Triadin Deletion Induces Impaired Skeletal Muscle Function

Triadin is a multiple proteins family, some isoforms being involved in muscle excitation-contraction coupling, and some having still unknown functions. To obtain clues on triadin functions, we engineered a triadin knock-out mouse line and characterized the physiological effect of triadin ablation on skeletal muscle function. These mice presented a reduced muscle strength, which seemed not to alter their survival and has been characterized in the present work. We first checked in these mice the expression level of the different proteins involved in calcium homeostasis and observed in fast muscles an increase in expression of dihydropyridine receptor, with a large reduction in calsequestrin expression. Electron microscopy analysis of KO muscles morphology demonstrated the presence of triads in abnormal orientation and a reduction in the sarcoplasmic reticulum terminal cisternae volume. Using calcium imaging on cultured myotubes, we observed a reduction in the total amount of calcium stored in the sarcoplasmic reticulum. Physiological studies have been performed to evaluate the influence of triadin deletion on skeletal muscle function. Muscle strength has been measured both on the whole animal model, using hang test or electrical stimulation combined with NMR analysis and strength measurement, or on isolated muscle using electrical stimulation. All the results obtained demonstrate an important reduction in muscle strength, indicating that triadin plays an essential role in skeletal muscle function and in skeletal muscle structure. These results indicate that triadin alteration leads to the development of a myopathy, which could be studied using this new animal model.     

Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R_h 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21–25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.Malaria caused by Plasmodium falciparum is a serious global health issue, resulting in an estimated 1.5 million deaths annually, primarily among infants and young children. Ongoing multifaceted global intervention strategies to control malaria include drug treatment, insecticide usage, bed-net use, and vaccine development. However, parasite and mosquito control measures have met with limited success resulting from an increased drug and insecticide resistance within the Plasmodia and mosquito populations, respectively. Vaccine development represents an encouraging approach given that previous animal and human studies using irradiated sporozoites demonstrated the feasibility of producing an efficacious vaccine (1–3). Although the exact immunologic correlates of protection remain elusive, an abundance of evidence indicates that protection against liver stage parasites is complex, involving multiple immune mechanisms (4–11).To date, the majority of the pre-erythrocytic stage vaccine development has focused on the circumsporozoite protein (CSP),² the predominant surface antigen on sporozoites. CSP can be segmented into three regions as follows: the N-terminal region containing region I; the central repeat region; and the C-terminal region containing the thrombospondin-like type I repeat (TSR). Initial CSP vaccine development focused on the central repeat region that contains the immunodominant B cell epitope (12). However, vaccine constructs quickly evolved to incorporate both the central repeat region containing the B cell epitopes and the C terminus containing the TSR domain, T cell epitopes, and B cell epitopes (13, 14). Currently, the most advanced and moderately effective malaria vaccine, RTS,S, is composed of a portion of the central repeat and the C-terminal regions linked to the hepatitis B surface antigen (15). However, recent studies have highlighted the physiological importance of the N-terminal region (16–19). Rathore et al. (19) not only demonstrated the role of the N-terminal region in liver cell attachment but also identified along with Ancsin and Kisilevsky (16) an epitope within the N-terminal region that interacted with liver cells through heparin sulfate (18). Moreover, this epitope was not only found to be immunogenic but the resulting antibodies were determined to be inhibitory in a sporozoite invasion assay (18). Peptides corresponding to the N-terminal region (PpCS-(22–110) and PpCS-(65–110)) were also recognized by sera obtained from individuals living in malaria-endemic regions (17).To better understand the structure of CSP and to produce good quality recombinant protein for human vaccine-directed studies, we generated full-length and near full-length recombinant CSP. We examined two expression systems, Escherichia coli and Pichia pastoris, to determine their feasibility to generate CSP. To assist the characterization of the rCSPs, we generated a panel of monoclonal antibodies (mAbs) that were characterized biologically prior to being used to examine the rCSPs. Additionally, each of the rCSP molecules was extensively biochemically and biophysically characterized. The results collated together have enabled the molecular modeling of CSP as a long flexible, rod-like protein.     

A Saccharomyces cerevisiae-based bioassay for assessing pesticide toxicity

This study evaluates the toxic effect of three pesticides (Azoxystrobin, Cymoxanil, and Diuron) on the yeast Saccharomyces cerevisiae for the development of a new bioassay based on inhibition of S. cerevisiae metabolic activity at the level of adenosine-5-triphosphate (ATP) synthesis, as compared with two different toxicity tests based on inhibition of Daphnia magna mobility (NF EN ISO 6341) and inhibition of Vibrio fisheri activity (NF EN ISO 11348). The S. cerevisiae bioassay is cheaper and 96 times faster than the D. magna toxicity bioassay, but has lower sensitivity. It is as fast as the V. fisheri bioassay and more sensitive. Thus, this new toxicity test can be proposed for rapid detection of pesticide residues in environmental samples as a complement to the more expensive and time-consuming D. magna toxicity test.     

The effect of re-dissolution solvents and HPLC columns on the analysis of mycosporine-like amino acids in the eulittoral macroalgae <Emphasis Type="Italic">Prasiola crispa</Emphasis> and <Emphasis Type="Italic">Porphyra umbilicalis</Emphasis>

Many macroalgal species that are regularly exposed to high solar radiation such as the eulittoral green alga Prasiola crispa and the red alga Porphyra umbilicalis synthesize and accumulate high concentrations of mycosporine-like amino acids (MAAs) as UV-sunscreen compounds. These substances are typically extracted with a widely used standard protocol following quantification by various high performance liquid chromatography (HPLC) techniques. However, further preparation steps prior to HPLC analysis as well as different HPLC column types have not been systematically checked regarding separation quality and reproducibility. Therefore pure methanol, distilled water and HPLC eluent were evaluated as re-dissolution solvent for dried Prasiola and Porphyra extracts, which were subsequently analyzed on three reversed-phase C8 and C18 HPLC columns. The data indicate that distilled water and the HPLC eluent gave almost identical peak patterns and MAA contents on the C8 and C18 columns. In contrast, the application of the widely used methanol led to double peaks or even the loss of specific peaks as well as to a strong decline in total MAA amounts ranging from about 35% of the maximum in P. crispa to 80% of the maximum in P. umbilicalis. Consequently, methanol should be avoided as re-dissolution solvent for the HPLC sample preparation. An improved protocol for the MAA analysis in macroalgae in combination with a reliable C18 column is suggested.