Highly conserved cysteines of mouse core 2 beta1,6-N-acetylglucosaminyltransferase I form a network of disulfide bonds and include a thiol that affects enzyme activity

Core 2 beta1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59-Cys413, Cys100-Cys172, Cys151-Cys199, and Cys372-Cys381. The only non-conserved residue within the beta1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 --> Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage beta-glucosyltransferase fold.     

Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response

Anumber of proteins are recruited to nuclear foci upon exposure to double-strand DNA damage, including 53BP1 and Rad51, but the precise role of these DNA damage-induced foci remain unclear. Here we show in a variety of human cell lines that histone deacetylase (HDAC) 4 is recruited to foci with kinetics similar to, and colocalizes with, 53BP1 after exposure to agents causing double-stranded DNA breaks. HDAC4 foci gradually disappeared in repair-proficient cells but persisted in repair-deficient cell lines or cells irradiated with a lethal dose, suggesting that resolution of HDAC4 foci is linked to repair. Silencing of HDAC4 via RNA interference surprisingly also decreased levels of 53BP1 protein, abrogated the DNA damage-induced G2 delay, and radiosensitized HeLa cells. Our combined results suggest that HDAC4 is a critical component of the DNA damage response pathway that acts through 53BP1 and perhaps contributes in maintaining the G2 cell cycle checkpoint.     

Hierarchical genetic algorithm for near optimal feedforward neural network design

In this paper, we propose a genetic algorithm based design procedure for a multi layer feed forward neural network. A hierarchical genetic algorithm is used to evolve both the neural networks topology and weighting parameters. Compared with traditional genetic algorithm based designs for neural networks, the hierarchical approach addresses several deficiencies, including a feasibility check highlighted in literature. A multi objective cost function is used herein to optimize the performance and topology of the evolved neural network simultaneously. In the prediction of Mackey Glass chaotic time series, the networks designed by the proposed approach prove to be competitive, or even superior, to traditional learning algorithms for the multi layer Perceptron networks and radial basis function networks. Based upon the chosen cost function, a linear weight combination decision making approach has been applied to derive an approximated Pareto optimal solution set. Therefore, designing a set of neural networks can be considered as solving a two objective optimization problem.     

The S. cerevisiae architectural HMGB protein NHP6A complexed with DNA: DNA and protein conformational changes upon binding

NHP6A is a non-sequence-specific DNA-binding protein from Saccharomyces cerevisiae which belongs to the HMGB protein family. Previously, we have solved the structure of NHP6A in the absence of DNA and modeled its interaction with DNA. Here, we present the refined solution structures of the NHP6A-DNA complex as well as the free 15bp DNA. Both the free and bound forms of the protein adopt the typical L-shaped HMGB domain fold. The DNA in the complex undergoes significant structural rearrangement from its free form while the protein shows smaller but significant conformational changes in the complex. Structural and mutational analysis as well as comparison of the complex with the free DNA provides insight into the factors that contribute to binding site selection and DNA deformations in the complex. Further insight into the amino acid determinants of DNA binding by HMGB domain proteins is given by a correlation study of NHP6A and 32 other HMGB domains belonging to both the DNA-sequence-specific and non-sequence-specific families of HMGB proteins. The resulting correlations can be rationalized by comparison of solved structures of HMGB proteins.     

A new synnematous species of Penicillium from soil in Taiwan

A synnematous species of Penicillium, P. calidicanium, is described and illustrated. The fungus was isolated from soil in Taiwan. Penicillium calidicanium can be placed in subgenus Biverticillium because of its symmetrical, biverticillate penicilli, ampulliform to acerose phialides, and ability to produce abundant synnemata in Czapek yeast extract agar, malt extract agar, and Czapek's solution agar. It is close to P. duclauxii and P. vulpinum, but differs in colony morphology, growth rate, morphology of the synnemata, and ornamentation of the conidial wall.     

