Differences in HERV-K LTR insertions in orthologous loci of humans and great apes

The classification of the long terminal repeats (LTRs) of the human endogenous retrovirus HERV-K (HML-2) family was refined according to diagnostic differences between the LTR sequences. The mutation rate was estimated to be approximately equal for LTRs belonging to different families and branches of human endogenous retroviruses (HERVs). An average mutation rate value was calculated based on differences between LTRs of the same HERV and was found to be 0.13% per million years (Myr). Using this value, the ages of different LTR groups belonging to the LTR HML-2 subfamily were found to vary from 3 to 50Myr. Orthologous potential LTR-containing loci from different primate species were PCR amplified using primers corresponding to the genomic sequences flanking LTR integration sites. This allowed us to calculate the phylogenetic times of LTR integrations in primate lineages in the course of the evolution and to demonstrate that they are in good agreement with the LTR ages calculated from the mutation rates. Human-specific integrations for some very young LTRs were demonstrated. The possibility of LTRs and HERVs involvement in the evolution of primates is discussed.     

Centrosome-directed translocation of Weibel-Palade bodies is rapidly induced by thrombin, calyculin A, or cytochalasin B in human aortic endothelial cells

To examine the possible role of the cytoskeleton in exocytosis of Weibel-Palade bodies (WPBs), we used double immunofluorescence and electron microscopy to study the spatial relationships between WPBs and main cytoskeletal elements in endothelial cells treated with secretagogue, such as thrombin, or cytoskeleton-damaging agents. Unexpectedly, we have found that WPBs undergo rapid translocation towards the centrosome both in cells treated with thrombin and in those treated with cytochalasin B or calyculin A. Typically, 3 or 5 min after agent addition compact cluster of WPBs became visible near the microtubule-organizing center (MTOC) in most endothelial cells in which a fivefold increase in WPBs localized in close proximity to the mother centriole had been detected. In both thrombin- and cytochalasin-treated cells that exhibit a noticeable depletion in WPBs compared to control cells, WPBs located at the cell periphery were found to colocalize with vimentin intermediate filaments, but not with microtubules. In contrast, there was precise colocalization observed between WPBs and microtubules in calyculin-treated cells in which all WPBs undergo centrosome-directed translocation within 15 min after the agent addition. When vimentin filaments were induced to collapse to a perinuclear location by the microtubule-disrupting agent demecolcine, WPBs also translocated to the perinuclear region, where numerous WPBs were found to be localized within the bundles of intermediate-sized filaments. The data provide the first direct evidence that secretory granules utilize microtubule-based transport system to move in retrograde direction, i.e., away from the plasma membrane, towards the centrosome. We suggest that anterograde movement of WPBs is primarily dependent on their interaction with vimentin intermediate filaments.     

The anti-apoptotic effects of nordihydroguaiaretic acid: inhibition of cPLA(2) activation during TNF-induced apoptosis arises from inhibition of calcium signaling

Nordihydroguaiaretic acid (NDGA) is a plant lignan produced by Larrea tridentata, the creosote bush of the American southwest. In this report we examine the mechanism underlying the ability of NDGA to inhibit TNF-induced apoptosis. Our results show that NDGA blocks many key indicators of apoptosis. Caspase cleavage, mitochondrial inactivation, externalization of phosphatidyl serine, and (51)Cr-release were all blocked by low micromolar concentrations of NDGA. NDGA also inhibited the cPLA(2)-dependent release of (3)H-arachidonic acid. We investigated this activity and found that NDGA prevented the rise in intracellular calcium necessary for the apoptotic activation of cPLA(2). On the other hand, NDGA did not interfere with the TNF-induced phosphorylation of cPLA(2), indicating that NDGA does not block all TNF-dependent signaling. Finally, we asked whether the anti-apoptotic effect of NDGA could be attributed to its anti-oxidant activity. Comparison with the effects of butylated hydroxyanisole (BHA) did not completely support this hypothesis. While BHA strongly inhibited caspase activation and partially blocked the release of (51)Cr, it was unable to significantly block the calcium response or the release of (3)H-arachidonic acid associated with TNF-induced apoptosis. The anti-oxidant activity of NDGA may, therefore, explain some but not all of its anti-apoptotic activity.     

