Contribution of CD8+ T cells to containment of viral replication and emergence of mutations in Mamu-A*01-restricted epitopes in Simian immunodeficiency virus-infected rhesus monkeys

Here, we investigated the containment of virus replication in simian immunodeficiency virus (SIV) infection by CD8(+) lymphocytes. Escape mutations in Mamu-A*01 epitopes appeared first in SIV Tat TL8 and then in SIV Gag p11C. The appearance of escape mutations in SIV Gag p11C was coincident with compensatory changes outside of the epitope. Eliminating CD8(+) lymphocytes from rhesus monkeys during primary infection resulted in more rapid disease progression that was associated with preservation of canonical epitopes. These results confirm the importance of cytotoxic T cells in controlling viremia and the constraint on epitope sequences that require compensatory changes to go to fixation.     

Identification of halosalicylamide derivatives as a novel class of allosteric inhibitors of HCV NS5B polymerase

Halosalicylamide derivatives were identified from high-throughput screening as potent inhibitors of HCV NS5B polymerase. The subsequent structure and activity relationship revealed the absolute requirement of the salicylamide moiety for optimum activity. Methylation of either the hydroxyl group or the amide group of the salicylamide moiety abolished the activity while the substitutions on both phenyl rings are acceptable. The halosalicylamide derivatives were shown to be non-competitive with respect to elongation nucleotide and demonstrated broad genotype activity against genotype 1-3 HCV NS5B polymerases. Inhibitor competition studies indicated an additive binding mode to the initiation pocket that is occupied by the thiadiazine class of compounds and an additive binding mode to the elongation pocket that is occupied by diketoacids, but a mutually exclusive binding mode with respect to the allosteric thumb pocket that is occupied by the benzimidazole class of inhibitors. Therefore, halosalicylamides represent a novel class of allosteric inhibitors of HCV NS5B polymerase.     

Cryo-electron tomography of cells: connecting structure and function

Cryo-electron tomography (cryo-ET) allows the visualization of cellular structures under close-to-life conditions and at molecular resolution. While it is inherently a static approach, yielding structural information about supramolecular organization at a certain time point, it can nevertheless provide insights into function of the structures imaged, in particular, when supplemented by other approaches. Here, we review the use of experimental methods that supplement cryo-ET imaging of whole cells. These include genetic and pharmacological manipulations, as well as correlative light microscopy and cryo-ET. While these methods have mostly been used to detect and identify structures visualized in cryo-ET or to assist the search for a feature of interest, we expect that in the future they will play a more important role in the functional interpretation of cryo-tomograms.     

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary

Background  

The aging of reproductive organs is not only a major social issue, but of special interest in aging research. A long-standing view of 'immortal germ line versus mortal soma' poses an important question of whether the reproductive tissues age in similar ways to the somatic tissues. As a first step to understand this phenomenon, we examine global changes in gene expression patterns by DNA microarrays in ovaries and testes of C57BL/6 mice at 1, 6, 16, and 24 months of age. In addition, we compared a group of mice on ad libitum (AL) feeding with a group on lifespan-extending 40% calorie restriction (CR).     

An expert system to estimate the pesticide contamination of small streams using benthic macroinvertebrates as bioindicators II. The knowledge base of LIMPACT

The development and the evaluation of a biological indicator system for pesticide pollution in streams are presented. For small headwater streams with an agricultural catchment area, the expert system LIMPACT estimates the pesticide contamination according to the four classes: Not Detected (ND), Low (L), Moderate (M) and High (H) contamination without any specification of the chemical agents. The input parameters are the abundance data of benthic macroinvertebrate taxa within four time frames in a year (March/April, May/June, July/August, September/October) and nine basic water-quality and morphological parameters. The heuristic knowledge base was developed with the shell-kit D3 and contains 921 diagnostic rules with scores either to establish or to de-establish a diagnosis. The 418 rules had less than three symptoms, and only 47 rules had more than four symptoms in their rule condition. We differentiate between positive indicator (PI) taxa, which indicate contamination by high abundance values and positive abundance dynamics, and negative indicator (NI) taxa, a high abundance of which rules out contamination and indicates an uncontaminated site. We analysed 39 taxa and found 13 positive and 24 negative indicators. The database comprises 157 investigations per stream and year with rainfall event-controlled pesticide sampling and repeated benthic sampling as described in Part 1 [Ecol. Indicators, this issue]. For the evaluation of LIMPACT, we used the same cases. The correct class for the 157 investigations per stream and year is established by LIMPACT in 66.7–85.5% of the cases, with better results for uncontaminated sites. The overall alpha error probability (false positive) is 9.6% while the beta error probability (false negative) varied between 0 and 8% depending on the contamination class. If each stream is considered only once in the system (n=104), the correct diagnosis is established by LIMPACT in 51.9–88.6% of the cases. In most of the remaining cases no diagnosis is established instead of a wrong one.     

The Janus face of the archaeal Cdc48/p97 homologue VAT: protein folding versus unfolding.

Members of the AAA family of ATPases have been implicated in chaperone-like activities. We used the archaeal Cdc48/p97 homologue VAT as a model system to investigate the effect of an AAA protein on the folding and unfolding of two well-studied, heterologous substrates, cyclophilin and penicillinase. We found that, depending on the Mg2+ concentration, VAT assumes two states with maximum rates of ATP hydrolysis that differ by an order of magnitude. In the low-activity state, VAT accelerated the refolding of penicillinase, whereas in the high-activity state, it accelerated its unfolding. Both reactions were ATP-dependent. In its interaction with cyclophilin, VAT was ATP-independent and only promoted refolding. The N-terminal domain of VAT, which lacks ATPase activity, also accelerated the refolding of cyclophilin but showed no effect on penicillinase. VAT appears to be structurally equivalent over its entire length to Sec18/NSF, suggesting that these results apply more broadly to group II AAA proteins.