutagens and refractory to DNA repair have not been previously studied for their effects on helicases. In this study, DNA repair and replication helicases were examined for unwinding a forked duplex DNA substrate harboring a single isopropyl PTE specifically positioned in the helicase-translocating or -nontranslocating strand within the double-stranded region. A comparison of SF2 helicases (RecQ, RECQ1, WRN, BLM, FANCJ, and ChlR1) with a SF1 DNA repair helicase (UvrD) and two replicative helicases (MCM and DnaB) demonstrates unique differences in the effect of the PTE on the DNA unwinding reactions catalyzed by these enzymes. All of the SF2 helicases tested were inhibited by the PTE lesion, whereas UvrD and the replication fork helicases were fully tolerant of the isopropyl backbone modification, irrespective of strand. Sequestration studies demonstrated that RECQ1 helicase was trapped by the PTE lesion only when it resided in the helicase-translocating strand. Our results are discussed in light of the current models for DNA unwinding by helicases that are likely to encounter sugar phosphate backbone damage during biological DNA transactions.     

Repellent,antifeedant and toxic effects of Ageratum conyzoides (Linnaeus) (Asteraceae) extract against Helicovepra armigera (Hübner) (Lepidoptera: Noctuidae)

Repellent, antifeedant and toxic effect of crude hexane extract of Ageratum conyzoides were investigated against Helicoverpa armigera. In orientation bioassay, the extract exhibited dose-dependent repellency against neonates. Extract significantly increased the mortality and decreased growth of different larval stages when administrated orally in artificial diet. EC₅₀ value was at 0.11% for larval growth inhibition. Toxicity of the extract was manifested by high mortality of first instar larvae after 7 days of feeding on diet containing 0.05–0.4% of extract with LC₅₀ of 0.17%. Under choice bioassay, extract showed strong antifeedant activity against fifth instar larvae with DI₅₀ of 0.21%. In nutritional bioassay, extract significantly reduced RCR, RGR, ECI and ECD of fifth instar larvae with increased AD. When RGR were plotted against RCR, the growth efficiency of larvae fed on treated diet was significantly lower than the control fed larvae suggesting the antifeedant and toxic effect of extract.