Phosphorylation of the human Fhit tumor suppressor on tyrosine 114 in Escherichia coli and unexpected steady state kinetics of the phosphorylated forms

The human tumor suppressor Fhit is a homodimeric histidine triad (HIT) protein of 147 amino acids which has Ap(3)A hydrolase activity. We have recently discovered that Fhit is phosphorylated in vivo and is phosphorylated in vitro by Src kinase [Pekarsky, Y., Garrison, P. N., Palamarchuk, A., Zanesi, N., Aqeilan, R. I., Huebner, K., Barnes, L. D., and Croce, C. M. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 3775-3779]. Now we have coexpressed Fhit with the elk tyrosine kinase in Escherichia coli to generate phosphorylated forms of Fhit. Unphosphorylated Fhit, Fhit phosphorylated on one subunit, and Fhit phosphorylated on both subunits were purified to apparent homogeneity by column chromatography on anion-exchange and gel filtration resins. MALDI-TOF and HPLC-ESI tandem mass spectrometry of intact Fhit and proteolytic peptides of Fhit demonstrated that Fhit is phosphorylated on Y(114) on either one or both subunits. Monophosphorylated Fhit exhibited monophasic kinetics with K(m) and k(cat) values approximately 2- and approximately 7-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. Diphosphorylated Fhit exhibited biphasic kinetics. One site had K(m) and k(cat) values approximately 2- and approximately 140-fold lower, respectively, than the corresponding values for unphosphorylated Fhit. The second site had a K(m) approximately 60-fold higher and a k(cat) approximately 6-fold lower than the corresponding values for unphosphorylated Fhit. The unexpected kinetic patterns for the phosphorylated forms suggest the system may be enzymologically novel. The decreases in the values of K(m) and k(cat) for the phosphorylated forms in comparison to those of unphosphorylated Fhit favor the formation and lifetime of the Fhit-Ap(3)A complex, which may enhance the tumor suppressor activity of Fhit.     

Protein microarrays for multiplex analysis of signal transduction pathways

We have developed a multiplexed reverse phase protein (RPP) microarray platform for simultaneous monitoring of site-specific phosphorylation of numerous signaling proteins using nanogram amounts of lysates derived from stimulated living cells. We first show the application of RPP microarrays to the study of signaling kinetics and pathway delineation in Jurkat T lymphocytes. RPP microarrays were used to profile the phosphorylation state of 62 signaling components in Jurkat T cells stimulated through their membrane CD3 and CD28 receptors, identifying a previously unrecognized link between CD3 crosslinking and dephosphorylation of Raf-1 at Ser259. Finally, the potential of this technology to analyze rare primary cell populations is shown in a study of differential STAT protein phosphorylation in interleukin (IL)-2-stimulated CD4(+)CD25(+) regulatory T cells. RPP microarrays, prepared using simple procedures and standard microarray equipment, represent a powerful new tool for the study of signal transduction in both health and disease.     

Ballistic seed projection in two herbaceous species

We found that the majority of ballistic seeds tested [N = 74 of 78 in Vicia sativa ssp. nigra (Fabaceae); N = 25 of 27 in Croton capitatus var. capitatus (Euphorbiaceae)] were projected at angles that would yield a greater distance than the average of seeds with the same initial speed projected at random angles. In addition, the median of fractional distance error (maximum distance - seed distance)/(maximum distance), of the seeds were 0.11 and 0.04 for V. sativa and C. capitatus, respectively. Seed projection distance was modeled by using initial projection angle, initial speed, and measured drag, along with other seed data. We improved upon previous such studies by using dual-angle high-speed stroboscopic photography to determine initial projection angle and speed. We also measured seed drag in a low-turbulence wind tunnel. Seed projection positions on the plant, which also affect seed projection distance, were found to be primarily from the top of the plant, with 98 of 137 and 407 of 407 fruits in the upper half of the plant for V. sativa and C. capitatus, respectively. Our findings are significant because they suggest that in addition to the ballistic projection mechanism itself, the species studied have additional adaptations that result in enhanced seed projection distance from the parent plant.     

