Phospholipase of rat intestine: mode of action]

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.     

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.     

Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia

The photorespiratory activity of mesophyll protoplasts of Nicotiana plumbaginifolia has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important CO₂-sensitive O₂ uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO₂ fixation was 60% lower at 50% O₂ compared to that measured at 2% O₂. Using ¹⁸O₂ and mass spectrometry, we measured O₂ exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO₂ compensation point (47.4 micromoles O₂ per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO₂ concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the O₂ uptake to the same extent as CO₂ saturation. We conclude from these results that the major part of the CO₂-sensitive O₂ uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO₂ saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and 1 millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO₂ compensation point at 20% O₂), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% O₂, but was stronger at 50% O₂ (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar l-methionine-dl-sulfoximine (l-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO₂ compensation point at 20% O₂ (30% for BHB and 20% for l-MSO). From the sites of action of the four inhibitors tested, we suggest the inhibition of photosynthesis occurring after a period of active photorespiration to be due to the toxic accumulation of nonmetabolized phosphoglycolate.     

Simultaneous analysis of skin response to the different L.E.T. regions of a 55 MeV helium ion beam

Summary Rat tail epidermis was used to analyze the in vivo response of a biological system to heavy particle irradiation. The conical configuration of the rat tail gives rise to a variable energy degradation of the beam thus yielding information on the damage elicited by 2 different L.E.T. regions of the helium beam at different sites on the same sample. Cytochrome oxidase activity and epidermal thickness were used to analyze the metabolic and structural radioinduced response. Quantitative evaluation of radiation damage revealed marked variations within a few micrometers of tissue.     

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.     

Atrazine and diuron resistant plants from photoautotrophic protoplast-derived cultures of Nicotiana plumbaginifolia

Atrazine and diuron resistant clones were isolated from diploid photoautotrophic protoplastderived colonies of Nicotiana plumbaginifolia. Protoplasts were mutagenised with 0.1 mM N-ethyl-N-nitrosourea and colonies were screened for resistance after plating. Selection of calli was carried out on their ability to grow and green on a selective medium containing either atrazine or diuron. Plants were regenerated from most tolerant calli. Herbicide spray showed that plants of 6 and 4 clones were resistant to atrazine and diuron, respectively.Abbreviations Atrazine 2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine - diuron 3-(3,4-dichlorophenyl)-1,1-dimethylurea - NEU N-ethyl-N-nitrosourea - PSII photosystem II