Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex

Apicomplexan parasites depend on the invasion of host cells for survival and proliferation. Calcium-dependent signaling pathways appear to be essential for micronemal release and gliding motility, yet the target of activated kinases remains largely unknown. We have characterized calcium-dependent phosphorylation events during Toxoplasma host cell invasion. Stimulation of live tachyzoites with Ca²⁺-mobilizing drugs leads to phosphorylation of numerous parasite proteins, as shown by differential 2-DE display of ³²[P]-labeled protein extracts. Multi-dimensional Protein Identification Technology (MudPIT) identified ∼546 phosphorylation sites on over 300 Toxoplasma proteins, including 10 sites on the actomyosin invasion motor. Using a Stable Isotope of Amino Acids in Culture (SILAC)-based quantitative LC-MS/MS analyses we monitored changes in the abundance and phosphorylation of the invasion motor complex and defined Ca²⁺-dependent phosphorylation patterns on three of its components - GAP45, MLC1 and MyoA. Furthermore, calcium-dependent phosphorylation of six residues across GAP45, MLC1 and MyoA is correlated with invasion motor activity. By analyzing proteins that appear to associate more strongly with the invasion motor upon calcium stimulation we have also identified a novel 15-kDa Calmodulin-like protein that likely represents the MyoA Essential Light Chain of the Toxoplasma invasion motor. This suggests that invasion motor activity could be regulated not only by phosphorylation but also by the direct binding of calcium ions to this new component.     

Prevalence of intron gain over intron loss in the evolution of paralogous gene families

The mechanisms and evolutionary dynamics of intron insertion and loss in eukaryotic genes remain poorly understood. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals and plants revealed numerous gains and losses of introns. In all analyzed lineages, the number of acquired new introns was substantially greater than the number of lost ancestral introns. This trend held even for lineages in which vertical evolution of genes involved more intron losses than gains, suggesting that gene duplication boosts intron insertion. However, dating gene duplications and the associated intron gains and losses based on the molecular clock assumption showed that very few, if any, introns were gained during the last ~100 million years of animal and plant evolution, in agreement with previous conclusions reached through analysis of orthologous gene sets. These results are generally compatible with the emerging notion of intensive insertion and loss of introns during transitional epochs in contrast to the relative quiet of the intervening evolutionary spans.     

Monitoring in vivo changes in lung microstructure with ³He MRI in Sendai virus-infected mice

Recently, a Sendai virus (SeV) model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human chronic obstructive pulmonary disease (COPD) and asthma, but the effect on distal lung parenchyma has not been investigated. The aim of our study is to image the time course and regional distribution of mouse lung microstructural changes in vivo after SeV infection. (1)H and (3)He diffusion magnetic resonance imaging (MRI) were successfully performed on five groups of C57BL/6J mice. (1)H MR images provided precise anatomical localization and lung volume measurements. (3)He lung morphometry was implemented to image and quantify mouse lung geometric microstructural parameters at different time points after SeV infection. (1)H MR images detected the SeV-induced pulmonary inflammation in vivo; spatially resolved maps of acinar airway radius R, alveolar depth h, and mean linear intercept Lm were generated from (3)He diffusion images. The morphometric parameters R and Lm in the infected group were indistinguishable from PBS-treated mice at day 21, increased slightly at day 49, and were increased with statistical significance at day 77 (p = 0.02). Increases in R and Lm of infected mice imply that there is a modest increase in alveolar duct radius distal to airway inflammation, particularly in the lung periphery, indicating airspace enlargement after virus infection. Our results indicate that (3)He lung morphometry has good sensitivity in quantifying small microstructural changes in the mouse lung and that the Sendai mouse model has the potential to be a valid murine model of COPD.     

The role of glucagon in the regulation of blood glucose: Model studies

Mathematical models afford a procedure of unifying concepts and hypotheses by expressing quantitative relationships between observables. The model presented indicates the roles of both insulin and glucagon as regulators of blood glucose, albeit in different ranges of the blood glucose concentrations. Insulin secretion is induced during hyperglycemia, while glucagon secretion results during hypoglycemia. These are demonstrated by simulations of a mathematical model conformed to data from the oral glucose tolerance test and the insulin infusion test in normal control subjects and stable and unstable diabetic patients. The model studies suggest the parameters could prove of value in quantifying the diabetic condition by indicating the degree of instability. Presented at the Society for Mathematical Biology Meeting, University of Pennsylvania, Philadelphia, August 19–21, 1976.     

MICROSPECTROPHOTOMETRIC ANALYSIS OF NUCLEAR DNA IN CHARA ZEYLANICA

Microspectrophotometric analysis of the DNA content of nuclei in various parts of Chara zeylanica Willd. revealed that the amount of DNA in the nucleus of an internodal cell equals twice the amount of DNA in the nucleus of a sperm, while the half-anaphase stage of the same nodal cells contains the same amount of DNA as the nuclei of the male gametes. The DNA content of the nuclei of internodal cells may rise as much as 50 times higher than that of the gametes. However, in the oldest (most basal) internodal cells, the DNA content of the minute nuclei falls again to the basic (1 C) amount. Measurements of sister nuclei derived by amitosis indicated that both nuclei have equal amounts of DNA; this was interpreted as further evidence that amitosis is not a disorganized process or manifestation of degeneration. The bearing of these analyses on the question of the site of meiosis in these plants is discussed.     

The Reactions of Hemoglobin in Various Media

The rate constants for the association of oxygen and of carbon monoxide with reduced hemoglobin and for the dissociation of oxygen from hemoglobin were studied in a variety of media. All three of these rates were essentially independent of diffusion rate and dielectric constant throughout a wide range. These results are very different from those found for other heme proteins whose rates are diffusion-controlled in very viscous solvents.

The interpretation of the average measured rates described herein in terms of the four rate constants discussed by Gibson and Roughton indicates that our measurements reflect most strongly the constants for the first reacting molecule in each case. For the purposes of these reactions, the reactants may be regarded as neutral molecules.

     

Encapsulated in silica: genome, proteome and physiology of the thermophilic bacterium Anoxybacillus flavithermus WK1

Background

Gram-positive bacteria of the genus Anoxybacillus have been found in diverse thermophilic habitats, such as geothermal hot springs and manure, and in processed foods such as gelatin and milk powder. Anoxybacillus flavithermus is a facultatively anaerobic bacterium found in super-saturated silica solutions and in opaline silica sinter. The ability of A. flavithermus to grow in super-saturated silica solutions makes it an ideal subject to study the processes of sinter formation, which might be similar to the biomineralization processes that occurred at the dawn of life.

Results

We report here the complete genome sequence of A. flavithermus strain WK1, isolated from the waste water drain at the Wairakei geothermal power station in New Zealand. It consists of a single chromosome of 2,846,746 base pairs and is predicted to encode 2,863 proteins. In silico genome analysis identified several enzymes that could be involved in silica adaptation and biofilm formation, and their predicted functions were experimentally validated in vitro. Proteomic analysis confirmed the regulation of biofilm-related proteins and crucial enzymes for the synthesis of long-chain polyamines as constituents of silica nanospheres.

Conclusions

Microbial fossils preserved in silica and silica sinters are excellent objects for studying ancient life, a new paleobiological frontier. An integrated analysis of the A. flavithermus genome and proteome provides the first glimpse of metabolic adaptation during silicification and sinter formation. Comparative genome analysis suggests an extensive gene loss in the Anoxybacillus/Geobacillus branch after its divergence from other bacilli.     

The discovery of furo[2,3-c]pyridine-based indanone oximes as potent and selective B-Raf inhibitors

Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.