“Ordered” structure in solutions and gels of a globular protein as studied by small angle neutron scattering

Small-angle neutron scattering measurements were used for structural investigation of β-lactoglobulin solutions and heat-set gels in conditions of strong double layer repulsions. At pH 9 and low ionic strength, a correlation peak was observed on the scattering curves of the solutions whatever the protein concentration C used (in the range C = 0.02–0.10 g/mL). The wave vector value q_max corresponding to these maxima scaled as C^0.25. This exponent value is in agreement with those reported in the literature for other globular proteins in the same concentration range. Increasing the ionic strength decreased the peak which vanished without changing position at 0.1M NaCl. This polyelectrolyte-like behaviour suggests a local structure in the protein solution due to double layer repulsions. In the case of heat-set aggregates and gels (0.02–0.13 g/mL) formed at pH 9 and low ionic strength, a peak in the scattering curves was also observed indicating that even after gelation a correlation is still present; q_max varied as C^0.5. As in the case of the solutions, the correlation peak decreased with increasing ionic strength, and it vanished at 0.06M NaCl. The dilution of the aggregates in order to determine their intraparticle structure factor showed that the correlations were lost and that the aggregates displayed the same internal structure as the elementary subunit in the gels. At high ionic strength, fractal structures of the aggregates down to a length scale of about 40 Å were observed with d_f = 1.3–1.75 ± 0.05, increasing with protein concentration. Subsequent dilution didn't change the fractal dimension of these structures. © 1996 John Wiley & Sons, Inc.     

In most Bradyrhizobium groups sequence comparison of 16S-23S rDNA internal transcribed spacer regions corroborates DNA-DNA hybridizations

In an extension of a previous small-scale test to assess the use of 16S-23S rDNA internal transcribed spacer (ITS) sequences for rapid grouping of bradyrhizobia, we have sequenced the ITS region of 32 isolates of Bradyrhizobium that had previously been studied using AFLP and DNA-DNA hybridizations. We also included representatives of Afipia and Rhodopseudomonas. Our results indicate that ITS sequences are very diverse among bradyrhizobia. Nevertheless, for most of the bradyrhizobia, the grouping of ITS sequences was in line with AFLP results and DNA-DNA hybridization data. Strains that have at least 95.5% ITS sequence similarity belong to the same genospecies, i.e. they have more than 60% DNA-DNA hybridization values. The ITS sequences can therefore provide a relatively fast way to guide strain identification and aid selection of the reference groups that should be included in DNA-DNA hybridization experiments for precise genotypic identification. The Bradyrhizobium strains isolated from Aeschynomene species showed a much larger diversity in ITS sequences than other bradyrhizobia, possibly as a result of lateral exchange. The above ITS sequence similarity criterion for genospecies therefore does not apply to them, but they can easily be distinguished from other Bradyrhizobium genospecies because they have a distinct tRNA(ala) gene.     

Antigen presentation by CD1d contributes to the amplification of Th2 responses to Schistosoma mansoni glycoconjugates in mice

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.     

Effect of hepatocyte growth factor on assembly of zonula occludens-1 protein at the plasma membrane

Hepatocyte growth factor (HGF) is a paracrine cytokine that influences epithelial morphogenesis by modulating cell–cell adhesion and cell polarity. We have examined the role of HGF in the tight junction (TJ) formation. We followed the assembly and disassembly at the plasma membrane of the major component of the TJ, zonula occludens-1 (ZO-1) protein, after HGF treatment. We applied HGF to the basolateral compartment of MDCK cell monolayers grown on transwell filters to analyze the effect of HGF on polarized cells. Confocal laser scanning microscopy showed that HGF caused a marked reduction of ZO-1 at the lateral sites and a concomitant increase in the cytoplasm. We used the calcium switch assay to analyze the effect of HGF in early TJ development. In MDCK cells cultured in low calcium levels, ZO-1 is distributed intracellularly. The presence of HGF greatly retarded the movement of ZO-1 from the cytosol to the membrane after restoration of normal (1.8 mM) calcium levels for 1.5 and 3 hr. The presence of HGF during the calcium switch caused increased tyrosine phosphorylation of β-catenin. The incubation of MDCK cells with vanadate, a potent tyrosine-specific phosphatase inhibitor, also affected the ZO-1 localization at the plasma membrane during the calcium switch. This was concomitant with increased tyrosine phosphorylation of β-catenin. These results suggest that HGF affects the TJ assembly, and this phenomenon may be important in loosening of intercellular junctions and migration of epithelial cells during HGF-induced morphogenesis. J. Cell. Physiol. 176:465–471, 1998. © 1998 Wiley-Liss, Inc.     