Export of L-isoleucine from Corynebacterium glutamicum: a two-gene-encoded member of a new translocator family

Bacteria possess amino acid export systems, and Corynebacterium glutamicum excretes L-isoleucine in a process dependent on the proton motive force. In order to identify the system responsible for L-isoleucine export, we have used transposon mutagenesis to isolate mutants of C. glutamicum sensitive to the peptide isoleucyl-isoleucine. In one such mutant, strong peptide sensitivity resulted from insertion into a gene designated brnF encoding a hydrophobic protein predicted to possess seven transmembrane spanning helices. brnE is located downstream of brnF and encodes a second hydrophobic protein with four putative membrane-spanning helices. A mutant deleted of both genes no longer exports L-isoleucine, whereas an overexpressing strain exports this amino acid at an increased rate. BrnF and BrnE together are also required for the export of L-leucine and L-valine. BrnFE is thus a two-component export permease specific for aliphatic hydrophobic amino acids. Upstream of brnFE and transcribed divergently is an Lrp-like regulatory gene required for active export. Searches for homologues of BrnFE show that this type of exporter is widespread in prokaryotes but lacking in eukaryotes and that both gene products which together comprise the members of a novel family, the LIV-E family, generally map together within a single operon. Comparisons of the BrnF and BrnE phylogenetic trees show that gene duplication events in the early bacterial lineage gave rise to multiple paralogues that have been retained in alpha-proteobacteria but not in other prokaryotes analyzed.     

Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis. A Fourier transform infrared spectroscopy study

A technique based on Fourier transform infrared (FT-IR) spectrometry was developed to detect the corresponding changes in chemical composition associated with the rapid changes in sodium and water content in 200 mM NaCl-stressed halophyte ice plants (Mesembryanthemum crystallinum). The changes in glycophyte Arabidopsis stressed with 50 mM NaCl were also examined for comparison. The obtained IR spectra were further processed by deconvolution and curve fitting to examine the chemical nature of the responding sources in the leaves. Using three stages of ice plant leaves, absorption bands corresponding to carbohydrates, cell wall pectin, and proteins were identified, with distinct IR spectra representing each developmental stage. Within 48 h of mild salt stress, the absorption band intensities in the fingerprint region increased continuously in both plants, suggesting that the carbon assimilation was not affected at the early stage of stress. The intensities of ester and amide I absorption bands decreased slightly in Arabidopsis but increased in ice plant, suggesting that the cell expansion and protein synthesis ceased in Arabidopsis but continued in ice plant. In both plants, the shift in amide I absorption band was observed hourly after salt stress, indicating a rapid conformational change of cellular proteins. Analyses of the ratio between major and minor amide I absorption band revealed that ice plant was able to maintain a higher-ordered form of proteins under stress. Furthermore, the changes in protein conformation showed a positive correlation to the leaf sodium contents in ice plant, but not in Arabidopsis.     

Tetrahydrobiopterin improves vascular endothelial function in ovariectomized rats

The goal of the present study is to investigate the role of tetrahydrobiopterin (BH4) in the vascular response in ovariectomized rats. Rats were randomly assigned to two groups: (1) sham group: sham-operated female rats, and (2) Ovx group: rats were ovariectomized. Our results have shown that the plasma 17 beta-estradiol levels in the Ovx group at the end of the experiment were significantly lower than in the sham group. Vasoreactivity assessed with intact aortic rings indicated that the phenylephrine-induced vasocontractile response to aortic rings from the Ovx group was greater than that of the sham group. In contrast, the vasodilator responses to acetylcholine and L-arginine (L-Arg) in the sham group were significantly greater than in the Ovx group. Differences in vasoreactivity in denuded aorta between the two groups were not noted. Moreover, exogenous BH4 significantly restored L-Arg-induced vasodilator responses in the Ovx group. However, this improvement effect was not found in the sham group. In addition, there were significant increases in superoxide anion production in aortic tissue and significant decreases in plasma nitric oxide levels in the Ovx group. Furthermore, BH4 contents in the aorta in the Ovx group were significantly decreased compared with the sham group. In conclusion, the present study demonstrates that the impairment of vascular reactivity was found in the ovariectomized rats. The possible mechanism of this defect may have resulted from the deficiency of available BH4. Thus, this study may provide a novel therapeutic strategy for the treatment of postmenopausal cardiovascular disorders.