Red blood cells imaging and antigen-antibody interaction measurement

In the present study the atomic force microscope (AFM) was used to image the surface morphology of red blood cells (RBC) for the first time. The AFM yielded very reproducible images without appreciable modifications of the sample surfaces. In addition to this topographical imaging, we have developed an experimental approach to measure the binding strength between antibody (anti-A), and the RBC antigen A, when reversible bonds between specific molecules such as antigen and antibody mediate the adhesion. The experimental results suggest that the procedure established here may be used for specific antibody detection. This study has also enhanced our understanding under physiological conditions of molecular interaction in particular antigen-antibody.     

Responses of Arabidopsis thaliana to bicarbonate-induced iron deficiency

Morpho-physiological and biochemical responses of Arabidopsis thaliana (accession N1438) to bicarbonate-induced iron deficiency were investigated. Plants were grown in cabinet under controlled conditions, in a nutrient solution containing 5 μM Fe, added or not with 10 mM NaHCO₃. After 30 days, bicarbonate-treated plants displayed significantly lower biomass, leaf number and leaf surface area as compared to control plants, and slight yellowing of their younger leaves was observed. Potassium (K⁺) content was not modified by bicarbonate treatment in roots, whereas it was significantly diminished in shoots. Their content in ferrous iron (Fe²⁺) and in leaf total chlorophylls was noticeably lower than in control plants. Root Fe(III)-chelate reductase and phosphoenolpyruvate carboxylase (PEPC) activities were significantly enhanced, but leaf ribulose 1.5-bisphosphate carboxylase (Rubisco) activity was decreased.     

Individual rotavirus-like particles containing 120 molecules of fluorescent protein are visible in living cells

Rotaviruses are large, complex icosahedral particles consisting of three concentric capsid layers. When the innermost capsid protein VP2 is expressed in the baculovirus-insect cell system it assembles as core-like particles. The amino terminus region of VP2 is dispensable for assembly of virus-like particles (VLP). Coexpression of VP2 and VP6 produces double layered VLP. We hypothesized that the amino end of VP2 could be extended without altering the auto assembly properties of VP2. Using the green fluorescent protein (GFP) or the DsRed protein as model inserts we have shown that the chimeric protein GFP (or DsRed)-VP2 auto assembles perfectly well and forms fluorescent VLP (GFP-VLP2/6 or DsRed-VLP2/6) when coexpressed with VP6. The presence of GFP inside the core does not prevent the assembly of the outer capsid layer proteins VP7 and VP4 to give VLP2/6/7/4. Cryo-electron microscopy of purified GFP-VLP2/6 showed that GFP molecules are located at the 5-fold vertices of the core. It is possible to visualize a single fluorescent VLP in living cells by confocal fluorescent microscopy. In vitro VLP2/6 did not enter into permissive cells or in dendritic cells. In contrast, fluorescent VLP2/6/7/4 entered the cells and then the fluorescence signal disappear rapidly. Presented data indicate that fluorescent VLP are interesting tools to follow in real time the entry process of rotavirus and that chimeric VLP could be envisaged as "nanoboxes" carrying macromolecules to living cells.     

Antibiotic resistance and survival of faecal coliforms in activated sludge system in a semi-arid region (Beni Mellal,Morocco)

The activated sludge process is one of the biological treatment methods used in many countries to reduce the high levels of organic and mineral pollutants and pathogenic micro-organisms present in wastewater. The present work was undertaken to study the dynamic and antibiotic-resistance of faecal coliforms (FC) in the activated sludge system of Beni Mellal. This work has also as objective the study of the survival of FC, protozoan cysts, helminth eggs and FC antibiotic resistance in the sludge dehydrated in drying beds in order to know if the agricultural usage of sludge presents any problems to public health. The activated sludge treatment of Beni Mellal resulted in an average reduction of FC and faecal streptococci of 90.75 and 91.06%, respectively. The overall resistance (resistance to at least one antibiotic) of 111 FC strains isolated from the system was 72.07%. This treatment system did not increase the incidence of FC antibiotic resistance in treated wastewaters. The antibiotic resistance of FC was found to be similar in both raw (71.05%) and treated sewage (77.77%). High levels of antibiotic resistance were towards streptomycin (54.05%), ampicillin (42.34%), amoxicillin (42.34%) and amoxicillin–clavulanic acid (31.53%). The treatment of sludge in drying beds appeared to be efficient in eliminating pathogenic micro-organisms: FC, protozoan cysts and helminth eggs. Moreover, the FC antibiotic resistance did not change over time in sludge-drying bed. According to the standard norms, agricultural utilization of this sludge cannot be excluded. However, it is important to study in the receptor environment the survival and the behaviour of antibiotic-resistant FC present in sludge and water.     

Different MHC class I heavy chains compete with each other for folding independently of beta 2-microglobulin and peptide

We reported previously that different MHC class I molecules can compete with each other for cell surface expression in F(1) hybrid and MHC class I transgenic mice. In this study, we show that the competition also occurs in transfected cell lines, and investigate the mechanism. Cell surface expression of an endogenous class I molecule in Chinese hamster ovary (CHO) cells was strongly down-regulated when the mouse K(d) class I H chain was introduced by transfection. The competition occurred only after K(d) protein translation, not at the level of RNA, and localization studies of a CHO class I-GFP fusion showed that the presence of K(d) caused retention of the hamster class I molecule in the endoplasmic reticulum. The competition was not for beta(2)-microglobulin, because a single chain version of K(d) that included mouse beta(2)-microglobulin also had a similar effect. The competition was not for association with TAP and loading with peptide, because a mutant form of the K(d) class I H chain, not able to associate with TAP, caused the same down-regulation of hamster class I expression. Moreover, K(d) expression led to a similar level of competition in TAP2-negative CHO cells. Competition for cell surface expression was also found between different mouse class I H chains in transfected mouse cells, and this competition prevented association of the H chain with beta(2)-microglobulin. These unexpected new findings show that different class I H chains compete with each other at an early stage of the intracellular assembly pathway, independently of beta(2)-microglobulin and peptide.     

Varied tolerance to NaCl salinity is related to biochemical changes in two contrasting lettuce genotypes

Salt stress perturbs a multitude of physiological processes such as photosynthesis and growth. To understand the biochemical changes associated with physiological and cellular adaptations to salinity, two lettuce varieties (Verte and Romaine) were grown in a hydroponics culture system supplemented with 0, 100 or 200 mM NaCl. Verte displayed better growth under 100 mM NaCl compared to Romaine, but both genotypes registered relatively similar reductions in growth under 200 mM NaCl treatment. Both varieties showed differences in net photosynthetic activity in the absence of salt and 8 days after salt treatment. These differences diminished subsequently under prolonged salt stress (14 days). Verte showed enhanced leaf proline and restricted total cations especially Na⁺, lesser malondialdehyde (MDA) formation and lignification in the roots under 100 mM NaCl salinity. Membrane damage estimated by electrolyte leakage increased with elevated salt concentrations in roots of both varieties, but Verte had significantly lower electrolyte leakage relative to Romaine under 100 mM NaCl. Moreover, Verte also accumulated greater levels of carotenoids under increasing NaCl concentrations compared to Romaine. Taken together, these findings suggest that the greater tolerance of Verte to 100 mM NaCl is related to the more restricted accumulation of total cations and toxic Na⁺ in the roots and enhanced levels of antioxidative metabolites in root and leaf tissue.     

Application of municipal solid waste compost reduces the negative effects of saline water in Hordeum maritimum L

The efficiency of composted municipal solid wastes (MSW) to reduce the adverse effects of salinity was investigated in Hordeum maritimum under greenhouse conditions. Plants were cultivated in pots filled with soil added with 0 and 40tha(-1) of MSW compost, and irrigated twice a week with tap water at two salinities (0 and 4gl(-1) NaCl). Harvests were achieved at 70 (shoots) and 130 (shoots and roots) days after sowing. At each cutting, dry weight (DW), NPK nutrition, chlorophyll, leaf protein content, Rubisco (ribulose-bisphosphate carboxylase/oxygenase) capacity, and contents of potential toxic elements were determined. Results showed that compost supply increased significantly the biomass production of non salt-treated plants (+80%). This was associated with higher N and P uptake in both shoots (+61% and +80%, respectively) and roots (+48% and +25%, respectively), while lesser impact was observed for K+. In addition, chlorophyll and protein contents as well as Rubisco capacity were significantly improved by the organic amendment. MSW compost mitigated the deleterious effect of salt stress on the plant growth, partly due to improved chlorophyll and protein contents and Rubisco capacity (-15%, -27% and -14%, respectively, in combined treatment, against -45%, -84% and -25%, respectively, in salt-stressed plants without compost addition), which presumably favoured photosynthesis and alleviated salt affect on biomass production by 21%. In addition, plants grown on amended soil showed a general improvement in their heavy metals contents Cu2+, Pb2+, Cd2+, and Zn2+ (in combined treatment: 190%, 53%, 168% and 174% in shoots and 183%, 42%, 42% and 114% in roots, respectively) but remained lower than phytotoxic values. Taken together, these findings suggest that municipal waste compost may be safely applied to salt-affected soils without adverse effects on plant physiology.     

Influence of nitrate—ammonium ratio on growth and nutrition of Arabidopsis thaliana

We investigated the microbial community structure and population size of arboreal soils—including canopy and bromeliad epiphytic leaf-tank soils—and ground soils in a tropical lowland rainforest in Costa Rica using molecular and cultivation methods. PCR-DGGE analysis of 16S rRNA and 18S rRNA gene fragments and quantitative real-time PCR were applied to survey the bacteria, ammonia-oxidizing bacteria (AOB), and fungi. Bacteria from epiphytic tank soils were isolated and identified. Bacillaceae, Pseudomonadaceae and Micrococcaceae were the most abundant families. According to cluster analysis of DGGE fingerprints a significant difference among the three soil types was evident for bacterial communities. In addition, the microbial communities of canopy and tank soils clustered apart from ground soils. The fungal and AOB communities were diverse but non-specific for the soil types analyzed.     

Effects of NaCl on the growth, ion accumulation and photosynthetic parameters of Thellungiella halophila

Thellungiella halophila seedlings grown on a solid substrate for 25 days on standard medium were challenged with NaCl. Growth, tissue hydration, ion accumulation, photosynthesis, lipid peroxidation and antioxidant enzymatic activities were studied on rosette leaves. Three accessions of Arabidopsis thaliana were cultivated under the same conditions. During the first two weeks of salt treatment, the growth of T. halophila leaves was restricted by NaCl. No significant difference appeared between T. halophila and A. thaliana concerning biomass deposition, or hydric and ionic parameters. However, all A. thaliana plants displayed foliar damage, and died during the third week of salt (50mM NaCl) treatment. Almost all (94%) T. halophila plants remained alive, but did not display any sign of altered physiological condition. Tissue hydration, chlorophyll content, stomatal conductance, photosynthetic quantum yield, and photosynthetic rate were very similar to those of control plants. Lipid peroxidation, estimated from thermoluminescence, was very low and insensitive to salt treatment. Only slight changes occurred in antioxidant enzymatic activities (SOD, several peroxidases, and catalase). From the absence of physiological disorder symptoms, we infer that salt was efficiently compartmentalized in leaf vacuoles. In salt-treated A. thaliana, the photosynthetic quantum yield was diminished, and lipid peroxidation was augmented. These observations reinforce the conclusion that T. halophila could accumulate salt in its leaves without damage, in contrast to A. thaliana.