All-atom homology model of the Escherichia coli 30S ribosomal subunit

Understanding the structural basis of ribosomal function requires close comparison between biochemical and structural data. Although a large amount of biochemical data are available for the Escherichia coli ribosome, the structure has not been solved to atomic resolution. Using a new RNA homology procedure, we have modeled the all-atom structure of the E. coli 30S ribosomal subunit. We find that the tertiary structure of the ribosome core, including the A-, P- and E-sites, is highly conserved. The hypervariable regions in our structure, which differ from the structure of the 30S ribosomal subunit from Thermus thermophilus, are consistent with the cryo-EM map of the E. coli ribosome.     

Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth

We have previously demonstrated that concomitant activation of receptor tyrosine kinases and certain G protein-coupled receptors (GPCRs) can promote a synergistic increase in the rate of airway smooth muscle cell (ASM) proliferation. Here we clarify the role of p70S6 kinase (p70S6K) as an integrator of receptor tyrosine kinase and GPCR signaling that augments ASM DNA synthesis by demonstrating that specific p70S6K phosphorylation sites receive distinct regulatory input from GPCRs that promotes sustained kinase activity critical to mitogenesis. Prolonged stimulation of ASM cells with EGF and thrombin induced a greater than additive effect in levels of p70S6K phosphorylated at residue T389, whereas a significant but more modest increase in the level of T229 and T421/S424 phosphorylation was also observed. The augmenting effects of thrombin could be dissociated from p42/p44 MAPK activation, as selective inhibition of thrombin-stimulated p42/p44 failed to alter the profile of cooperative p70S6K T389 phosphorylation, p70S6K kinase activity, or ASM [(3)H]thymidine incorporation. Thrombin stimulated a sustained increase in the level of Akt phosphorylation and also augmented EGF-stimulated Akt phosphorylation. The cooperative effects of thrombin on Akt/p70S6K phosphorylation and [(3)H]thymidine incorporation were all attenuated by heterologous expression of Gbetagamma sequestrants. These data suggest that PI3K-dependent T389/T229 phosphorylation is limiting in late-phase p70S6K activation by EGF and contributes to the cooperative effect of GPCRs on p70S6K activity and cell growth.     

FAMCS： Finding All Maximal Common Substructures in Proteins

Finding the common substructures shared by two proteins is considered as one of the central issues in computational biology because of its usefulness in understanding the structure-function relationship and application in drug and vaccine design. In this paper, we propose a novel algorithm called FAMCS （Finding All Maximal Common Substructures） for the common substructure identification problem. Our method works initially at the protein secondary structural element （SSE） level and starts with the identification of all structurally similar SSE pairs. These SSE pairs are then merged into sets using a modified Apriori algorithm, which will test the similarity of various sets of SSE pairs incrementally until all the maximal sets of SSE pairs that deemed to be similar are found. The maximal common substructures of the two proteins will be formed from these maximal sets. A refinement algorithm is also proposed to fine tune the alignment from the SSE level to the residue level. Comparison of FAMCS with other methods on various proteins shows that FAMCS can address all four requirements and infer interesting biological discoveries.     

Systematic identification and analysis of exonic splicing silencers

Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.     

Hydrogen peroxide is necessary for abscisic acid-induced senescence of rice leaves

The role of H₂O₂ in abscisic acid (ABA)-induced rice leaf senescence is investigated. ABA treatment resulted in H₂O₂ production in rice leaves, which preceded the occurrence of leaf senescence. Dimethylthiourea, a chemical trap for H₂O₂, was observed to be effective in inhibiting ABA-induced senescence, ABA-increased malondialdehyde (MDA) content, ABA-increased antioxidative enzyme activities (superoxide dismutase, ascorbate peroxidase, glutathione reductase and catalase), and ABA-decreased antioxidant contents (ascorbic acid and reduced glutathione) in rice leaves. Diphenyleneiodonium chloride (DPI) and imidazole (IMD), inhibitors of NADPH oxidase, and KCN and NaN₃, inhibitors of peroxidase, prevented ABA-induced H₂O₂ production, suggesting NADPH oxidase and peroxidase are H₂O₂-generating enzymes in ABA-treated rice leaves. DPI, IMD, KCN, and NaN₃ also inhibited ABA-promoted senescence, ABA-increased MDA contents, ABA-increased antioxidative enzyme activities, and ABA-decreased antioxidants in rice leaves. These results suggest that H₂O₂ is involved in ABA-induced senescence of rice leaves.     

