ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.     

A FTZ-F1-containing yeast artificial chromosome recapitulates expression of steroidogenic factor 1 in vivo

Steroidogenic factor 1 (SF-1/Nr5a1) is an orphan nuclear receptor encoded by the Ftz-F1 gene and is required for gonad and adrenal development and regulation of hormone production within the reproductive and adrenal axes. To extend our understanding of Ftz-F1 and its role in SF-1 expression, we identified and characterized a yeast artificial chromosome (YAC) containing Ftz-F1. Within this YAC, Ftz-F1 is centrally located and flanked by genes encoding a second orphan nuclear receptor, germ cell nuclear factor, and proteasome (prosome, macropain) subunit beta type 7. Three lines of transgenic mice carrying the YAC were generated and in two lines (lines 7 and 14), RT-PCR and ribonuclease protection analysis showed that expression of transgenic SF-1 mimicked that of endogenous SF-1, both spatially and quantitatively. In the third line (line 15), pituitary and hypothalamic expression were absent. Comparison of the integrated transgenes revealed that line 15 was truncated at the end of intron 4 and revealed a region within the locus that is responsible for SF-1 expression in the pituitary and hypothalamus. The line 14 transgene was introduced into a mouse strain lacking functional SF-1. Examination of SF-1-deficient, transgene-positive mice revealed that the YAC was able to rescue adrenal and gonad development, which normally arrests in the SF-1-null embryos and showed that the 153-kb transgene integrated in line 14 is sufficient to properly direct SF-1 expression and support its biological activity. Thus, the study defines a region of Ftz-F1 that contains the requisite set of regulatory elements to direct SF-1 cell-specific expression and all temporal and quantitative changes need for its biological activity.     

Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells

Villin is an actin-binding protein present in intestinal and kidney brush borders. Villin has been shown to present in vitro Ca(2+)-dependent bundling and severing F-actin properties. The study of villin knock-out mice allowed us to show that while bundling of F-actin microfilaments is unaffected, this protein is important for the reorganization of the actin cytoskeleton elicited by various signals during both physiological and pathological conditions. Here, we studied the role of villin during infection by Shigella flexneri, the causative agent of bacillary dysentery. This bacterium induces the reorganization of the host actin cytoskeleton to penetrate into epithelial cells and spread from cell to cell. In vivo, we show that unlike newborn vil+/+ mice, which are sensitive to Shigella invasion, resulting in a destructive inflammatory response of the intestinal mucosa following intragastric inoculation, newborn vil-/- mice appear fully resistant to infection. Using primary cultures of intestinal epithelial cells derived from vil+/+ or vil -/- mice, we demonstrate that villin plays an essential role in S. flexneri entry and cell-to-cell dissemination. Villin expression is thus critical for Shigella infection through its ability to remodel the actin cytoskeleton.     

Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity

Increasing numbers of bacterial strains being resistant to conventional antibiotics emphasize the urgent need for new antimicrobial agents. One strategy is based on host defence peptides that can be found in every organism including humans. We have studied the antimicrobial peptide LF11, derived from the pepsin cleavage product of human lactoferrin, known for its antimicrobial and lipid A-binding activity, and peptide C12LF11, the N-lauryl-derivative of LF11, which has owing to the attached hydrocarbon chain an additional hydrophobic segment. The influence of this hydrocarbon chain on membrane selectivity was studied using model membranes composed of dipalmitoylphosphatidylglycerol (DPPG), mimicking bacterial plasma membranes, and of dipalmitoylphosphatidylcholine (DPPC), a model system for mammalian membranes. A variety of biophysical techniques was applied. Thereby, we found that LF11 did not affect DPPC bilayers and showed only moderate effects on DPPG membranes in accordance with its non-hemolytic and weak antimicrobial activity. In contrast, the introduction of the N-lauryl group caused significant changes in the phase behaviour and lipid chain packing in both model membrane systems. These findings correlate with the in vitro tests on methicillin resistant S. aureus, E. coli, P. aeruginosa and human red blood cells, showing increased biological activity of C12LF11 towards these test organisms. This provides evidence that both electrostatic and hydrophobic interactions are crucial for biological activity of antimicrobial peptides, whereas a certain balance between the two components has to be kept, in order not to loose the specificity for bacterial membranes.     

