HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,detergent-resistant,raft membrane domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56(lck). Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.     

Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats

Transplantation of spermatogonial stem cells into syngeneic or immunosuppressed recipient mice or rats can result in donor-derived spermatogenesis and fertility. Recently, this approach has been employed to introduce a transgene into the male germline. Germ-cell transplantation in species other than laboratory rodents, if successful, holds great promise as an alternative to the inefficient methods currently available to generate transgenic farm animals that can produce therapeutic proteins in their milk or provide organs for transplantation to humans. To explore whether germ-cell transplantation could result in donor-derived spermatogenesis and fertility in immunocompetent recipient goats, testis cells were transplanted from transgenic donor goats carrying a human alpha-1 antitrypsin expression construct to the testes of sexually immature wild-type recipient goats. After puberty, sperm carrying the donor-derived transgene were detected in the ejaculates of two out of five recipients. Mating of one recipient resulted in 15 offspring, one of which was transgenic for the donor-derived transgene. This is the first report of donor cell-derived sperm production and transmission of the donor haplotype to the next generation after germ-cell transplantation in a nonrodent species. Furthermore, these results indicate that successful germ-cell transplantation is feasible between immunocompetent, unrelated animals. In the future, transplantation of genetically modified germ cells may provide a more efficient alternative for production of transgenic domestic animals.     

The respiratory metabolism of a lizard (Lacerta vivipara) in supercooled and frozen states

We investigated the respiratory metabolism of the overwintering lizard Lacerta vivipara while in either supercooled or frozen states. With a variable pressure and volume microrespirometer and a chromatograph, we show that the oxygen consumption of the supercooled animals showed a nonlinear relationship with temperature and an aerobic metabolism demand between 0.5 and -1.5 degrees C. A significant increase in the respiratory quotient (RQ) values indicated an increasing contribution by the anaerobic pathways with decreasing temperature. In the frozen state, two phases are easily detectable and are probably linked to the ice formation within the body. During the first 5-6 h, the animals showed an oxygen consumption of 3.52 +/- 0.28 microl. g(-1). h(-1) and a RQ value of 0.52 +/- 0.09. In contrast, after ice equilibrium, oxygen consumption decreased sharply (0.55 +/- 0.09 microl. g(-1). h(-1)) and the RQ values increased (2.49 +/- 0.65). The present study confirms the fact that supercooled invertebrates and vertebrates respond differently to subzero temperatures, in terms of aerobic metabolism, and it shows that aerobic metabolism persists under freezing conditions.     

In vitro tests for haematotoxicity: prediction of drug-induced myelosuppression by the CFU-GM assay

In a prevalidation study, a standard operating procedure (SOP) for human and mouse in vitro tests was developed, for evaluating the potential haematotoxicity of xenobiotics in terms of their direct, adverse effects on the myeloid colony-forming unit (CFU-GM). Based on the adjustment of the mouse-derived maximum tolerated dose (MTD), a prediction model was set up to calculate the human MTD, and an international blind trial was designed to apply this model to the clinical neutropenia of 23 drugs including 17 antineoplastics. The model correctly predicted the human MTD for 20 drugs out of the 23 (87%). This high percentage of predictivity, and the reproducibility of the SOP testing, confirmed the scientific validation of this model, and suggest promising applications for developing and validating other in vitro methods for use in haematotoxicology.     

Validation and use of an enzymic time-temperature integrator to monitor thermal impacts inside a solid/liquid model food

Heat denaturation kinetics of Bacillus licheniformis alpha-amylase, equilibrated at 81% equilibrium relative humidity at 4 degrees C (BLA81), was studied with help of isothermal and nonisothermal conditions by monitoring the decrease in enthalpy associated with the heat denaturation of the enzyme. Due to its low water content, BLA81 denaturation could be studied in the range of 118-124 degrees C. Two batches of BLA81 were successfully validated under nonisothermal conditions allowing the determinations of process values (reference temperature of 121.1 degrees C) in the range of 1-15 min. In a second step, BLA81 was used as a time-temperature integrator (TTI) to investigate potential differences of process values received by freely moving spherical particles as compared to a centrally fixed particle (single-position impact) inside cans containing water as brine. Results showed that the process value received by freely moving particles can be from 5.6% (4 rpm) to 19.7% (8 rpm) smaller than the process value received by the centrally fixed sphere. This means that evaluating the process value by means of a particle fixed at the critical point in a package can lead to potentially overestimations of the actual process value with possible hazardous quality/safety implications. These results highlight the potentials of the TTI technology to monitor the safety of heat-processed agitated solid/liquid foodstuffs.     

Intestinal preconditioning prevents systemic inflammatory response in hemorrhagic shock. Role of HO-1

Intestinal ischemia-reperfusion has been implicated in the systemic inflammatory response and organ injury in hemorrhagic shock, but the exact role of the intestine has never been directly demonstrated. Preconditioning (PC) with brief periods of intermittent ischemia is a known potent anti-ischemic intervention and thus can be used as a tool to assess the role of local intestinal ischemia-reperfusion injury in systemic inflammatory response. Thus rats were first subjected to sham surgery or intestinal preconditioning with four cycles of 1-min ischemia and 10 min of reperfusion 24 h before hemorrhagic shock followed by resuscitation. PC reduced fluid requirements, lung edema, and lactate and tumor necrosis factor-alpha production. These effects were abolished by the heme-oxygenase-1 (HO-1) inhibitor tin protoporphyrin (Sn-PP). PC induced more than fivefold in intestinal HO-1 expression. These results suggest that intestinal ischemia-reperfusion is a major trigger for inflammatory response and organ injury in nonseptic shock. HO-1 appears to play an important role in the protective effect of intestinal preconditioning.     

Protein stability and plasticity of the hydrophobic cavity in wheat ns-LTP

Plant ns-LTPs display an original structure with four helices and a flexible C-terminus, maintained together by four disulphide bridges and delineating an elongated central hydrophobic cavity. In order to relate these structural features to the protein stability and plasticity, combined molecular mechanics and simulated annealing calculations were undertaken on a wheat ns-LTP "mutant" with Cys-Ala replacement and with the application of core inter-residue restraints up to 2 A, reducing the cross-section size of the hydrophobic cavity. Analysis of the energy-minimized structures shows that removal of the disulphide bridges results in structures with a lower total energy and a smaller cavity volume. A 1-ns MD simulation at 300K in water, underlines that, despite the absence of a well-packed hydrophobic core, the native structure is extremely stable at room temperature and the cavity is not hydrated. This confirms that the disulphide bridges are essential for the existence of the cavity, whereas its plasticity depends both on the hydrophobic chain lining the cavity and on the C-terminal flexibility. A high temperature (500K) MD simulation confirms the stability of the secondary structure elements and the flexibility of the loops and of the C-terminal segment. Two important structural transitions during this simulation are discussed and possible routes for the insertion and release of hydrophobic ligands are suggested.     

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Résumé

À l'inverse de la situation des vertébrés et de la majorité des insectes, chez qui un gène unique code pour l'acétylcholinestérase (AChE), quatre gènes d'AChE ont été clones et caractérisés chez Caenorhabditis elegans. Le gène ace-1 (localisé sur le chromosome X) et le gène ace-2 (chromosome I) assurent respectivement l'expression de l'AChE dans les tissus musculaire (ace-1) et nerveux (ace-2). Les gènes ace-x et ace-y ne sont séparés que de quelques centaines de paires de bases sur le chromosome II et leur rôle est pour l'instant inconnu.