Nucleosome dynamics

In the 30 years since the discovery of the nucleosome, our picture of it has come into sharp focus. The recent high-resolution structures have provided a wealth of insight into the function of the nucleosome, but they are inherently static. Our current knowledge of how nucleosomes can be reconfigured dynamically is at a much earlier stage. Here, recent advances in the understanding of chromatin structure and dynamics are highlighted. The ways in which different modes of nucleosome reconfiguration are likely to influence each other are discussed, and some of the factors likely to regulate the dynamic properties of nucleosomes are considered.     

Redox potentials of the blue copper sites of bilirubin oxidases

The redox potentials of the multicopper redox enzyme bilirubin oxidase (BOD) from two organisms were determined by mediated and direct spectroelectrochemistry. The potential of the T1 site of BOD from the fungus Myrothecium verrucaria was close to 670 mV, whereas that from Trachyderma tsunodae was >650 mV vs. NHE. For the first time, direct electron transfer was observed between gold electrodes and BODs. The redox potentials of the T2 sites of both BODs were near 390 mV vs. NHE, consistent with previous finding for laccase and suggesting that the redox potentials of the T2 copper sites of most blue multicopper oxidases are similar, about 400 mV.     

Facile synthesis toward the construction of an activity probe library for glycosidases

Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for beta-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including alpha- and beta-d-Glc, alpha- and beta-d-Gal, alpha-d-Man, and alpha-l-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target alpha-d-glucosidase, alpha-d-galactosidase, and alpha-l-fucosidase, respectively.     

Accelerated induction of apoptosis in insect cells by baculovirus-expressed SARS-CoV membrane protein

It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.     

Focal adhesions: what's new inside

The cytoplasmic side of focal adhesions is comprised of large molecular complexes that link transmembrane receptors, such as integrins, to the actin cytoskeleton and mediate signals modulating cell attachment, migration, proliferation, differentiation, and gene expression. These complexes are heterogeneous and dynamic structures that are apparent targets of regulatory signals that control the function of focal adhesions. Recent studies using genetic approaches in invertebrate and vertebrate systems have begun to reveal the structure and function of these complexes in vivo.     

Caspase-9 holoenzyme is a specific and optimal procaspase-3 processing machine

Caspase-9 activation is critical for intrinsic cell death. The activity of caspase-9 is increased dramatically upon association with the apoptosome, and the apoptosome bound caspase-9 is the caspase-9 holoenzyme (C9Holo). In this study, we use quantitative enzymatic assays to fully characterize C9Holo and a leucine-zipper-linked dimeric caspase-9 (LZ-C9). We surprisingly show that LZ-C9 is more active than C9Holo for the optimal caspase-9 peptide substrate LEHD-AFC but is much less active than C9Holo for the physiological substrate procaspase-3. The measured Km values of C9Holo and LZ-C9 for LEHD-AFC are similar, demonstrating that dimerization is sufficient for catalytic activation of caspase-9. The lower activity of C9Holo against LEHD-AFC may be attributed to incomplete C9Holo assembly. However, the measured Km of C9Holo for procaspase-3 is much lower than that of LZ-C9. Therefore, in addition to dimerization, the apoptosome activates caspase-9 by enhancing its affinity for procaspase-3, which is important for procaspase-3 activation at the physiological concentration.     

New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 1. Investigation of the influence of structural parameters on the extent of degradation

Six methyl celluloses (MCs), one with a degree of substitution (DS) of 1.32 and five with DS between 1.83 and 1.88, were thoroughly investigated. Monomer composition and methyl distribution in the polymer chain were analyzed after total or partial random hydrolysis and appropriate derivatization with gas chromatography (GC) and mass spectrometry (MS), respectively, and used as reference data. The same MCs were then hydrolyzed with an enzyme preparation of Trichoderma longibrachiatum and further investigated with size-exclusion chromatography with multiangle light scattering and refractive index detection (SEC-MALS/RI) and MS. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) in combination with various MS analyzers were compared with respect to quantification of the degradation products directly and after perdeuteriomethylation. The methyl group distribution in the oligomeric fractions and the average DS as a function of chain length were calculated from ESI mass spectra. With help of the reference analysis, patterns could be corrected for the unspecific contribution of end groups. By labeling and ESI tandem MS, our knowledge about the tolerance of the enzymes' sub-sites with respect to the number of methyl groups could be improved.     

New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 2. Comparison of various enzyme preparations

In this part of our studies, dealing with new approaches to the analysis of enzymatically hydrolyzed methyl cellulose, five different enzymes or enzyme preparations containing endoglucanases (from Bacillus agaradhaerens Cel 5A, Trichoderma reesei, Trichoderma viride, and two obtained from Trichoderma longibrachiatum) were used to hydrolyze six different methyl celluloses (MCs). The main goal was to investigate whether enzymes could be used for determination of the heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity, it was necessary to gather information on how the enzymes affect hydrolysis. Size exclusion chromatography with multi-angle light scattering and refractive index detection (SEC-MALS/RI) was used to estimate the molar mass distribution of the MCs before and after hydrolysis. A novel internal standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-ITMS) was used to determine the amount of formed oligomers. Two MCs, one with a degree of substitution (DS) of 1.8 and one with DS 1.3, were hydrolyzed with all of the five enzymes. The yield of summarized di- and trisaccharides was approximately 2% of the hydrolysis products for the MC with DS 1.8, whereas the product mixture, obtained from a MC with a DS of 1.3, contained 7-16% di- and trisaccharides. By a novel sample preparation method in combination with ESI-IT tandem MS, outlined in part 1 of this work, it was shown that the enzymes produced oligomers with the reducing end bearing no or only one substituent. Comparison of the methyl pattern at the nonreducing ends of the dimers and trimers indicated that the -2 subsite of the active complex is less tolerant than subsites -3 and +1. All enzymes had similar general selectivity toward the methyl substituents but also showed some differences. From both SEC-MALS/RI and ESI-ITMS, differences with respect to substituent distribution of MCs could be recognized but not for each enzyme used. Basic considerations for enzymatic hydrolysis and analysis of methyl cellulose were listed as a consequence of the results from the work.     

Modification of the pyridine moiety of non-peptidyl indole GnRH receptor antagonists

The synthesis of a number of indole GnRH antagonists is described. Oxidation of the pyridine ring nitrogen, combined with alkylation at the two position, led to a compound with an excellent in vitro activity profile as well as oral bioavailability in both rats and dogs.