Impact of the HIV-1 env genetic context outside HR1-HR2 on resistance to the fusion inhibitor enfuvirtide and viral infectivity in clinical isolates

Resistance mutations to the HIV-1 fusion inhibitor enfuvirtide emerge mainly within the drug's target region, HR1, and compensatory mutations have been described within HR2. The surrounding envelope (env) genetic context might also contribute to resistance, although to what extent and through which determinants remains elusive. To quantify the direct role of the env context in resistance to enfuvirtide and in viral infectivity, we compared enfuvirtide susceptibility and infectivity of recombinant viral pairs harboring the HR1-HR2 region or the full Env ectodomain of longitudinal env clones from 5 heavily treated patients failing enfuvirtide therapy. Prior to enfuvirtide treatment onset, no env carried known resistance mutations and full Env viruses were on average less susceptible than HR1-HR2 recombinants. All escape clones carried at least one of G36D, V38A, N42D and/or N43D/S in HR1, and accordingly, resistance increased 11- to 2800-fold relative to baseline. Resistance of full Env recombinant viruses was similar to resistance of their HR1-HR2 counterpart, indicating that HR1 and HR2 are the main contributors to resistance. Strictly X4 viruses were more resistant than strictly R5 viruses, while dual-tropic Envs featured similar resistance levels irrespective of the coreceptor expressed by the cell line used. Full Env recombinants from all patients gained infectivity under prolonged drug pressure; for HR1-HR2 viruses, infectivity remained steady for 3/5 patients, while for 2/5 patients, gains in infectivity paralleled those of the corresponding full Env recombinants, indicating that the env genetic context accounts mainly for infectivity adjustments. Phylogenetic analyses revealed that quasispecies selection is a step-wise process where selection of enfuvirtide resistance is a dominant factor early during therapy, while increased infectivity is the prominent driver under prolonged therapy.     

Dietary myristic acid at physiologically relevant levels increases the tissue content of C20:5 n-3 and C20:3 n-6 in the rat

This study was designed to investigate the effect of myristic acid on the biosynthesis and metabolism of highly unsaturated fatty acids, when it is supplied in a narrow physiological range in the diet of the rat (0.2-1.2% of total dietary energy). Three experimental diets were designed, containing 22% of total dietary energy as lipids and increasing doses of myristic acid (0.71, 3.00 and 5.57% of total fatty acids). Saturated fat did not exceed 31% of total fat and the C18:3 n-3 amount in each diet was strictly equal (1.6% of total fatty acids). After 7 weeks, the diets had no effect on plasma cholesterol level but greatly modified the liver, plasma and adipose tissue saturated, monounsaturated and polyunsaturated fatty acid profiles. Firstly, daily intakes of myristic acid resulted in a dose-dependent tissue accumulation of myristic acid itself. Palmitic acid was significantly increased in the tissues of the rats fed the higher dose of myristic acid. A dose-response accumulation of tissue C16:1 n-7 as a function of dietary C14:0 was also shown. Secondly, a main finding was that, among n-3 and n-6 polyunsaturated fatty acids, a dose-response accumulation of liver and plasma C20:5 n-3 and C20:3 n-6 (two precursors of eicosanoids) as a function of dietary C14:0 was shown. This result suggests that dietary myristic acid may participate in the regulation of highly unsaturated fatty acid biosynthesis and metabolism.     

The microRNA pathway controls germ cell proliferation and differentiation in C. elegans

The discovery of the miRNA pathway revealed a new layer of molecular control of biological processes. To uncover new functions of this gene regulatory pathway, we undertook the characterization of the two miRNA-specific Argonaute proteins in Caenorhabditis elegans, ALG-1 and ALG-2. We first observed that the loss-of-function of alg-1 and alg-2 genes resulted in reduced progeny number. An extensive analysis of the germline of these mutants revealed a reduced mitotic region, indicating fewer proliferating germ cells. We also observed an early entry into meiosis in alg-1 and alg-2 mutant animals. We detected ALG-1 and ALG-2 protein expressions in the distal tip cell (DTC), a specialized cell located at the tip of both C. elegans gonadal arms that regulates mitosis-meiosis transition. Re-establishing the expression of alg-1 specifically in the DTC of mutant animals partially rescued the observed germline defects. Further analyses also support the implication of the miRNA pathway in gametogenesis. Interestingly, we observed that disruption of five miRNAs expressed in the DTC led to similar phenotypes. Finally, gene expression analysis of alg-1 mutant gonads suggests that the miRNA pathway is involved in the regulation of different pathways important for germline proliferation and differentiation. Collectively, our data indicate that the miRNA pathway plays a crucial role in the control of germ cell biogenesis in C. elegans.