The distribution of the urban mosquitoes, Culex pipiens pipiens f. molestus (Diptera: Culicidae) in Petrozavodsk City

The distribution of the urban mosquitoes, Culex pipiens pipiens f. molestus in Petrozavodsk city was studied for the first time. Immature mosquito stages were found in 21 of 68 basements flooded with polluted waters, the occurrence of the mosquitoes varied in different districts of the sity. The mean siphonal index of the mosquito larvae from 6 underground populations varied from 3.8 up to 4.2, i. e. within the range typical for C. p. p. f. molestus.     

The low density lipoprotein receptor regulates the level of central nervous system human and murine apolipoprotein E but does not modify amyloid plaque pathology in PDAPP mice

Apolipoprotein E (apoE), a chaperone for the amyloid beta (Abeta) peptide, regulates the deposition and structure of Abeta that deposits in the brain in Alzheimer disease (AD). The primary apoE receptor that regulates levels of apoE in the brain is unknown. We report that the low density lipoprotein receptor (LDLR) regulates the cellular uptake and central nervous system levels of astrocyte-derived apoE. Cells lacking LDLR were unable to appreciably endocytose astrocyte-secreted apoE-containing lipoprotein particles. Moreover, cells overexpressing LDLR showed a dramatic increase in apoE endocytosis and degradation. We also found that LDLR knock-out (Ldlr-/-) mice had a significant, approximately 50% increase in the level of apoE in the cerebrospinal fluid and extracellular pools of the brain. However, when the PDAPP mouse model of AD was bred onto an Ldlr-/- background, we did not observe a significant change in brain Abeta levels either before or after the onset of Abeta deposition. Interestingly, human APOE3 or APOE4 (but not APOE2) knock-in mice bred on an Ldlr-/- background had a 210% and 380% increase, respectively, in the level of apoE in cerebrospinal fluid. These results demonstrate that central nervous system levels of both human and murine apoE are directly regulated by LDLR. Although the increase in murine apoE caused by LDLR deficiency was not sufficient to affect Abeta levels or deposition by 10 months of age in PDAPP mice, it remains a possibility that the increase in human apoE3 and apoE4 levels caused by LDLR deficiency will affect this process and could hold promise for therapeutic targets in AD.     

Dual modes of modification of hepatitis A virus 3C protease by a serine-derived beta-lactone: selective crystallization and formation of a functional catalytic triad in the active site

Hepatitis A virus (HAV) 3C proteinase is a member of the picornain cysteine proteases responsible for the processing of the viral polyprotein, a function essential for viral maturation and infectivity. This and its structural similarity to other 3C and 3C-like proteases make it an attractive target for the development of antiviral drugs. Previous solution NMR studies have shown that a Cys24Ser (C24S) variant of HAV 3C protein, which displays catalytic properties indistinguishable from the native enzyme, is irreversibly inactivated by N-benzyloxycarbonyl-l-serine-beta-lactone (1a) through alkylation of the sulfur atom at the active site Cys172. However, crystallization of an enzyme-inhibitor adduct from the reaction mixture followed by X-ray structural analysis shows only covalent modification of the epsilon2-nitrogen of the surface His102 by the beta-lactone with no reaction at Cys172. Re-examination of the heteronuclear multiple quantum coherence (HMQC) NMR spectra of the enzyme-inhibitor mixture indicates that dual modes of single covalent modification occur with a >/=3:1 ratio of S-alkylation of Cys172 to N-alkylation of His102. The latter product crystallizes readily, probably due to the interaction between the phenyl ring of the N-benzyloxycarbonyl (N-Cbz) moiety and a hydrophobic pocket of a neighboring protein molecule in the crystal. Furthermore, significant structural changes are observed in the active site of the 3C protease, which lead to the formation of a functional catalytic triad with Asp84 accepting one hydrogen bond from His44. Although the 3C protease modified at Cys172 is catalytically inactive, the singly modified His102 N(epsilon2)-alkylated protein displays a significant level of enzymatic activity, which can be further modified/inhibited by N-iodoacetyl-valine-phenylalanine-amide (IVF) (in solution and in crystal) or excessive amount of the same beta-lactone inhibitor (in solution). The success of soaking IVF into HAV 3C-1a crystals demonstrates the usefulness of this new crystal form in the study of enzyme-inhibitor interactions in the proteolytic active site.     

