Contribution of peaks of virus load to simian immunodeficiency virus pathogenesis

The mechanisms causing AIDS and subsequently death in human immunodeficiency virus type 1 infection are not yet fully understood. Nonetheless, correlates of accelerated progression to disease based on immunological and virological markers have been identified. The best correlate identified to date is the baseline virus load or the so-called viral set point. By focusing on a virus load measurement from a restricted time range, however, we ignore valuable information contained in the long-term profile of the virus load. Here, we investigate the relationship between virus load and survival with the aid of a statistical model. The model takes into consideration the virus load at every stage of the disease. In particular, we aim to determine the effect of peaks of virus load on disease progression. We fit our model to unique sequential viral load data of 12 simian immunodeficiency virus mac251-infected rhesus macaques which contain frequent measurements throughout the entire course of the infection until the development of simian AIDS. Our model enables us to predict the survival times of the animals more accurately than an equivalent model which considers the viral set point only. Furthermore, we find that peaks of the virus load contribute less to disease progression than phases of low virus load with the same amount of viral turnover. Our analysis implies that the total viral turnover is not the best correlate of survival. As a consequence, the direct cytopathic effects of virus replication may, by themselves, have less of an impact on disease progression than previously thought.     

A chimeric human T-cell lymphotropic virus type 1 with the envelope glycoprotein of Moloney murine leukemia virus is infectious for murine cells

We constructed a chimeric human T-cell lymphotropic virus type 1 (HTLV-1) provirus in which the original envelope precursor sequence was replaced by that of ecotropic Moloney murine leukemia virus (Mo-MuLV). Chimeric particles produced by transient transfection of this chimeric provirus were infectious for murine cells, such as NIH 3T3 fibroblasts, lymphoid EL4 cells, and primary CD4(+) T lymphocytes, whereas HTLV-1 particles were not. The infectivity of chimeric particles increased 10 times when the R peptide located at the carboxy terminus of the MuLV envelope glycoprotein was deleted. Primary murine CD4(+) T lymphocytes, infected by the Delta R chimeric virus, released particles that could spread the infection to other naive murine lymphoid cells. This chimeric virus, with the Mo-MuLV envelope glycoprotein and the replication characteristics of HTLV-1, should be useful in studying the pathogenesis of HTLV-1 in a mouse model.     

Theiler's virus infection of primary cultures of bone marrow-derived monocytes/macrophages

Theiler's virus, a murine picornavirus, causes a persistent infection of macrophage/microglial cells in the central nervous systems of SJL/J mice. Viral replication is restricted in the majority of infected cells, whereas a minority of them contain large amounts of viral RNA and antigens. For the present work, we infected primary cultures of bone marrow monocytes/macrophages from SJL/J mice with Theiler's virus. During the first 10 h postinfection (p.i.), infected monocytes/macrophages were round and covered with filopodia and contained large amounts of viral antigens throughout their cytoplasm. Later on, they were large, flat, and devoid of filopodia and they contained only small amounts of viral antigens distributed in discrete inclusions. These two types of infected cells were very reminiscent of the two types of infected macrophages found in the spinal cords of SJL/J mice. At the peak of virus production, the viral yield per cell was approximately 200 times lower than that for BHK-21 cells. Cell death occurred in the culture during the first 24 h p.i. but not thereafter. No infected cells could be detected after 4 days p.i., and the infection never spread to 100% of the cells. This restriction was unchanged by treating the medium at pH 2 but was abolished by treating it with a neutralizing alpha/beta interferon antiserum, indicating a role for this cytokine in limiting virus expression in monocyte/macrophage cultures. The role of alpha/beta interferon was confirmed by the observation that monocytes/macrophages from IFNA/BR(-/-) mice were fully permissive.     

Insight into the factors influencing the backbone dynamics of three homologous proteins,dendrotoxins I and K,and BPTI: FTIR and time-resolved fluorescence investigations

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, combined with hydrogen/deuterium exchange technique and time-resolved fluorescence spectroscopy, has been used to investigate the changes in structure and dynamics that underlie the thermodynamic stability differences observed for three closely homologous proteins: dendrotoxins I and K, and bovine pancreatic trypsin inhibitor (BPTI). The experiments were performed on proteins under their native state and a modified form, obtained by selective reduction of a disulfide bond at the surface of the molecule, increasing slightly the backbone flexibility without changing the average structure. The data confirmed the high local as well as global rigidity of BPTI. In protein K, the exchange process was slow during the first 2 h of exchange, presumably reflecting a compact three-dimensional conformation, and then increased rapidly, the internal amide protons of the beta-strands exchanging 10-fold faster than in BPTI or protein I. The most probable destabilizing element was identified as Pro32, in the core of the beta-sheet. Protein I was found to present a 10% more expanded volume than protein K or BPTI, and there is a possible correlation between the resulting increased flexibility of the molecule and the lower thermodynamic stability observed for this protein. Interestingly, the interior amide protons of the beta-sheet structure were found to be as protected against exchange in protein I as in BPTI, suggesting that, although globally more flexible than that of Toxin K or BPTI, the structure of Toxin I could be locally quite rigid. The structural factors suspected to be responsible for the differences in internal flexibility of the two toxins could play a significant role in determining their functional properties.     

