Junctional form of acetylcholinesterase restored at nerve-free endplates.

Rat soleus muscles were ectopically innervated by implanting a foreign nerve in an endplate-free region of muscle and, 2–3 weeks later, cutting the original nerve. The junctional, 16 S form of acetylcholinesterase (AChE) and focal staining for AChE disappeared from the old endplate region within a few days after denervation. In muscles with an ectopic nerve, but not in paired control muscles, 16 S AChE and focal staining were restored in the old endplate region 1–2 weeks after denervation even though nerve fibers could not be detected in that region. These results suggest that the nerve exerts a local effect, specifying the site at which junctional AChE appears, and a nonlocal effect, perhaps mediated by muscle activity, regulating the amount of junctional AChE.     

Exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV

We have investigated the exposure and electronic interaction of tyrosine and tryptophan residues in human apolipoprotein A-IV (apo A-IV). Differential absorption spectroscopy and chemical titration demonstrated that human apo A-IV contains six tyrosine residues, four of which are buried in the hydrophobic interior of the protein and two of which are exposed on the protein surface. Denaturation of the protein by guanidinium chloride caused progressive exposure of the buried tyrosines. The fluorescence emission spectra of apo A-IV were characterized by a blue-shifted tryptophan emission with a low relative quantum yield of 0.37 and a tyrosine emission with a relative quantum yield of 0.62. Fluorescence quenching studies demonstrated a low fractional exposure of tryptophan in the native state. Denaturation of apo A-IV was accompanied by an increase in the relative quantum yield which peaked at the denaturation midpoint. Fluorescence excitation techniques demonstrated energy transfer from tyrosine residues with a transfer efficiency of 0.40 in the native state; the efficiency was conformation dependent and decreased with protein unfolding. Fluorescence studies of tetranitromethane-modified apo A-IV suggested that a significant fraction of energy transfer proceeds from the exposed tyrosine residues. These data demonstrate the existence of intramolecular fluorescence energy transfer and tryptophan quenching in human apolipoprotein A-IV and suggest that the amino terminus of this protein is situated in a hydrophobic domain within energy-transfer range of nonvicinal tyrosine residues.     

Revising the Absolute Configurations of Coatlines via Density Functional Theory Calculations of Electronic Circular Dichroism Spectra

Coatline A ( 1 ) and α‐epi‐coatline A ( 4 ) co‐occur in the trunk extract of Andira coriacea. Inspection of their chiroptical properties led to intriguing results. After a careful examination of the experimental data used for the previously reported absolute configuration of these compounds, some uncertainties were identified. A combined theoretical approach including conformational analyses and calculation of electronic circular dichroism (ECD) spectra, in addition with experimental data obtained for schoepfin A ( 5 ) and the new schoepfin D ( 6 ) isolated from Senna quinquangulata, allowed the revision of the absolute configuration of coatlines A ( 1 ) and B ( 2 ). Chirality 25:180–184, 2013. © 2012 Wiley Periodicals, Inc.     

Microdomains in Forebrain Spines: an Ultrastructural Perspective

Glutamatergic axons in the mammalian forebrain terminate predominantly onto dendritic spines. Long-term changes in the efficacy of these excitatory synapses are tightly coupled to changes in spine morphology. The reorganization of the actin cytoskeleton underlying this spine “morphing” involves numerous proteins that provide the machinery needed for adaptive cytoskeletal remodeling. Here, we review recent literature addressing the chemical architecture of the spine, focusing mainly on actin-binding proteins (ABPs). Accumulating evidence suggests that ABPs are organized into functionally distinct microdomains within the spine cytoplasm. This functional compartmentalization provides a structural basis for regulation of the spinoskeleton, offering a novel window into mechanisms underlying synaptic plasticity.     

Structural and Thermodynamic Characterization of the TYK2 and JAK3 Kinase Domains in Complex with CP-690550 and CMP-6

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

Random generation of RNA secondary structures according to native distributions

Background

Random biological sequences are a topic of great interest in genome analysis since, according to a powerful paradigm, they represent the background noise from which the actual biological information must differentiate. Accordingly, the generation of random sequences has been investigated for a long time. Similarly, random object of a more complicated structure like RNA molecules or proteins are of interest.

