Automatic imaging of Drosophila embryos with light sheet fluorescence microscopy on chip

We present a microscope on chip for automated imaging of Drosophila embryos by light sheet fluorescence microscopy. This integrated device, constituted by both optical and microfluidic components, allows the automatic acquisition of a 3D stack of images for specimens diluted in a liquid suspension. The device has been fully optimized to address the challenges related to the specimens under investigation. Indeed, the thickness and the high ellipticity of Drosophila embryos can degrade the image quality. In this regard, optical and fluidic optimization has been carried out to implement dual-sided illumination and automatic sample orientation. In addition, we highlight the dual color investigation capabilities of this device, by processing two sample populations encoding different fluorescent proteins. This work was made possible by the versatility of the used fabrication technique, femtosecond laser micromachining, which allows straightforward fabrication of both optical and fluidic components in glass substrates.     

Protein kinase of bacteriophage T7. 2. Properties, enzyme synthesis in vitro and regulation of enzyme synthesis and activity in vivo.

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3':5'-AMP and/or cyclic 3':5'-GMP. The T7 protein kinase carries -- SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested.     

Immunoglobulin secreting cells in lymphocytes of patients with IgA deficiency or hyper-IgA

To assess the ability of immunoglobulin production in vivo, we enumerated the immunoglobulin secreting cells in the peripheral blood of patients with an IgA deficiency and of those with hyper-IgAemia. All seven patients with primary IgA deficiency and two of the three patients with secondary IgA deficiency had low numbers of IgA secreting cells. In all five patients with hyper-IgA the number of IgA secreting cells was increased. Our results suggest that measurement of immunoglobulin secreting cells in PBMCs is useful in the assessment of ability of immunoglobulin production in vivo.Abbreviations PBMCs peripheral blood mononuclear cells - MEM minimum essential medium - PFC plaque forming cells - VH immunoglobulin heavy chain variable region - CH immunoglobulin heavy chain constant region     

Orotidine Monophosphate Decarboxylase——A Fascinating Workhorse Enzyme with Therapeutic Potential

Orotidine 50-monophosphate decarboxylase(ODCase) is known as one of the most proficient enzymes. The enzyme catalyzes the last reaction step of the de novo pyrimidine biosynthesis, the conversion from orotidine 50-monophosphate(OMP) to uridine 50-monophosphate. The enzyme is found in all three domains of life, Bacteria, Eukarya and Archaea. Multiple sequence alignment of 750 putative ODCase sequences resulted in five distinct groups. While the universally conserved Dx Kxx Dx motif is present in all the groups,depending on the groups, several characteristic motifs and residues can be identified. Over 200 crystal structures of ODCases have been determined so far. The structures, together with biochemical assays and computational studies, elucidated that ODCase utilized both transition state stabilization and substrate distortion to accelerate the decarboxylation of its natural substrate. Stabilization of the vinyl anion intermediate by a conserved lysine residue at the catalytic site is considered the largest contributing factor to catalysis, while bending of the carboxyl group from the plane of the aromatic pyrimidine ring of OMP accounts for substrate distortion. A number of crystal structures of ODCases complexed with potential drug candidate molecules have also been determined, including with 6-iodouridine, a potential antimalarial agent.     

Mutagenicity of N-hydroxylamines and N-hydroxycarbamates towards strains of Escherichia coli and Salmonella typhimurium

The mutagenic activity of several arylamines, alkyl- and arylcarbamates and their corresponding N-hydroxylated derivatives towards Escherichia coli WP2uvrA was investigated using the fluctuation test without a metabolic activation system. None of the parent amines or carbamates were mutagenic while several arylhydroxylamines and N-hydroxycarbamates were direct-acting base-pair substitution mutagens. With the exception of n-hexyl-N-hydroxycarbamate, the mutagenic activity of the N-hydroxycarbamates increased with increase in the length of alkyl substituent. Some arylamines and arylhydroxylamines were further examined, again without a metabolic activation system, using a plate test in conjunction with bacterial strains which detect either base-pair or frameshift mutagens. The arylhydroxylamines were found to cause both base-pair and frameshift mutations but were more active as frameshift mutagens. Possible reasons for the observed mutagenic activity are considered.