Use of ethidium bromide fluorescence enhancement to detect duplex DNA and DNA bacteriophages during zone sedimentation in sucrose gradients: molecular weight of DNA as a function of sedimentation rate

Duplex DNA molecules and DNA bacteriophages have been sedimented through 5--25% sucrose gradients containing ethidium bromide. The location of DNA within the gradients has been determined by illuminating gradients with ultraviolet light and observing the ethidium bromide fluorescence enhancement induced by the DNA. The relative sedimentation rates of linear, duplex DNAs from bacteriophages T4, T5, T7 and an 8.3% T7 deletion mutant have been determined. The distances sedimented by DNA have been corrected, when necessary, for a progressive decrease in sedimentation rate that occurs after the DNA has traversed 40% of the sucrose gradient. The corrected distances sedimented by two DNA molecules, r1' and r2', are related to the DNA molecular weights, m1 and m2, by the equation: r1'/r2' = (m1/m2)0.38 when 0.025--0.70 microgram of each type of DNA is sedimented. Intact bacteriophages were also sedimented in ethidium bromide--sucrose gradients and detected by fluorescence enhancement.     

Does change in blood pressure predict heart disease?

Whether a change in blood pressure is related to the occurrence of cardiovascular disease, independent of blood pressure level, was investigated using data collected in the Framingham heart study on 5209 subjects. The follow up period of 26 years was divided into a first period of 12 years in which the blood pressure change was computed for each individual and a subsequent period of 14 years in which the risk of cardiovascular disease was determined. Blood pressure change was positively related to risk of cardiovascular disease. This association remained when blood pressure level at the start of the study was taken into account but disappeared when the level attained after the first 12 years was taken into consideration. For the clinician this suggests that the decision to treat high blood pressure is best guided by the actual level of pressure and not by its long term trend in the past.     

A Multiplex PCR/LDR Assay for the Simultaneous Identification of Category A Infectious Pathogens: Agents of Viral Hemorrhagic Fever and Variola Virus

CDC designated category A infectious agents pose a major risk to national security and require special action for public health preparedness. They include viruses that cause viral hemorrhagic fever (VHF) syndrome as well as variola virus, the agent of smallpox. VHF is characterized by hemorrhage and fever with multi-organ failure leading to high morbidity and mortality. Smallpox, a prior scourge, has been eradicated for decades, making it a particularly serious threat if released nefariously in the essentially non-immune world population. Early detection of the causative agents, and the ability to distinguish them from other pathogens, is essential to contain outbreaks, implement proper control measures, and prevent morbidity and mortality. We have developed a multiplex detection assay that uses several species-specific PCR primers to generate amplicons from multiple pathogens; these are then targeted in a ligase detection reaction (LDR). The resultant fluorescently-labeled ligation products are detected on a universal array enabling simultaneous identification of the pathogens. The assay was evaluated on 32 different isolates associated with VHF (ebolavirus, marburgvirus, Crimean Congo hemorrhagic fever virus, Lassa fever virus, Rift Valley fever virus, Dengue virus, and Yellow fever virus) as well as variola virus and vaccinia virus (the agent of smallpox and its vaccine strain, respectively). The assay was able to detect all viruses tested, including 8 sequences representative of different variola virus strains from the CDC repository. It does not cross react with other emerging zoonoses such as monkeypox virus or cowpox virus, or six flaviviruses tested (St. Louis encephalitis virus, Murray Valley encephalitis virus, Powassan virus, Tick-borne encephalitis virus, West Nile virus and Japanese encephalitis virus).     

Molecular simulation of Tyr105 phosphorylated pyruvate kinase M2 to understand its structure and dynamics

Microstructure of dibenzo-18-crown-6 (DB18C6) and DB18C6/Li⁺ complex in different solvents (water, methanol, chloroform, and nitrobenzene) have been analyzed using radial distribution function (RDF), coordination number (CN), and orientation profiles, in order to identify the role of solvents on complexation of DB18C6 with Li⁺, using molecular dynamics (MD) simulations. In contrast to aqueous solution of LiCl, no clear solvation pattern is found around Li⁺ in the presence of DB18C6. The effect of DB18C6 has been visualized in terms of reduction in peak height and shift in peak positions of g_Li-Ow. The appearance of damped oscillations in velocity autocorrelation function (VACF) of complexed Li⁺ described the high frequency motion to a “rattling” of the ion in the cage of DB18C6. The solvent-complex interaction is found to be higher for water and methanol due to hydrogen bond (HB) interactions with DB18C6. However, the stability of DB18C6/Li⁺ complex is found to be almost similar for each solvent due to weak complex-solvent interactions. Further, Li⁺ complex of DB18C6 at the liquid/liquid interface of two immiscible solvents confirm the high interfacial activity of DB18C6 and DB18C6/Li⁺ complex. The complexed Li⁺ shows higher affinity for water than organic solvents; still they remain at the interface rather than migrating toward water due to higher surface tension of water as compared to organic solvents. These simulation results shed light on the role of counter-ions and spatial orientation of species in pure and hybrid solvents in the complexation of DB18C6 with Li⁺. Graphical Abstract