Results

In this article, we present a new general framework for deriving algorithms for the non-uniform random generation of combinatorial objects according to the encoding and probability distribution implied by a stochastic context-free grammar. Briefly, the framework extends on the well-known recursive method for (uniform) random generation and uses the popular framework of admissible specifications of combinatorial classes, introducing weighted combinatorial classes to allow for the non-uniform generation by means of unranking. This framework is used to derive an algorithm for the generation of RNA secondary structures of a given fixed size. We address the random generation of these structures according to a realistic distribution obtained from real-life data by using a very detailed context-free grammar (that models the class of RNA secondary structures by distinguishing between all known motifs in RNA structure). Compared to well-known sampling approaches used in several structure prediction tools (such as SFold) ours has two major advantages: Firstly, after a preprocessing step in time for the computation of all weighted class sizes needed, with our approach a set of m random secondary structures of a given structure size n can be computed in worst-case time complexity while other algorithms typically have a runtime in . Secondly, our approach works with integer arithmetic only which is faster and saves us from all the discomforting details of using floating point arithmetic with logarithmized probabilities.

Conclusion

A number of experimental results shows that our random generation method produces realistic output, at least with respect to the appearance of the different structural motifs. The algorithm is available as a webservice at http://wwwagak.cs.uni-kl.de/NonUniRandGen and can be used for generating random secondary structures of any specified RNA type. A link to download an implementation of our method (in Wolfram Mathematica) can be found there, too.     

Proteomic signatures of human oral epithelial cells in HIV-infected subjects

The oral epithelium, the most abundant structural tissue lining the oral mucosa, is an important line of defense against infectious microorganisms. HIV infected subjects on highly active antiretroviral therapy (HAART) are susceptible to comorbid viral, bacterial and fungal infections in the oral cavity. To provide an assessment of the molecular alterations of oral epithelia potentially associated with susceptibility to comorbid infections in such subjects, we performed various proteomic studies on over twenty HIV infected and healthy subjects. In a discovery phase two Dimensional Difference Gel Electrophoresis (2-D DIGE) analyses of human oral gingival epithelial cell (HOEC) lysates were carried out; this identified 61 differentially expressed proteins between HIV-infected on HAART subjects and healthy controls. Down regulated proteins in HIV-infected subjects include proteins associated with maintenance of protein folding and pro- and anti-inflammatory responses (e.g., heat-shock proteins, Cryab, Calr, IL-1RA, and Galectin-3-binding protein) as well as proteins involved in redox homeostasis and detoxification (e.g., Gstp1, Prdx1, and Ero1). Up regulated proteins include: protein disulfide isomerases, proteins whose expression is negatively regulated by Hsp90 (e.g., Ndrg1), and proteins that maintain cellular integrity (e.g., Vimentin). In a verification phase, proteins identified in the protein profiling experiments and those inferred from Ingenuity Pathway Analysis were analyzed using Western blotting analysis on separate HOEC lysate samples, confirming many of the discovery findings. Additionally in HIV-infected patient samples Heat Shock Factor 1 is down regulated, which explains the reduced heat shock responses, while activation of the MAPK signal transduction cascade is observed. Overall, HAART therapy provides an incomplete immune recovery of the oral epithelial cells of the oral cavity for HIV-infected subjects, and the toxic side effects of HAART and/or HIV chronicity silence expression of multiple proteins that in healthy subjects function to provide robust innate immune responses and combat cellular stress.     

Isolation of a putative progenitor subpopulation of alveolar epithelial type 2 cells

Alveolar epithelial type 2 cells (AEC2) isolated from hyperoxia-treated animals exhibit increases in both proliferation and DNA damage in response to culture. AEC2 express the zonula adherens proteins E-cadherin, -, - and -catenin, desmoglein, and pp120, as demonstrated by Western blotting. Immunohistochemical analysis of cultured AEC2 showed expression of E-cadherin on cytoplasmic membranes varying from strongly to weakly staining. When cultured AEC2 placed in suspension were labeled with fluorescent-tagged antibodies to E-cadherin, cells could be sorted into at least two subpopulations, either dim or brightly staining for this marker. With the use of antibody to E-cadherin bound to magnetic beads, cells were physically separated into E-cadherin-positive and -negative subpopulations, which were then analyzed for differences in proliferation and DNA damage. The E-cadherin-positive subpopulation contained the majority of damaged cells, was quiescent, and expressed low levels of telomerase activity, whereas the E-cadherin-negative subpopulation was undamaged, proliferative, and expressed high levels of telomerase activity.     

Spectrometric studies on stability of tenuazonic acid (TeA) solution in organic solvents

The stability of tenuazonic acid solution at different temperatures and storage times was studied using methanol, methanol-water (8:2 v/v), benzene and benzene-acetonitrile (98:2 v/v) as solvents. Solutions were analysed by a spectrometric method TeA U.V.-spectrum was recorded. Results indicated that the optimum temperature for long-time storage period of tenuazonic acid solution in any solvent assayed is -20°C. Benzene and benzene-acetonitrile (98:2 v/v) could be advised to make tenuazonic acid solution which will be stored less than 2 months at 4°C. Methanol and methanolwater (8:2 v/v) are not recommended because a low stability of TeA solution in this solvents.