Wild type and <Emphasis Type="Italic">H43Y</Emphasis> variant of human <Emphasis Type="Italic">TRIM5α</Emphasis> show similar anti-human immunodeficiency virus type 1 activity both in vivo and in vitro

Polymorphisms in human genes have been shown to affect the rate of disease progression to acquired immune deficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Recently, tripartite motif 5α (TRIM5α) was identified as a factor that confers resistance to HIV-1 infection in Old World monkey cells. Subsequently, Sawyer et al. (Curr Biol 16:95–100, 2006) reported a single nucleotide polymorphism (H43Y) in the human TRIM5α gene and TRIM5α protein with 43Y was found to lose its ability to restrict HIV-1. In the present study, we reevaluated effects of this allele on in vitro anti-HIV-1 activity as well as on HIV-1 disease progression in European and Asian cohorts of HIV-1-infected individuals. Our epidemiological and molecular biological findings clearly indicate H43Y has a very minor effect on anti-HIV-1 activity of TRIM5α, suggesting that this allele is immaterial, at least in HIV-1-infected Europeans and Asians.     

Discovery of (-)-6-[2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone--a potent NR2B-selective N-methyl D-aspartate (NMDA) antagonist for the treatment of pain

(-)-6-[2-[4-(3-Fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl]-3,4-dihydro-2(1H)-quinolinone was identified as an orally active NR2B-subunit selective N-methyl-d-aspartate (NMDA) receptor antagonist. It has very high selectivity for NR2B subunits containing NMDA receptors versus the HERG-channel inhibition (therapeutic index=4200 vs NR2B binding IC(50)). This compound has improved pharmacokinetic properties compared to the prototype CP-101,606.     

Small molecular CD4 mimics as HIV entry inhibitors

Derivatives of CD4 mimics were designed and synthesized to interact with the conserved residues of the Phe43 cavity in gp120 to investigate their anti-HIV activity, cytotoxicity, and CD4 mimicry effects on conformational changes of gp120. Significant potency gains were made by installation of bulky hydrophobic groups into the piperidine moiety, resulting in discovery of a potent compound with a higher selective index and CD4 mimicry. The current study identified a novel lead compound 11 with significant anti-HIV activity and lower cytotoxicity than those of known CD4 mimics.     

Natural hybridization of Japanese <Emphasis Type="Italic">Rhododendron</Emphasis> section <Emphasis Type="Italic">Brachycaryx</Emphasis> in Mount Kintoki in eastern Japan and concerns for genetic diversity in restoring their habitat

Native Rhododendrons section Brachycaryx in eastern Japan are decreasing in their natural habitats and the need to restore these habitats is increasing. Conservation of genetic diversity in restoring habitat requires clarification of the balance of interspecies genetic exchange which occurs in their natural habitats. In well-preserved natural habitats of Rhododendron dilatatum, R. kiyosumense, and R. wadanum and their natural hybrids R.×kuratanum and R.×hasegawai we investigated their geographical distribution, frequency, and flowering period. DNA analysis of the internal transcribed spacer (ITS) region was also conducted to confirm the species related to hybridization. Our findings in the field survey were: (1) Hybridizations occur in the overlap zones of related species. (2) R.×hasegawai occurs more frequently than R.×kuratanum, probably because the flowering seasons of R. kiyosumense and R. wadanum overlap longer than those of R. dilatatum and R. kiyosumense. (3) Natural hybrid occurrence is, nevertheless, under 9% of all related Rhododendrons section Brachycalyx. Analysis of the ITS region suggested that the two hybrids are generated from interspecific gene exchange, i.e., (4) R. dilatatum and R. kiyosumense relate to the formation of R.×kuratanum. (5) R.×hasegawai is a hybrid of R. wadanum and some species other than R. wadanum. On the basis of these findings we delineated several guidelines for restoring habitats of Rhododendrons of Section Brachycaryx with concerns for genetic diversity: (1) Before use, identify plant materials by morphological traits to determine whether they are original species or hybrids. (2) Investigate the distribution of remnant Rhododendrons section Brachycaryx before restoration. (3) Combine plant materials of original species in the natural distribution.     

