Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3= splice site (3=ss) A1 but lack splicing at 5=ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3=ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3=ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5=ss D2. Here we show that an intronic G run (G I2 -1) represses the use of a second 5=ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G I2 -1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vifto-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here. R eplication of human immunodeficiency virus type 1 (HIV-1) is counteracted by four major classes of host-encoded restriction factors: APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), TRIM5␣ (tripartite motif 5␣), tetherin (BST-2, CD317, or HM1.24), and SAMHD1 (1-4). A3G (5) belongs to a family of cytidine deaminases that includes seven members (A3A to A3D and A3F to A3H) located in a gene cluster on chromosome 22 (6-8). It is encapsidated into newly assembled virions and introduces C-to-U substitutions during minus-strand synthesis, resulting in G-to-A hypermutations and aberrant DNA ends in the HIV-1 genome. Furthermore, A3G, independent of its deaminase activity, inhibits reverse transcriptase-mediated minus-strand elongation by direct binding to the viral RNA (9). This leads to massive impairment of viral replication (10). However, the HIV-1-encoded accessory protein Vif counteracts A3G by direct interaction, by inducing proteasomal degradation, and by repression of mRNA synthesis (10). Whereas HIV-1 is able to replicate efficiently in A3G-expressing cells, Vifdeficient virus strains are completely suppressed (5). Nevertheless, a narrowly restricted level of Vif is crucial for optimal HIV-1 replication since proteolytic processing of the Gag precursor at the p2/nucleocapsid processing site is inhibited by high levels of Vif (11).During the course of infection, the HIV-1 9-kb single-sense pre-mRNA is processed into more than 40 alternatively spliced m...

Prolonged impaired lung function after major abdominal surgery is not ameliorated by low tidal volume ventilation.

Mammalian CYP4B1 enzymes are cytochrome P450 monooxygenases that are responsible for the bioactivation of several exogenous pro-toxins including 4-ipomeanol (4-IPO). In contrast to the orthologous rabbit enzyme, we show here that native human CYP4B1 with a serine at position 427 is unable to bio-activate 4-IPO and does not cause cytotoxicity in HepG2 cells and primary human T-cells that overexpress these enzymes. We also demonstrate that a proline residue in the meander region at position 427 in human CYB4B1 and 422 in rabbit CYP4B1 is important for protein stability and rescues the 4-IPO bioactivation of the human enzyme, but is not essential for the catalytic activity of the rabbit CYP4B1 protein. Systematic substitution of native and p.S427P human CYP4B1 with peptide regions from the highly active rabbit enzyme reveals that 18 amino acids in the wild-type rabbit CYP4B1 protein are key for conferring high 4-IPO metabolizing activity. Introduction of 12 of the 18 amino acids that are also present at corresponding positions in other human CYP4 family members into the p.S427P human CYP4B1 protein results in a mutant human enzyme (P+12) that is as stable and as active as the rabbit wild-type CYP4B1 protein. These 12 mutations cluster in the predicted B–C loop through F-helix regions and reveal new amino acid regions important to P450 enzyme stability. Finally, by minimally re-engineering the human CYP4B1 enzyme for efficient activation of 4-IPO, we have developed a novel human suicide gene system that is a candidate for adoptive cellular therapies in humans.

Ageing, the progressive functional decline of virtually all tissues, affects numerous living organisms. Main phenotypic alterations of human skin during the ageing process include reduced skin thickness and elasticity which are related to extracellular matrix proteins. Dermal fibroblasts, the main source of extracellular fibrillar proteins, exhibit complex alterations during in vivo ageing and any of these are likely to be accompanied or caused by changes in gene expression. We investigated gene expression of short term cultivated in vivo aged human dermal fibroblasts using RNA-seq. Therefore, fibroblast samples derived from unaffected skin were obtained from 30 human donors. The donors were grouped by gender and age (Young: 19 to 25 years, Middle: 36 to 45 years, Old: 60 to 66 years). Two samples were taken from each donor, one from a sun-exposed and one from a sun-unexposed site. In our data, no consistently changed gene expression associated with donor age can be asserted. Instead, highly correlated expression of a small number of genes associated with transforming growth factor beta signalling was observed. Also, known gene expression alterations of in vivo aged dermal fibroblasts seem to be non-detectable in cultured fibroblasts.