Effects of different green manures on soil biological properties and maize yield

The utilization of green manures as alternatives to reduce the use of mineral fertilizers is considered a good agricultural practice. However, the effect of each green manure on soil properties and crop yield depends upon its chemical composition. The main objective of this work was to study the effect of incorporating three green manures originating from residues of Trifolium pratense, L. (TP), Brassica napus, L. (BN), and the mixture of TP+BN at rates of 5384 and 8973 kg C ha(-1), on soil biological properties (soil microbial biomass-C, soil respiration and soil enzymatic activities), nutrition (leaf N, P and K concentration, pigments and soluble carbohydrate concentrations) and yield parameters of maize (Zea mays cv. Tundra) crop for four years on an Typic Xerofluvent located near Sevilla (Guadalquivir Valley, Andalusia, Spain). All green manures had a positive effect on the soil biological properties, plant nutrition an crop yield parameters, although at the end of the experimental period and at the high organic matter rate, the soil microbial biomass and dehydrogenase, urease, beta-glucosidase, phosphatase and arylsulfatase activities increased more significantly in the TP amended soils (79.2%, 92.1%, 93.9%, 99.3%, 87.9% and 96%, respectively) respect to the control soil, followed by TP+BN amended soils (77.3%, 90.9%, 92.8%, 99.1%, 84.4% and 95.7%, respectively) and BN amended soils (76%, 90.1%, 91.7%, 99%, 83.2% and 95.2%, respectively). Since these soil enzymatic activities measured are responsible for important cycles such as C, N, P and S, an increase of leaf N, P an K contents and pigments and soluble carbohydrate contents were highest in TP amended soils, followed by TP+BN and BN treatments. The application of TP in soils at high doses increased the grain protein concentration, number of grains corncob(-1) and crop yield 44.6%, 6.3% and 22.1%, respectively, compared with the control soil, followed by TP+BN treatment (41.7%, 5.7% and 20.8%, respectively) and BN treatment (39%, 5.3% and 20%, respectively). The explanation of these results can be a consequence to the different chemical composition of the green manures applied to the soils and its mineralization, aspect controlled by the soil C/N ratio.     

Orally administered tryptophan and experimental type 2 diabetes

There is a link between diabetes and oxidative stress. Hyperglycaemia leads to free radical generation and alterations of endogenous antioxidants. Our aim is to study the effect of orally administered L-tryptophan (TRP), the melatonin precursor, an endogenous antioxidant, on circulating levels of glycaemia, insulin and melatonin, and on the superoxide dismutase and catalase antioxidant systems in non-diabetic (ND) and type 2 diabetic (n5-STZ) male Wistar rats.At 19:30 every day for 15 days, TRP (125 mg/kg body weight) was administered orally. At 09:00 every two days the glycaemia was measured and every day the intake of food and water was recorded. At the beginning and end of treatment (at 09:00; 21:00; 02:00) plasma insulin and melatonin levels were measured, and (at 09:00) the enzymatic activities of catalase and superoxide dismutase (SOD) in erythrocytes were also measured. Glycaemia values were greater (p < 0.01) in n5-STZ rats than in ND rats, while insulin levels were lower (p < 0.05) at all times studied and these parameters were not altered by the TRP administration. Melatonin levels at 02:00 were lower in n5-STZ than in ND rats (p < 0.05). The TRP administration did not modify the circulating melatonin levels in ND rats, but raised (p < 0.01) the levels at 02:00 in the treated n5-STZ group. In ND rats after TRP administration there was a decline in catalase activity (p < 0.05), while in n5-STZ rats there was a rise (p < 0.01) at the end of treatment. However, there were no significant changes in SOD activity. There was increased food intake (g/day) in the treated n5-STZ group (p < 0.01). In conclusion, the oral administration of TRP did not modify glycaemia or insulinaemia levels, but raised melatonin levels in diabetic rats at 02:00, lowered catalase activity in ND rats but raised it in n5-STZ rats, and increased food intake in n5-STZ rats. (Mol Cell Biochem 261: 57–61, 2004)     

Culture of human corpus cavernosum endothelium

Summary A method for culturing endothelial cells (HCC-EC) from surgical specimens of human corpus cavernosum has been developed. The approach involves selective endothelial outgrowth from explants and may be generally applicable to tissue whose endothelium is not amenable to isolation by routine mechanical or enzymatic methods. The tissue is minced into pieces which are placed onto gelatin-or fibronectin-coated tissue culture plastic, and grown in medium suitable for microvascular endothelial cell growth (Carson and Haudenschild, In Vitro 22:344–354, 1986). By Days 5 to 7 EC colonies are found. Within a day or two after the appearance of the EC colonies, a non-EC cell type appears and, if undisturbed, quickly overgrows the EC. An exploitable temporal separation between the emergence of EC and non-EC is obtained when both conditioned medium (from bovine aortic endothelium) and retinal extract are present during the outgrowth period. Explants are removed by pipetting at the first sign of the emergence of the non-EC cell type. Once isolated, HCC-EC do not require conditioned medium but do require either retinal extract or acidic fibroblast growth factor for survival and growth. Approximately 60% of the first passage cultures are at least 80% EC as judged by DiI-Ac-LDL labeling. One corpus (0.3×0.3×0.5 cm) usually produces 120 cm² of primary culture within 2 wk. These EC form contact-inhibited monolayers and stain positively for Factor VIII. They have a doubling time at 6th passage of 48 h and a plateau density of 5 to 7×10⁴ cells/cm². The availability of such cultures should facilitate the study of endothelium-mediated responses which play an important role in the erectile function of human penile corpus cavernosum. Supported by NIH R01 HL23567-09, DK-39080-01, DK40025-01, DK40487-01.