Light-induced proton slip and proton leak at the thylakoid membrane

A treatment of leaves of Spinacia oleracea L. with light or with the thiol reagent dithiothreitol in the dark led to partly uncoupled thylakoids. After induction in intact leaves, the partial uncoupling was irreversible at the level of isolated thylakoids. We distinguish between uncoupling by proton slip, which means a decrease of the H⁺/e⁻-ratio due to less efficient proton pumping, and proton leak as defined by enhanced kinetics of proton efflux. Proton slip and proton leak made about equal contributions to the total uncoupling. The enhanced proton efflux kinetics corresponded to reduction of subunit CF₁-γ of the ATP synthase as shown by fluorescence labeling of thylakoid proteins with the sulfhydryl probe 5-iodoacetamido fluorescein. The maximum value of the fraction of reduced CF₁-γ was only 36%, which indicates that in vivo the reduction of CF₁-γ could be limited by fast reoxidation and/or restricted accessibility of CF₁-γ to thioredoxin. Measurements of the ratio ATP/2e indicated that only the uncoupling related to less efficient proton pumping led to a decrease in the ATP yield.     

Size optimization of synthetic graft copolymers for in vivo angiogenesis imaging

Angiogenesis is a critical step in tumor development and more than 25 angiogenesis inhibitors are currently in clinical trials. Noninvasive in vivo imaging of angiogenesis represents a unique opportunity of repeatedly quantitating microvascular parameters prior to and during anti-angiogenic treatments. While several imaging tracers have been proposed for MR and nuclear imaging, there does not exist any consensus of what constitutes an ideal size of an imaging agent. A series of synthetic pegylated DOTA derivatized graft copolymers (30, 60, 120 kDa) were synthesized and their in vivo behavior tested in two breast cancer models differing in vascular endothelial growth factor (VEGF) expression. Polymers were labeled with different lanthanides (Eu, Gd, Dy) and absolute blood and tumor concentrations were determined by ICP-AES measurements. DOTA and the 30 kDa polymers underwent renal clearance resulting in low plasma levels. Slow leakage across neovasculature into tumor interstitium was clearly dependent on the molecular mass of all tested agents in MCF-7 tumors. However, a cutoff was observed with minimal extravasation occurring at and above 120 kDa in well differentiated MCF-7 tumors. VEGF overexpression caused detectable differences in extravasation of all polymers, including the 120 kDa compound. We conclude that large molecular weight contrast agents with a molecular mass of <120 kDa extravasate from experimental tumor neovasculature and may not be an accurate marker for measuring true blood volume fractions when in vivo imaging is performed in the steady state.     

In vivo molecular target assessment of matrix metalloproteinase inhibition

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.     

An early oxygen-dependent step is required for dexamethasone-induced apoptosis of immature mouse thymocytes

The roles of oxygen and reactive oxygen intermediates in apoptosis are unclear at present. Although oxygen and reactive oxygen intermediates are not required for the execution of apoptosis, oxygen may be involved in at least some forms of apoptosis. In this study we show that dexamethasone (Dex)-induced apoptosis of immature mouse thymocytes is completely inhibited by hypoxic culture. In contrast, anti-CD95 thymocyte apoptosis is unaffected by hypoxia, indicating the existence of two forms of thymocyte apoptosis: an oxygen-dependent pathway (Dex induced) and an oxygen-independent pathway (anti-CD95 induced). Furthermore, hypoxia inhibited mitochondrial permeability transition (PT) in Dex-treated, but not in anti-CD95-treated, thymocytes, suggesting that the oxygen-sensitive step is upstream of mitochondria. Both Dex- and anti-CD95-induced PT and apoptosis were dependent on activation of IL-converting enzyme-like protease, as PT and apoptosis were inhibited by preincubation with Cbz-Val-Ala-Asp-fluoromethyl ketone, an irreversible inhibitor of IL-converting enzyme-like proteases. In addition, hypoxia inhibited the activation by Dex of caspase-3 (CPP32)-like proteases. Our data show that the private signaling pathways of Dex (oxygen dependent) and anti-CD95 (oxygen independent) both converge upstream of mitochondrial changes. The oxygen-dependent step in Dex-induced apoptosis lies upstream of caspase-3-like protease activation. Our observations support a model of apoptosis signaling in which independent pathways (oxygen dependent and oxygen independent) particular to each stimuli converge at a central point in the apoptotic cascade.