Simultaneous suppression of multiple genes by single transgenes. Down-regulation of three unrelated lignin biosynthetic genes in tobacco

Many reports now describe the manipulation of plant metabolism by suppressing the expression of single genes. The potential of such work could be greatly expanded if multiple genes could be coordinately suppressed. In the work presented here, we test a novel method for achieving this by using single chimeric constructs incorporating partial sense sequences for multiple genes to target suppression of two or three lignin biosynthetic enzymes. We compare this method with a more conventional approach to achieving the same end by crossing plants harboring different antisense transgenes. Our results indicate that crossing antisense plants is less straightforward and predictable in outcome than anticipated. Most progeny had higher levels of target enzyme activity than predicted and had lost the expected modifications to lignin structure. In comparison, plants transformed with the chimeric partial sense constructs had more consistent high level suppression of target enzymes and had significant changes to lignin content, structure, and composition. It was possible to suppress three target genes coordinately using a single chimeric construct. Our results indicate that chimeric silencing constructs offer great potential for the rapid and coordinate suppression of multiple genes on diverse biochemical pathways and that the technique therefore deserves to be adopted by other researchers.     

Conversion of a postocellar into an ocellar region as a transdetermination event occurring in adult ribbonworms

Transections and grafting experiments performed in Lineus ruber rostral ends allowed us to generate ribbonworms with a duplication of the postocellar region combined with a deletion of the ocellar region. In such homeotically reconstructed animals, the syngeneic postocellar region transdifferentiated into an ocellar region with functional eyes while the allogeneic postocellar region underwent no transformation. In this case, transdifferentiation is a morphogenetic process leading to the restoration of the normal antero-posterior (A-P) axis pattern in adult worms. This regulative conversion of one adult body region into another, which so far has not been described in any bilaterian animal, is comparable with transdetermination of larval imaginal discs in Drosophila. Under certain conditions, Drosophila, wing imaginal disc cells express the eyeless master control gene and give rise to eyes. Here, we show in Lineus that the transposition of postocellar tissue into the ocellar location causes expression of the eyeless ortholog LsPax-6 and results in eye development. Our results in Lineus clearly suggest that transdifferentiation of adult body regions moved to a different position along the A-P axis is similar to transdetermination of the larval imaginal disc cells which are determined, but not yet differentiated.     

Muscle fibre ontogenesis in farm animal species

In farm animals (bovine, ovine, swine, rabbit and poultry), muscle fibre characteristics play a key role in meat quality. The present review summarises the knowledge on muscle fibre characteristics and ontogenesis in these species. Myofibre ontogenesis begins very early during embryonic life, with the appearance of two or three successive waves of myoblasts which constitute the origin of the different types of muscle fibres. In small animals (rodents and poultry), a primary and a secondary generation of fibres arise respectively during the embryonic and foetal stages of development. In the largest species (bovines, sheep, pigs) a third generation arises in the late foetal or early postnatal period. Following these two or three waves of myogenesis, the total number of fibres is fixed. This occurs during foetal life (bovines, ovines, pigs and poultry) or during the first postnatal month in rabbits. Contractile and metabolic differentiation proceed by steps in parallel to myogenesis and are partially linked to each other. In bovines and ovines, the main events occur during foetal life, whereas they occur soon after birth in the pig, poultry and rabbit, but some plasticity remains later in life in all species. This comparative survey shows that the cellular processes of differentiation are comparable between species, while their timing is usually species specific.     

Charged acrylamide copolymer gels as media for weak alignment

The use of mechanically strained acrylamide/acrylate copolymers is reported as a new alignment medium for biomacromolecules. Compared to uncharged, strained polyacrylamide gels, the negative charges of the acrylamide/acrylate copolymer strongly alter the alignment tensor and lead to pronounced electroosmotic swelling. The swelling itself can be used to achieve anisotropic, mechanical strain. The method is demonstrated for the alignment of TipAS, a 17 kDa antibiotic resistance protein, as well as for human ubiquitin, where alignment tensors with an A_ZZ,NH of up to 60 Hz are achieved at a gel concentration of 2% (w/v). The alignment can be modulated by the variation of pH, ionic strength, and gel concentration. The high mechanical stability of the swollen gels makes it possible to obtain alignment at polymer concentrations of less than 1% (w/v).     

Prenylated p-coumarates from the twigs of Phebalium rude subsp. amblycarpum (Rutaceae)

Phebarudol, a novel prenylated p-coumarate, was isolated from the twigs of Phebalium rude Bartl. subsp. amblycarpum (F. Muell.) P. G. Wilson (Rutaceae) together with the two already known related compounds, werneria chromene and methyl demethoxywutaiensate. The structure of phebarudol was established by spectroscopic methods.