The C-Terminal Domain of ERp29 Mediates Polyomavirus Binding, Unfolding, and Infection

Penetration of the endoplasmic reticulum (ER) membrane by polyomavirus (PyV) is a decisive step in virus entry. We showed previously that the ER-resident factor ERp29 induces the local unfolding of PyV to initiate the ER membrane penetration process. ERp29 contains an N-terminal thioredoxin domain (NTD) that mediates its dimerization and a novel C-terminal all-helical domain (CTD) whose function is unclear. The NTD-mediated dimerization of ERp29 is critical for its unfolding activity; whether the CTD plays any role in PyV unfolding is unknown. We now show that three hydrophobic residues within the last helix of the ERp29 CTD that were individually mutated to either lysine or alanine abolished ERp29's ability to stimulate PyV unfolding and infection. This effect was not due to global misfolding of the mutant proteins, as they dimerize and do not form aggregates or display increased protease sensitivity. Moreover, the mutant proteins stimulated secretion of the secretory protein thyroglobulin with an efficiency similar to that of wild-type ERp29. Using a cross-linking coimmunoprecipitation assay, we found that the physical interaction of the ERp29 CTD mutants with PyV is inefficient. Our data thus demonstrate that the ERp29 CTD plays a crucial role in PyV unfolding and infection, likely by serving as part of a substrate-binding domain.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-Hydroxy-1-methoxy-Δ-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

γ-glutamyl transpeptidase of spermatozoa may decrease oocyte glutathione content at fertilization in pigs

The presence of γ-glutamyl transpeptidase (GGT) in boar spermatozoa and the potential role of the GGT at sperm penetration were examined using in vitro matured porcine oocytes. In the first experiment, GGT of boar spermatozoa was examined using a histochemical stain. GGT was detected in the midpiece and the acrosome regions of boar spermatozoa. In the second experiment, porcine oocytes matured in vitro were injected with approximately 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT or 1 mM guanosine-5′-0-(3′-thiotriphosphate) (GTP-γ-S; G-protein activator). When GGT was injected into oocytes, the incidence of oocytes activated (23.7 ± 1.4%) was not different (P > 0.05) from HEPES-injected controls (24.9 ± 1.3%) at 6 h after injection. Injected GTP-γ-S, however, activated 76.0 ± 5.3% of oocytes at 6 h after injection, but extrusion of the second polar body was very low (2.8 ± 4.8%). Total content of glutathione (GSH) and glutathione disulfide (GSSG) did not differ (P > 0.05) between GTP-γ-S injected oocytes (4.2 ± 0.7 pmol/oocyte) and noninjected oocytes (4.0 ± 0.1 pmol/oocyte) at 6 h after injection. However, the total content of GSH and GSSG was lower (P < 0.01) in GGT-injected oocytes (2.1 ± 0.2 pmol/oocyte) than HEPES-injected oocytes (3.4 ± 0.2 pmol/oocyte) at 6 h after injection. In the third experiment, in vitro matured porcine oocytes were injected with about 40 pl of 10 mM HEPES solution alone or HEPES containing 0.5 U/ml GGT and then inseminated. At 12 h after insemination, the incidence of male pronuclear formation was significantly lower in oocytes injected with GGT as compared with injected control oocytes. These results demonstrated that (1) GGT was present on the surface of spermatozoa, (2) total oocyte content of GSH and GSSG was decreased by microinjection of GGT but not by that of GTP-γ-S, and (3) male pronuclear formation was inhibited in GGT-injected oocytes. These results suggest that sperm GGT may be a limiting factor for male pronuclear formation in polyspermic oocytes. © 1996 Wiley-Liss, Inc.     

Identification of S-hydroxylysyl-methionine as the covalent cross-link of the noncollagenous (NC1) hexamer of the alpha1alpha1alpha2 collagen IV network: a role for the post-translational modification of lysine 211 to hydroxylysine 211 in hexamer assembly

