The nucleotide sequence of the replication origin beta of the plasmid R6K

We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta. This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature. The nucleotide sequence of the region of replication origin beta has been determined. The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.     

Comparative Molecular Modeling Study of Arabidopsis NADPH-Dependent Thioredoxin Reductase and Its Hybrid Protein

2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region, ii) shifted Trx domain, and iii) different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

Intermittent Hypoxia Can Aggravate Motor Neuronal Loss and Cognitive Dysfunction in ALS Mice

Background

Patients with ALS may be exposed to variable degrees of chronic intermittent hypoxia. However, all previous experimental studies on the effects of hypoxia in ALS have only used a sustained hypoxia model and it is possible that chronic intermittent hypoxia exerts effects via a different molecular mechanism from that of sustained hypoxia. No study has yet shown that hypoxia (either chronic intermittent or sustained) can affect the loss of motor neurons or cognitive function in an in vivo model of ALS.

Objective

To evaluate the effects of chronic intermittent hypoxia on motor and cognitive function in ALS mice.

Methods

Sixteen ALS mice and 16 wild-type mice were divided into 2 groups and subjected to either chronic intermittent hypoxia or normoxia for 2 weeks. The effects of chronic intermittent hypoxia on ALS mice were evaluated using the rotarod, Y-maze, and wire-hanging tests. In addition, numbers of motor neurons in the ventral horn of the spinal cord were counted and western blot analyses were performed for markers of oxidative stress and inflammatory pathway activation.

Results

Compared to ALS mice kept in normoxic conditions, ALS mice that experienced chronic intermittent hypoxia had poorer motor learning on the rotarod test, poorer spatial memory on the Y-maze test, shorter wire hanging time, and fewer motor neurons in the ventral spinal cord. Compared to ALS-normoxic and wild-type mice, ALS mice that experienced chronic intermittent hypoxia had higher levels of oxidative stress and inflammation.

Conclusions

Chronic intermittent hypoxia can aggravate motor neuronal death, neuromuscular weakness, and probably cognitive dysfunction in ALS mice. The generation of oxidative stress with activation of inflammatory pathways may be associated with this mechanism. Our study will provide insight into the association of hypoxia with disease progression, and in turn, the rationale for an early non-invasive ventilation treatment in patients with ALS.     

S100A7, S100A10, and S100A11 are transglutaminase substrates

S100 proteins are a family of 10-14 kDa EF-hand-containing calcium binding proteins that function to transmit calcium-dependent cell regulatory signals. S100 proteins have no intrinsic enzyme activity but bind in a calcium-dependent manner to target proteins to modulate target protein function. Transglutaminases are enzymes that catalyze the formation of covalent epsilon-(gamma-glutamyl)lysine bonds between protein-bound glutamine and lysine residues. In the present study we show that transglutaminase-dependent covalent modification is a property shared by several S100 proteins and that both type I and type II transglutaminases can modify S100 proteins. We further show that the reactive regions are at the solvent-exposed amino- and carboxyl-terminal ends of the protein, regions that specify S100 protein function. We suggest that transglutaminase-dependent modification is a general mechanism designed to regulate S100 protein function.     

Inhibition of cytochrome P450 isozymes and ornithine decarboxylase activities by polysaccharides from soybeans fermented with Phellinus igniarius or Agrocybe cylindracea

Polysaccharides (5, 10, 25, 50 and 100 microg ml(-1)) from soybeans and soybeans fermented with Phellinus igniarius or Agrocybe cylindracea inhibited cytochrome P450 1A1, cytochrome P450 1A2 and cytochrome P450 2B1 activities in rat liver microsomes. The polysaccharides (5, 10 and 25 microg ml(-1)) also suppressed 12-O-tetradecanoylphorbol-13-acetate-induced ornithine decarboxylase activity. The most potent inhibitors of cytochrome P450 isozymes and ornithine decarboxylase activities were the polysaccharides from soybeans fermented with Agrocybe cylindracea.     

Epimerization of hydroxyl group in the lupan series of triterpenes

Two methods of obtaining of 3 alpha-betulinic acid and related compounds from their 3 beta-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3-O-tosyllupeol or of the betulin hydroxyl by benzoyloxy group resulted only in delta 2, 3-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60:40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3 alpha-betulinic acid increased toward melanoma Bro cells and decreased toward melanoma MS cells.     

Epimerization of Hydroxyl Group in Lupan Series Triterpenoids

Two methods of obtaining 3-betulinic acid and related compounds from their 3-epimers were studied: the reaction of bimolecular substitution and the stereoselective reduction of 3-ketoderivatives. The substitution of acyloxy by formyloxy group in 3--tosyllupeol or of the belulin hydroxyl by benzoyloxy group resulted only in ^{2, 3}-elimination products, with none of the expected products of bimolecular substitution being found. The catalytic hydrogenation of betulonic acid over Raney nickel resulted only in reduction of the isopropenyl double bond, whereas the use of 5% Ru/C gave a 60 : 40 mixture of epimers of dihydrobetulinic acid. Practically the same mixture of betulinic acid epimers was obtained when reducing betulonic acid with L-Selectride. The cytotoxic activity of 3-betulinic acid increased toward the Bro melanoma cells and decreased toward the MS melanoma cells.     

Enzyme-linked immunosorbent assay for detection of chitooligosaccharides

In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 microg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100%, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40%, respectively.