Hydrocarbon-metabolizing activity of various mammalian cells in culture

Summary Two methods for determining the hydrocarbon-metabolizing enzyme activity of cultured mammalian cells were compared. The method designed to measure benzo[a]an-thracene-induced aryl hydrocarbon hydroxylase activity could detect and quantify enzyme activities in low passage rodent cells, but could not reproducibly detect levels in intermediate or high passage mouse, rat, or human cells. The method designed to measure the ability of a cell to convert benzo[a]pyrene from an organic-soluble to an aqueous acetone-soluble form proved to be more reproducible. This technique, when modified, was demonstrated to be an effective screening test for the detection of those lines with higher levels of hydrocarbon-metabolizing enzymes. Supported by the Council for Tobacco Research and Contract NIH 70-2068 within the Virus Cancer Program, National Cancer Institute, National Institutes of Health.     

2 A prebiotic RNA apparatus functions within the contemporary ribosome

Ribosomes, the universal cellular machines, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region, which was identified within all contemporary ribosomes. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA molecular apparatus. Hence, we named it the “proto-ribosome”. This prebiotic pocket-like RNA entity is suggested to be capable to accommodate substrates whose stereochemistry enables the creation of chemical bonds. It could have evolved from an earlier catalytic RNA entity that we named the “pre-proto-ribosome”, presumed to be a molecular machine capable of performing various essential tasks in the RNA world, which was snatched by the amino acid invaders for producing proteins.     

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.