DB18C6/Li⁺ complex in pure solvents (water, methanol, chloroform, and nitrobenzene) and water/nitrobenzene interface     

Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi

To better understand how airways produce thick airway mucus, nonvolatile solids were measured in liquid secreted by bronchi from normal pig, cystic fibrosis (CF) human, and non-CF human lungs. Bronchi were exposed to various secretagogues and anion secretion inhibitors to induce a range of liquid volume secretion rates. In all three groups, the relationship of solids concentration (percent nonvolatile solids) to liquid volume secretion rate was curvilinear, with higher solids concentration associated with lower rates of liquid volume secretion. In contrast, the secretion rates of solids mass and water mass as functions of liquid volume secretion rates exhibited positive linear correlations. The y-intercepts of the solids mass-liquid volume secretion relationships for all three groups were positive, thus accounting for the higher solids concentrations in airway liquid at low rates of secretion. Predictive models derived from the solids mass and water mass linear equations fit the experimental percent solids data for the three groups. The ratio of solids mass secretion to liquid volume secretion was 5.2 and 2.4 times higher for CF bronchi than for pig and non-CF bronchi, respectively. These results indicate that normal pig, non-CF human, and CF human bronchi produce a high-percent-solids mucus (>8%) at low rates of liquid volume secretion (≤1.0 μl·cm(-2)·h(-1)). However, CF bronchi produce mucus with twice the percent solids (~8%) of pig or non-CF human bronchi at liquid volume secretion rates ≥4.0 μl·cm(-2)·h(-1).     

Underwater sinkhole sediments sequester Lake Huron’s carbon

Lake Huron’s submerged sinkhole habitats are impacted by high-conductivity groundwater that allows photosynthetic cyanobacterial mats to form over thick, carbon-rich sediments. To better understand nutrient cycling in these habitats, we measured the stable isotopic content of carbon and nitrogen in organic and inorganic carbon pools in Middle Island sinkhole, a ~23 m deep feature influenced by both groundwater and overlying lake water. Two distinct sources of dissolved CO₂ (DIC) were available to primary producers. Lake water DIC (δ ¹³C = ?0.1 ‰) differed by +5.9 ‰ from groundwater DIC (δ ¹³C = ?6.0 ‰). Organic carbon fixed by primary producers reflected the two DIC sources. Phytoplankton utilizing lake water DIC were more enriched in ¹³C (δ ¹³C = ?22.2 to ?23.2 ‰) than mat cyanobacteria utilizing groundwater DIC (δ ¹³C = ?26.3 to ?30.0 ‰). Sinkhole sediments displayed an isotopic signature (δ ¹³C = ?23.1 ‰) more similar to sedimenting phytoplankton than the cyanobacterial mat. Corroborated by sediment C/N ratios, these data suggest that the carbon deposited in sinkhole sediments originates primarily from planktonic rather than benthic sources. ²¹⁰Pb/¹³⁷Cs radiodating suggests rapid sediment accumulation and sub-bottom imaging indicated a massive deposit of organic carbon beneath the sediment surface. We conclude that submerged sinkholes may therefore act as nutrient sinks within the larger lake ecosystem.     

CNP/cGMP signaling regulates axon branching and growth by modulating microtubule polymerization

The peptide hormone CNP has recently been found to positively regulate axon branching and growth via activation of cGMP signaling in embryonic dorsal root ganglion (DRG) neurons, but the cellular mechanisms mediating the regulation of these developmental processes have not been established. In this study, we provide evidence linking CNP/cGMP signaling to microtubule dynamics via the microtubule regulator CRMP2. First, phosphorylation of CRMP2 can be suppressed by cGMP activation in embryonic DRG neurons, and non‐phosphorylated CRMP2 promotes axon branching and growth. In addition, real time analysis of growing microtubule ends indicates a similar correlation of CRMP2 phosphorylation and its activity in promoting microtubule polymerization rates and durations in both COS cells and DRG neuron growth cones. Moreover, direct activation of cGMP signaling leads to increased assembly of dynamic microtubules in DRG growth cones. Finally, low doses of a microtubule depolymerization drug nocodazole block CNP/cGMP‐dependent axon branching and growth. Taken together, our results support a critical role of microtubule dynamics in mediating CNP/cGMP regulation of axonal development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 673–687, 2013