The role of Munc18-1 in docking and exocytosis of peptide hormone vesicles in the anterior pituitary

BACKGROUND INFORMATION: Many neurons secrete classical transmitters from synaptic vesicles as well as peptide transmitters from LDCVs (large dense-core vesicles). Little is known about the mechanistic differences between these two secretory pathways. The soluble protein Munc18-1 is essential for synaptic vesicle secretion [Verhage, Maia, Plomp, Brussaard, Heeroma, Vermeer, Toonen, Hammer, van den Berg, Missler, et al. (2000) Science 287, 864-869.]. RESULTS: In the present study, we tested if Munc18 genes are also involved in peptidergic secretion from LDCVs using the anterior pituitary as a model system. We show that Munc18-1 is the dominant isoform expressed in the anterior pituitary. In Munc18-1 null mutant mice, the anterior pituitary developed normally and the five major endocrine cell types had a normal distribution. However, circulating peptide hormone levels were decreased by up to 50-fold in the null mutant, whereas the intracellular levels were significantly higher than that in controls. Ultrastructural analysis using the tannic acid method revealed striking differences in the distribution of secretory vesicles: (i) the number of exocytotic figures was mostly decreased in the null mutants and (ii) the LDCVs accumulated near but not at their target membrane. This is in contrast with the apparently normal distribution of synaptic vesicles in developing synapses in the null mutant (Verhage et al., 2000). CONCLUSIONS: We conclude that Munc18-1 is involved in the secretion of peptide hormones and in the docking of LDCVs. These results unmask an apparent mechanistic difference between LDCVs and synaptic vesicles.     

A structural mechanism of integrin alpha(IIb)beta(3) "inside-out" activation as regulated by its cytoplasmic face

Activation of the ligand binding function of integrin heterodimers requires transmission of an "inside-out" signal from their small intracellular segments to their large extracellular domains. The structure of the cytoplasmic domain of a prototypic integrin alpha(IIb)beta(3) has been solved by NMR and reveals multiple hydrophobic and electrostatic contacts within the membrane-proximal helices of its alpha and the beta cytoplasmic tails. The interface interactions are disrupted by point mutations or the cytoskeletal protein talin that are known to activate the receptor. These results provide a structural mechanism by which a handshake between the alpha and the beta cytoplasmic tails restrains the integrin in a resting state and unclasping of this interaction triggers the inside-out conformational signal that leads to receptor activation.     

Regulation of human endometrial transforming growth factor beta1 and beta3 isoforms through menstrual cycle and medroxyprogesterone acetate treatment

The progesterone-induced differentiation of endometrial tissue from proliferative into secretory and decidua seems to be modulated by locally produced hormones and cytokines. Transforming growth factor beta (TGFbeta). a cytokine produced by endometrial cells, has been shown to modulate endometrial cell proliferation in vitro. Our aim was to evaluate the effects of medroxyprogesterone acetate (MPA) and the influence of menstrual cycle on the expression of TGFbeta1 and TGFbeta3 in human endometrium in vivo. In a double-blind, placebo-controlled trial, 46 healthy women with regular menstrual cycles received either MPA (10 mg/day) or placebo during 10 days. Endometrial and blood samples were collected 8-12 hours after the last MPA or placebo administration. Patients were classified into three groups according to biopsy dating and treatment: proliferative [tissue]/placebo, secretory [tissue]/placebo and secretory [tissue]/MPA. The immunohistochemical distribution of TGFbeta1 and TGFbeta1 mRNA was similar in all groups. Immunoreactive TGFbeta3 was present in the epithelium in 9.1% of proliferative samples, in 41.2% of secretory/placebo samples and in 87.5% of secretory/MPA samples (p=0.001). In the stroma, the frequency of TGFbeta3 staining was markedly increased after treatment with MPA (62.5%) compared to placebo (proliferative: 9.1%; secretory: 5.9%; p=0.005). The levels of TGFbeta3 mRNA increased during the secretory phase and were higher in the MPA-treated group, being directly correlated with morphological endometrial differentiation. It is concluded that MPA administration to healthy women increased TGFbeta3 but did not change TGFbeta1 gene and protein expression in the endometrium. This finding suggests that TGFbeta3 may be a local factor mediating progesterone- and progestogen-induced endometrial differentiation.     

A novel mode of sensory transduction in archaea: binding protein-mediated chemotaxis towards osmoprotectants and amino acids

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation.