Approach to the mechanism of Zajdela's hepatoma cell growth inhibition induced by concanavalin A and phytohaemagglutinin

Cell growth of tumour ascites cells was inhibited by concanavalin A, phytohaemagglutinin and Ricinus lectin at 2-100 micrograms/ml. As expected, the Ricinus lectin inhibited the protein synthesis estimated by leucine incorporation and decreased thymidine incorporation, whereas concanavalin A and phytohaemagglutinin stimulate the uptake and the incorporation of both leucine and thymidine, and thus, synthesis of protein and DNA. These results suggest that different mechanisms are involved in the hepatoma cell growth inhibition by the lectins. This difference was not related to the kinetic characteristics of the lectin interactions with the cells which represent a first and necessary step. It was showed that concanavalin A and phytohaemagglutinin as well as chloroquine inhibited the 14C-labelled asialofetuin degradation. We can conclude that Ricinus lectin present a toxic effect whereas both concanavalin A and phytohaemagglutinin show an anti-protease activity.     

Evidence for the expression of morphological and biochemical characteristics of C3-photosynthesis in chlorohyllous callus cultures of Zea mays

A Zea mays callus culture containing chlorophyll was established and grown photomixotrophically. Cell chloroplast structure, and pigment and soluble protein contents were examined. Expression of some key enzymes of C₄ carbon metabolism was compared with that of etiolated (heterotrophic) and green photoautotrophic leaves. Chlorophyll content of the callus was 15–20% that of green leaves. Soluble protein content of callus was half that of leaf cells. Electron microscopic observations showed that green callus cells contained only typical granal chloroplasts. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.38) activities in green callus were ca 30% those of green leaves but 2–3 times higher than in etiolated leaves. Quantitative enzyme protein determination, using antibodies specific to maize leaf Rubisco showed that the chloroplastic carboxylase represented about 7% of total soluble protein in green callus, in parallel to its low chlorophyll content. The specific activity of Rubisco in callus and leaves was unchanged. Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) activity in green callus was about 20% that of green leaves and similar to that measured in etiolated leaves. Apparent K_m (PEP) values (0.08 mM) for PEPC isolated from green callus and etiolated leaves were very different from values (0.5 mM) obtained with PEPC from green leaves. These kinetic characteristics together with the absence of inhibition by malate and activation by glucose-6-phosphate suggest that the properties of PEPC isolated from green callus and etiolated maize leaves are very similar to those of PEPPC from C₃ plants. Using PEPC antibodies specific to green maize leaf enzyme, immunotitration of PEPC preparations containing identical enzyme units allowed complete precipitation of the green leaf enzyme with increasing antibody volumes. In contrast, 60–70% of the activity of PEPC from etiolated and green callus was inhibited, suggesting low affinity for the maize green leaf PEPC antiserum (typical C₄ form). Ouchterlony double diffusion tests revealed only partial recognition of PEPC in green callus and etiolated leaves. NAD-malate dehydrogenase (NAD-MDH, EC 1.1.1.37) activity in callus was 2 and 3 times higher, respectively, than in etiolated and green leaves. NADP-malic enzyme (NADP-ME, EC 1.1.1.40) activity in callus cultures was much lower than in green leaves. All our data support the hypothesis that cultures of fully dedifferentiated chlorophyllous tissues of Zea mays possess a C₃-like metabolism.     

Sensitivity and Specificity of a New Vertical Flow Rapid Diagnostic Test for the Serodiagnosis of Human Leptospirosis

Background: Leptospirosis is a growing public health concern in many tropical and subtropical countries. However, its diagnosis is difficult because of non-specific symptoms and concurrent other endemic febrile diseases. In many regions, the laboratory diagnosis is not available due to a lack of preparedness and simple diagnostic assay or difficult access to reference laboratories. Yet, an early antibiotic treatment is decisive to the outcome. The need for Rapid Diagnostic Tests (RDTs) for bedside diagnosis of leptospirosis has been recognized. We developed a vertical flow immunochromatography strip RDT detecting anti-Leptospira human IgM and evaluated it in patients from New Caledonia, France, and French West Indies. Methodology/Principal Findings: Whole killed Leptospira fainei cells were used as antigen for the test line and purified human IgM as the control line. The mobile phase was made of gold particles conjugated with goat anti-human IgM. Standards for Reporting of Diagnostic Accuracy criteria were used to assess the performance of this RDT. The Microscopic Agglutination Test (MAT) was used as the gold standard with a cut-off titer of ≥400. The sensitivity was 89.8% and the specificity 93.7%. Positive and negative Likelihood Ratios of 14.18 and 0.108 respectively, and a Diagnostic Odds Ratio of 130.737 confirmed its usefulness. This RDT had satisfactory reproducibility, repeatability, thermal tolerance and shelf-life. The comparison with MAT evidenced the earliness of the RDT to detect seroconversion. When compared with other RDT, the Vertical Flow RDT developed displayed good diagnostic performances.

Conclusions/Significance

This RDT might be used as a point of care diagnostic tool in limited resources countries. An evaluation in field conditions and in other epidemiological contexts should be considered to assess its validity over a wider range of serogroups or when facing different endemic pathogens. It might prove useful in endemic contexts or outbreak situations.     

Somatic hybrids between potato <Emphasis Type="Italic">Solanum tuberosum</Emphasis> and wild species <Emphasis Type="Italic">Solanum verneï</Emphasis> exhibit a recombination in the plastome

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum verneï (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.     

The structure of the O-glycosidic oligosaccharide chains of the major Zajdela hepatoma ascites-cell-membrane glycoprotein

Glycoprotein MII2, the major cell surface glycoprotein (molecular mass 110 kDa) of Zajdela hepatoma ascites cells, contains about 25 O-glycosidic oligosaccharide chains per molecule. They were released as oligosaccharide-alditols by alkaline borohydride treatment of MII2, and purified by gel filtration on Bio-Gel P-6 followed by high-voltage paper electrophoresis. Four oligosaccharide-alditol fractions (A-D) were obtained in relative yields of 8:6:3:3. The structure of the components of fractions A-C was determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis, to be as follows. (A) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B1) NeuAc alpha(2----3)Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B2) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (C) NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol. On the basis of sugar composition and characteristics on Bio-Gel P-6 filtration, paper electrophoresis and thin-layer chromatography, the structure of the carbohydrate component of fraction D is proposed to be as follows. (D) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol     

Carbon metabolism in in vitro cultures of date palm: the role of carboxylases (PEPC and RubisCO)

While describing major trends of carbon metabolism during the initiation and expression of somatic embryogenesis in date palm (Phoenix dactylifera L., cv. Deglet Nour), we have investigated the role of two carboxylases, namely PEPC (Phosphoenolpyruvate carboxylase, EC 4.1.1.31) and RubisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39), in embryogenic and non-embryogenic cultures. The detection of PEPC activity on polyacrylamide native gels after electrophoresis revealed the presence of 3 active isoforms in crude extracts from the embryogenic (E) callus strain, whereas only a single band was present in the non-embryogenic (NE) one. The level of PEPC specific capacity was of the same order (3.9 ± 1.2 μmol CO₂ h⁻¹ mg⁻¹ TSP) in both types of cultures. Further changes in carboxylase (PEPC and RubisCO) activities during the growth and development of somatic embryo–derived plantlets were also analysed. The PEPC/RubisCO ratio was found to progressively decrease (from 17.7 to 0.2) throughout the in vitro development of plantlets, due to a substantial depletion of PEPC activity, which decreased from 5.3 to 1.2 μmol CO₂ h⁻¹ mg⁻¹ TSP. Concomitantly, RubisCO assumed greater importance (from 0.3 to 5.3 μmol CO₂ h⁻¹ mg⁻¹ _TSP ) and became the main route for inorganic carbon fixation. Western blot analysis using polyclonal antibodies raised against PEPC and RubisCO purified from tobacco leaves confirmed this trend in terms of relative enzyme abundance. This revised version was published online in June 2006 with corrections to the Cover Date.     

Potential use of Cu2+, K+ and Na+ for the destruction of Caulerpa taxifolia: differential effects on photosynthetic parameters

Chemical techniques were investigated in order to eradicate Caulerpa taxifolia, a green alga spreading at a remarkable rate in the Mediterranean Sea. The action of copper, potassium and sodium ions on survival rates and photosynthetic parameters was compared, in order to optimise the conditions of further in situ treatments. The lethal doses were determined and the impact of the studied cations on photosynthesis and respiration rates and PSII photochemistry was analysed from measurements of net oxygen exchanges and chlorophyll fluorescence. The Cu²⁺ concentrations required to obtain 100% mortality were 15 × 10² to 10⁴ times lower than those of K⁺ and Na⁺. Respiration was slightly affected whatever the salt concentration,while photosynthesis could be totally inhibited depending on the applied treatment. Changes in the structure of the Ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO, EC: 4.1.1.39) were also detected when C. taxifolia under went cation treatments (10 mg L^-1 Cu²⁺, 1h; 20 gL^-1 K⁺, 3 h; 20 g L^-1 Na⁺, 1 h). Given the high concentration and long incubation periods required with K⁺ and Na⁺ ions, these cations are not suitable to be used in situ. Our results make possible the utilisation of copper cations following technical approaches such asion-exchange textile covers, which allows a controlled release of cupric ions without dissemination in the marine environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Intra-epidemic genome variation in highly pathogenic African swine fever virus (ASFV) from the country of Georgia

Background

African swine fever virus (ASFV) causes an acute hemorrhagic infection in suids with a mortality rate of up to 100%. No vaccine is available and the potential for catastrophic disease in Europe remains elevated due to the ongoing ASF epidemic in Russia and Baltic countries. To date, intra-epidemic whole-genome variation for ASFV has not been reported. To provide a more comprehensive baseline for genetic variation early in the ASF outbreak, we sequenced two Georgian ASFV samples, G-2008/1 and G-2008/2, derived from domestic porcine blood collected in 2008.

Methods

Genomic DNA was extracted directly from low-volume ASFV PCR-positive porcine blood samples and subjected to next generation sequencing on the Illumina Miseq platform. De novo and mapped sequence assemblies were performed using CLCBio software. Genomic illustrations, sequence alignments and assembly figures were generated using Geneious v10.2.4. Sequence repeat architecture was analyzed using DNASTAR GeneQuest 14.1.0.

Results

The G-2008/1 and G-2008/2 genomes were distinguished from each other by coding changes in seven genes, including MGF 110-1?L, X69R, MGF 505-10R, EP364R, H233R, E199L, and MGF 360-21R in addition to eight homopolymer tract variations. The 2008/2 genome possessed a novel allele state at a previously undescribed intergenic repeat locus between genes C315R and C147L. The C315R/C147L locus represents the earliest observed variable repeat sequence polymorphism reported among isolates from this epidemic. No sequence variation was observed in conventional ASFV subtyping markers. The two genomes exhibited complete collinearity and identical gene content with the Georgia 2007/1 reference genome. Approximately 56 unique homopolymer A/T-tract variations were identified that were unique to the Georgia 2007/1 genome. In both 2008 genomes, within-sample sequence read heterogeneity was evident at six homopolymeric G/C-tracts confined to the known hypervariable ~?7?kb region in the left terminal region of the genome.

Conclusions

This is the first intra-epidemic comparative genomic analysis reported for ASFV and provides insight into the intra-epidemic microevolution of ASFV. The genomes reported here, in addition to the G-2007/1 genome, provide an early baseline for future genome-level comparisons and epidemiological tracing efforts.