High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

A high-throughput screening method has been developed which enables functional analysis of bacteriorhodpsin in whole cell pastes. Reflectance spectra, from as little as 5 ml of Halobacterium salinarum cells, show close correspondence to that obtained from the purified purple membrane (PM), containing bacteriorhodopsin (BR) as the sole protein component. We demonstrate accurate quantification of BR accumulation by ratiometric analysis of BR (A_max 568) and a membrane-bound cytochrome (A_max 410). In addition, ground-state light- and dark-adapted (LA and DA, respectively) spectral differences were determined with high accuracy and precision. Using cells expressing the BR mutant D85N, we monitored transitions between intermediate-state homologues of the reprotonation phase of the light-activated proton pumping mechanism. We demonstrate that phenotypes of three mutants (D85N/T170C, D85N/D96N, and D85N/R82Q) previously characterized for their effect on photocycle transitions are reproduced in the whole cell samples. D85N/T170C stabilizes accumulation of the N state while D85N/D96N accumulates no N state. D85N/R82Q was found to have perturbed the pK_a of M accumulation. These studies illustrate the correspondence between pH-dependent ground-state transitions accessed by D85N and the transitions accessed by the wild-type protein following photoexcitation. We demonstrate that whole cell reflectance spectroscopy can be used to efficiently characterize the large numbers of mutants generated by engineering strategies that exploit saturation mutagenesis.     

Safer one-pot synthesis of the ‘SHAPE’ reagent 1-methyl-7-nitroisatoic anhydride (1m7)

Estimating the reactivity of 2′-hydroxyl groups along an RNA chain of interest aids in the modeling of the folded RNA structure; flexible loops tend to be reactive, whereas duplex regions are generally not. Among the most useful reagents for probing 2′-hydroxyl reactivity is 1-methyl-7-nitroisatoic anhydride (1m7), but the absence of a reliable, inexpensive source has prevented widespread adoption. An existing protocol for the conversion of an inexpensive precursor 4-nitroisatoic anhydride (4NIA) recommends the use of NaH in dimethylformamide (DMF), a reagent combination that most molecular biology labs are not equipped to handle, and that does not scale safely in any case. Here we describe a safer, one-pot method for bulk conversion of 4NIA to 1m7 that reduces costs and bypasses the use of NaH. We show that 1m7 produced by this method is free of side products and can be used to probe RNA structure in vitro.     

Pharmacological Inhibition of Glycogen Synthase Kinase 3 Regulates T Cell Development In Vitro

The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3) thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol–3-kinase (PI3K) pathway. Glycogen Synthase Kinase 3 (GSK3) is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.     

Brucellosis in a Veterans' Hospital, 1963

In Canada, about 100 sporadically occurring cases of brucellosis are reported yearly. Three patients were admitted to one Montreal hospital in the first seven months of 1963; all were employed in or around a packing plant. One had pain and electrocardiographic changes suggestive of Brucella myocarditis; he recovered promptly. Symptoms of neurasthenia and anxiety are common and were observed in two of these three cases. Two had positive blood cultures; the third showed conclusive agglutination to Br. abortus (1:10,240). In some patients with frequent previous exposures to infection the agglutination response may be equivocal and difficult to interpret. All three patients responded well to tetracycline; streptomycin and corticosteroids also have a role in the therapy of some cases. There may also be some place for combined antibiotic and vaccine therapy. The program of control of the disease in cattle in Canada is aimed at eradication by 1967.     

Acrolysin,the aminoproteinase catalyzing the initial conversion of proacrosin to acrosin in mammalian fertilization

Extracts of mammalian sperm acrosomes and testes contain a proteinase, acrolysin, that hydrolyzes the aminopeptide bonds of hydrophobic aminoacyl residues. Acrolysin has hydrolytic specificity and properties similar to the proteinase, thermolysin (E.C. 3.4.24.4), that also catalyzes the conversion of proacrosin to acrosin (E.C. 3.4.21.10). Acrolysin is the apparent initiator of proacrosin activation in mammalian fertilization.