A central kinase domain of type I phosphatidylinositol phosphate kinases is sufficient to prime exocytosis: isoform specificity and its underlying mechanism

Exocytosis, a critical process for neuronal communication and hormonal regulation, involves several distinct steps including MgATP-dependent priming (which involves the synthesis of phosphatidylinositol 4,5-bisphosphate). Type I phosphatidylinositol phosphate kinases (PIPKIs) were purified biochemically as a priming factor. PIPKI consists of three domains: the N-terminal region, the central kinase domain, and the C-terminal region. Three isoforms (alpha, beta, and gamma) of PIPKI have been identified, and each is alternatively spliced at the C-terminal region. In the present study, we conducted a structure/function analysis of PIPKIs in the priming of exocytosis, and we found that recombinant PIPKIalpha and PIPKIgamma had priming activity. However, an unexpected finding of these results was that PIPKIbeta did not prime exocytosis. The N- or C-terminal region of PIPKIalpha and PIPKIgamma was not required for priming, which indicates that the central kinase domain is sufficient for this process. Alternative splicing in each isoform did not affect the isoform specificity in priming. Priming activity by isoforms is strongly correlated with their phosphatidylinositol phosphate kinase activity because PIPKIalpha and PIPKIgamma had higher kinase activity than PIPKIbeta. These results suggest that PIPKIalpha and PIPKIgamma are the critical priming factors for exocytosis; it also suggests that the levels of phosphatidylinositol phosphate kinase activity in producing phosphatidylinositol 4,5-bisphosphate specify the function of PIPKI isoforms in priming.     

RalA-exocyst interaction mediates GTP-dependent exocytosis

Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca2+-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. Here we show the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA (RalA(E38R)) and the Sec5 (Sec5(T11A)) fragment, which abolish RalA-Sec5 interaction also abolished the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalA(E38R), enhanced GTP-dependent exocytosis. In contrast the RalA and the Sec5 fragment showed no inhibition of Ca2+-dependent exocytosis, but cleavage of a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca2+-dependent exocytosis. Our results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca2+-dependent exocytosis use different sensors and effectors for triggering exocytosis whereas their final fusion steps are both SNARE-dependent.     

Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin

We describe an organotypic model of mouse skin consisting of a stratified sheet of epidermal keratinocytes and dermal fibroblasts within a contracted collagen gel. The model was designed to maintain the polarity of stratified keratinocytes and permit their long-term culture at an air-liquid interface. After air exposure, the thickness of the keratinocyte sheet transiently increased and then decreased to two cell layers at 2 weeks. The two-cell-layer structure is similar to that of the adult mouse epidermis. Cytokeratin 5 was localized in the lowest cell layer in the epithelial sheet, but cytokeratin 1 and loricrin were localized in the outer cell layers, resembling mouse skin. The expressions of interleukin 1alpha and 1beta in the keratinocytes and of keratinocyte growth factor 1 and 2 in the fibroblasts correlated with keratinocyte stratification. The mouse organotypic coculture is useful in studying epithelial cell-mesenchymal cell interactions in vitro.     

The PI3K/Akt pathway contributes to arenavirus budding

Several arenaviruses, chiefly Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a significant public health concern in regions where they are endemic. On the other hand, evidence indicates that the globally distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway participates in many cellular processes, including cell survival and differentiation, and also has been shown to play important roles in different steps of the life cycles of a variety of viruses. Here we report that the inhibition of the PI3K/Akt pathway inhibited budding and to a lesser extent RNA synthesis, but not cell entry, of LCMV. Accordingly, BEZ-235, a PI3K inhibitor currently in cancer clinical trials, inhibited LCMV multiplication in cultured cells. These findings, together with those previously reported for Junin virus (JUNV), indicate that targeting the PI3K/Akt pathway could represent a novel antiviral strategy to combat human-pathogenic arenaviruses.     

Immunogenic dialyzable factor derived from a ribosomal fraction of Salmonella typhimurium III. Analysis of resistance induced by dialyzable factors

Dialyzable factors (DF) were prepared from ribosomal fractions of several organisms including rough mutants of Salmonella typhimurium LT2, salmonella species of different serogroups, other enteric bacteria and gram-positive organisms, and tested for their immunogenicity against S. typhimurium infection in mice. All of them conferred local resistance on mice challenged intramuscularly with S. typhimurium LT2 in the early stage of immunization before the establishment of delayed-type hypersensitivity (DTH) to salmonella antigens. Although DFs of enteric bacteria including rough mutants of S. typhimurium induced DTH to salmonella antigens, only DF of a two-heptose mutant of S. typhimurium LT2 afforded significant mouse protection but others only prolonged the mean time to death. DF of Listeria monocytogenes induced the cross-reacting immunity which afforded the low level of mouse protection as well as an increase in mean time to death without inducing DTH. Passive transfer of anti-O antibody did not enhance the mouse protection provided by each DF. Resistance conferred by DF of S. typhimurium LT2 consisted of two phases: (i) nonspecific macrophage activation resulting in reduction of organisms at the infected site, which became active in the early stage of immunization and (ii) salmonella-specific immunity capable of preventing systemic infection, which became active in the late stage of immunization.