Collagen IV networks are present in all metazoans as components of basement membranes that underlie epithelia. They are assembled by the oligomerization of triple-helical protomers, composed of three alpha-chains. The trimeric noncollagenous domains (NC1) of each protomer interact forming a hexamer structure. Upon exposure to acidic pH or denaturants, the hexamer dissociates into monomer and dimer subunits, the latter reflect distinct interactions that reinforce/cross-link the quaternary structure of hexamer. Recently, the cross-link site of the alpha1alpha1alpha2 network was identified, on the basis of x-ray crystal structures at 1.9-A resolution, in which the side chains of Met93 and Lys211 were proposed to be connected by a novel thioether bond (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P., Bartunik, H. D., and Bode, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6607-6612); however, at the higher resolution of 1.5 A, we found no evidence for this cross-link (Vanacore, R. M., Shanmugasundararaj, S., Friedman, D. B., Bondar, O., Hudson, B. G., and Sundaramoorthy, M. (2004) J. Biol. Chem. 279, 44723-44730). Given this discrepancy in crystallographic findings, we sought chemical evidence for the location and nature of the reinforcement/cross-link site. Trypsin digestion of monomer and dimer subunits excised a approximately 5,000-Da complex that distinguished dimers from monomers; the complex was characterized by mass spectrometry, Edman degradation, and amino acid composition analyses. The tryptic complex, composed of two peptides of 44 residues derived from two alpha1 NC1 monomers, contained Met93 and Lys211 post-translationally modified to hydroxylysine (Hyl211). Truncation of the tryptic complex with post-proline endopeptidase reduced its size to 14 residues to facilitate characterization by tandem mass spectrometry, which revealed a covalent linkage between Met93 and Hyl211. The novel cross-link, termed S-hydroxylysyl-methionine, reflects at least two post-translational events in its formation: the hydroxylation of Lys211 to Hyl211 within the NC1 domain during the biosynthesis of alpha-chains and the connection of Hyl211 to Met93 between the trimeric NC1 domains of two adjoining triple-helical protomers, reinforcing the stability of collagen IV networks.     

Genome sequences of the ethanol-tolerant Lactobacillus vini strains LMG 23202T and JP7.8.9

We report on the genome sequences of Lactobacillus vini type strain LMG 23202(T) (DSM 20605) (isolated from fermenting grape musts in Spain) and the industrial strain L. vini JP7.8.9 (isolated from a bioethanol plant in northeast Brazil). All contigs were assembled using gsAssembler, and genes were predicted and annotated using Rapid Annotation using Subsystem Technology (RAST). The identified genome sequence of LMG 23202(T) had 2.201.333 bp, 37.6% G+C, and 1,833 genes, whereas the identified genome sequence of JP7.8.9 had 2.301.037 bp, 37.8% G+C, and 1,739 genes. The gene repertoire of the species L. vini offers promising opportunities for biotechnological applications.     

Enantioselective synthesis of 1-methoxy- and 1-deoxy-2'-methyl-delta8-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Two new series of cannabinoids were prepared and their affinities for the CB1 and CB2 receptors were determined. These series are the (2'R)- and (2'S)-1-methoxy- and 1-deoxy-3-(2'-methylalkyl)-delta8-tetrahydrocannabinols, with alkyl side chains of three to seven carbon atoms. These compounds were prepared by a route that employed the enantioselective synthesis of the resorcinol precursors to the cannabinoid ring system. All of these compounds have greater affinity for the CB2 receptor than the CB1 receptor and four of them, (2'R)-1-methoxy-3-(2'-methylbutyl)-delta8-THC (JWH-359), (2'S)-1-deoxy-3-(2'-methylbutyl)-delta8-THC (JWH-352), (2'S)-1-deoxy-3-(2'-methylpentyl)-delta8-THC (JWH-255), and (2'R)-1-deoxy-3-(2'-methylpentyl)-delta8-THC (JWH-255), have good affinity (K(i) = 13-47 nM) for the CB2 receptor and little affinity (K(i) = 1493 to >10,000 nM) for the CB1 receptor. In the 1-deoxy-3-(2'-methylalkyl)-delta8-THC series, the 2'S-methyl compounds in general have greater affinity for the CB2 receptor than the corresponding 2'R isomers.     

Caribbean corals in crisis: record thermal stress, bleaching, and mortality in 2005

Background

The rising temperature of the world''s oceans has become a major threat to coral reefs globally as the severity and frequency of mass coral bleaching and mortality events increase. In 2005, high ocean temperatures in the tropical Atlantic and Caribbean resulted in the most severe bleaching event ever recorded in the basin.

Methodology/Principal Findings

Satellite-based tools provided warnings for coral reef managers and scientists, guiding both the timing and location of researchers'' field observations as anomalously warm conditions developed and spread across the greater Caribbean region from June to October 2005. Field surveys of bleaching and mortality exceeded prior efforts in detail and extent, and provided a new standard for documenting the effects of bleaching and for testing nowcast and forecast products. Collaborators from 22 countries undertook the most comprehensive documentation of basin-scale bleaching to date and found that over 80% of corals bleached and over 40% died at many sites. The most severe bleaching coincided with waters nearest a western Atlantic warm pool that was centered off the northern end of the Lesser Antilles.

Conclusions/Significance

Thermal stress during the 2005 event exceeded any observed from the Caribbean in the prior 20 years, and regionally-averaged temperatures were the warmest in over 150 years. Comparison of satellite data against field surveys demonstrated a significant predictive relationship between accumulated heat stress (measured using NOAA Coral Reef Watch''s Degree Heating Weeks) and bleaching intensity. This severe, widespread bleaching and mortality will undoubtedly have long-term consequences for reef ecosystems and suggests a troubled future for tropical marine ecosystems under a warming climate.