Identification of Immunogenic Determinants of the Spike Protein of SARS-like Coronavirus

Bat SARS-like coronavirus (SL-CoV) has a genome organization almost identical to that of SARS-CoV, but the N-terminus of the Spike (S) proteins, which interacts with host receptor and is a major target of neutralizing antibodies against CoVs, of the two viruses has only 63-64% sequence identity. Although there have been reports studying the overall immunogenicity of SSL , knowledge on the precise location of immunodominant determinants for SSL is still lacking. In this study, using a series of truncated expressed SSL fragments and SSL specific mouse sera, we identified two immunogenic determinants for SSL . Importantly, one of the two regions seems to be located in a region not shared by known immunogenic determinants of the SSARS . This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.

Staurosporine-Induced Cell Death in Trypanosoma brucei and the Role of Endonuclease G during Apoptosis

Apoptosis in single-cell organisms like Trypanosoma or Leishmania was characterized in several studies in the last few years [1]-[4]. Cell death in these caspase lacking protozoa is still poorly understood and a conclusive apoptotic pathway has not been identified so far. In the work presented here, we studied the effects of prostaglandin D2 and staurosporine induced cell death in blood-forms of Trypanosoma brucei in a time dependent manner and focused on the role of a nuclease similar to endonuclease G of higher eukaryotes. We found that these parasites undergo apoptotic cell death as demonstrated by the appearance of several canonical hallmarks of apoptosis in higher eukaryotes, but that different stimuli induce remarkable differences in the way these cells die. We compared the effects of prostaglandin D2 and staurosporine in trypanosomes with and without endonuclease G overexpression by flow cytometric and electron microscopic methods with the result that endonuclease G overexpression led to a significant modification of intracellular organelles and accelerated apoptotic cell death in prostaglandin D2 or staurosporine treated cells. Our results demonstrate that different stimuli induce apoptosis even in these ancient organisms in different caspase-independent ways. Whereas central processes of apoptosis like ROS formation, loss of mitochondrial membrane potential, endonuclease G release, phosphatidylserine exposure and DNA fragmentation appeared in the same chronology during treatment with either one of both drugs, other effects like cell cycle arrest or change of cell shape occurred only in the case of prostaglandin D2 or staurosporine treatment. We conclude from these results that trypanosomes react to stimuli of apoptosis with the concerted action of cellular responses but cannot control the final outcome if additional stress, as in the case of staurosporine, is superimposed.

Proteomic Analysis of the Proplastid Envelope Membrane Provides Novel Insights into Small Molecule and Protein Transport across Proplastid Membranes

Proplastids are undifferentiated plastids of meristematic tissues that synthesize amino acids for protein synthesis, fatty acids for membrane lipid production, and purines and pyrimidines for DNA and RNA synthesis. Unlike chloroplasts, proplastids depend on supply, with reducing power, energy, and precursor metabolites from the remainder of the cell. Comparing proplastid and chloroplast envelope proteomes and the corresponding transcriptomes of leaves and shoot apex revealed a clearly distinct composition of the proplastid envelope. It is geared towards import of metabolic precursors and export of product metabolites for the rapidly dividing cell. The analysis also suggested a new role for the triosephosphate translocator in meristematic tissues, identified the route of organic nitrogen import into proplastids, and detected an adenine nucleotide exporter. The protein import complex contains the import receptors Toc120 and Toc132 and lacks the redox sensing complex subunits of Tic32, Tic55, and Tic62, which mirrors the expression patterns of the corresponding genes in leaves and the shoot apex. We further show that the protein composition of the internal membrane system is similar to etioplasts, as it is dominated by the ATP synthase complex and thus remarkably differs from that of chloroplast thylakoids.

Mutants, Overexpressors, and Interactors of Arabidopsis Plastocyanin Isoforms： Revised Roles of Plastocyanin in Photosynthetic Electron Flow and Thylakoid Redox State

Two homologous plastocyanin isoforms are encoded by the genes PETE1 and PETE2 in the nuclear genome of Arabidopsis thaliana. The PETE2 transcript is expressed at considerably higher levels and the PETE2 protein is the more abundant isoform. Null mutations in the PETE genes resulted in plants, designated pete1 and pete2, with decreased plastocyanin contents. However, despite reducing plastocyanin levels by over -90%, a pete2 null mutation on its own affects rates of photosynthesis and growth only slightly, whereas pete1 knockout plants, with about 60-80% of the wild-type plastocyanin level, did not show any alteration. Hence, plastocyanin concentration is not limiting for photosynthetic elec- tron flow under optimal growth conditions, perhaps implying other possible physiological roles for the protein. Indeed, plastocyanin has been proposed previously to cooperate with cytochrome C6A （Cyt C6A） in thylakoid redox reactions, but we find no evidence for a physical interaction between the two proteins, using interaction assays in yeast. We observed homodimerization of Cyt C6A in yeast interaction assays, but also Cyt C6A homodimers failed to interact with plastocyanin. Moreover, phenotypic analysis of atc6-1 pete1 and atc6-1 pete2 double mutants, each lacking Cyt C6A and one of the two piastocyanin-encoding genes, failed to reveal any genetic interaction. Overexpression of either PETE1 or PETE2 in the pete1 pete2 double knockout mutant background results in essentially wild-type photosynthetic performance, excluding the possibility that the two plastocyanin isoforms could have distinct functions in thylakoid electron flow.