Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Organization and nucleotide sequence of a gene cluster coding for eight ribosomal proteins in the archaebacterium Halobacterium marismortui

A DNA fragment containing the genes for the eight ribosomal proteins HmaL3, HL6, HmaL23, HmaL2, HmaS19, HmaL22, HmaS3, and HmaL29 from Halobacterium marismortui has been cloned and sequenced. The organization of this gene cluster in general corresponds to the S10 operon of Escherichia coli although there exists some differences between them. The sequence analysis of the 5'- and 3'-region of the gene cluster revealed three open reading frames (orf1, orf2, and orf3) which do not code for any ribosomal protein whose structure is known. A putative promoter is located upstream of orf1. Out of the eight ribosomal proteins five have counterparts in eubacteria only, two in both eubacteria and eukaryotes, and one is exclusively related to an eukaryotic ribosomal protein.     

Phylogenetic relationships among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> (Bacillaceae: Bacillales) strains based on a comparison of SSU rRNA sequences and genome profiling

Bacillus thuringiensis Berliner has previously been classified via the serological identification of flagellar antigens. However, the phylogenetic relationships among strains of B. thuringiensis cannot be investigated by serotyping. Furthermore, high levels of homology have been found in gene sequences among various strains, complicating the determination of their evolutionary relationships. In order to elucidate the phylogenetic relationships within B. thuringiensis, we analyzed 40 strains belonging to typical serotypes using two approaches: an analysis of small subunit (SSU) rRNA sequences and genome profiling (GP) based on temperature gradient gel electrophoresis of random PCR products. The SSU rRNA analysis resulted in all 40 strains forming a single cluster with Bacillus cereus Frankland & Frankland. The distances among the subclusters were too small to further classify the strains. On the other hand, the phylogenetic analysis based on GP resulted in three clusters of B. thuringiensis strains. These results suggest that GP is a better method for the determination of phylogenetic relationships within B. thuringiensis.     

Egg Deposition Behavior in the Haplodiploid Sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Symphyta: Tenthredinidae)

The egg deposition behavior of the turnip sawfly, Athalia rosae (Hymenoptera: Symphyta), is described. Both unmated and mated females lay eggs individually inside of fresh young leaves of cruciferous plants. During an oviposition event, females exhibit a distinct pause in abdominal contractions just before the actual egg deposition act. Unmated females show a longer pause (11.31 s on average) than mated females (4.38 s on overall average). By employing an eye color mutation, the sex of the eggs laid by females was ascertained. Females mated once lay mostly fertilized (diploid female) eggs initially but begin to lay a considerable number of unfertilized (haploid male) eggs later in life. The laying of an unfertilized egg is associated with a longer pause (6.98 s on average) than the laying of a fertilized egg (3.76 s on average). These results are in contrast to previous reports on apocritan Hymenoptera, where the presence of a pause or a longer pause during oviposition was associated with the deposition of fertilized eggs rather than unfertilized eggs. The possibility that mated Athalia rosae females control fertilization and its implications for sex allocation strategies are discussed.     

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.     

Quantum chemical studies for oxidation of morpholine by Cytochrome P450

Since morpholine oxidation has recently been shown to involve Cytochrome P450, the study on its mechanism at molecular level using quantum chemical calculations for the model of cytochrome active site is reported here. The reaction pathway is investigated for two electronic states, the doublet and the quartet, by means of density functional theory. The results show that morpholine hydroxylation occurs through hydrogen atom abstraction and rebound mechanism. However, in the low spin state, the reaction is concerted and hydrogen atom abstraction yields directly ferric-hydroxy morpholine complex without a distinct rebound step while in quartet state the reaction is stepwise. The presence of nitrogen in a morpholine heterocycle is postulated to greatly facilitate hydrogen abstraction. The hydroxylated product undergoes intramolecular hydrogen atom transfer from hydroxy group to nitrogen, leading to the cleavage of the C-N bond and the formation of 2-(2-aminoethoxy) acetaldehyde. The cleavage of the C-N bond is indicated as the rate-determining step for the studied reaction. The assistance of explicit water molecule is shown to lower the energy barrier for the C-N bond cleavage in enzymatic environment whereas solvent effects mimicked by COSMO solvent model have minor influence on relative energies along the pathway.     

Diel changes in resource use and diet overlap in temperate stream fishes

Interspecific variation in diel-scale temporal niches is common in natural communities. Such variation changes population dynamics via effects on the growth and reproduction of individuals. Also at the community level, theory predicts that animals can reduce competition for shared resources by changing diel activity in certain situations. However, the role of diel activity at the community-level has not been examined sufficiently. In this study, to examine whether the diel-scale temporal niche act as a competition-mitigating mechanism for stream fishes at the community level, we surveyed diel changes in microhabitat use and foraging, and the pattern of interspecific diet overlap in the middle reaches of a temperate stream where various fish species that seemed to be either nocturnal or diurnal coexisted. Our results suggest that the fishes forage during both daytime and night, but change their foraging mode at different times of the day, so that the foraging habits of these fish species cannot be divided simply into nocturnal and diurnal. Furthermore, fishes appeared to aggregate in the vicinity of common food resources during time zones with high availability of the resources, and therefore, inter-guild diet overlap was high during certain time zones. On the other hand, when inter-guild diet overlap was low, each fish species used foods or microhabitats that did not any have the potential to be used by species of another guild. Therefore, we conclude that variation in diel niche use is influenced by variation in the fundamental niche and food supply or availability rather than by competitive interaction between fishes in the stream fish community.     

Real-time detection of DNA hybridization on microarray using a CCD-based imaging system equipped with a rotated microlens array disk

This work describes a novel charge-coupled device (CCD)-based imaging system (MB Biochip Reader?) for real-time detection of DNA hybridization to DNA microarrays. The MB Biochip Reader? consisted of a laser light source (532 nm), a microlens array for generation of a multi-beam laser, and a CCD for 2-D signal imaging. The MB Biochip Reader? with a rotated microlens array, allowed large-field imaging (6.2 mm × 7.6 mm with 6.45 μm resolution) with fast time-resolution at 0.2 s without speckle noise. Furthermore, real-time detection of DNA hybridization, which is sufficient to obtain accurate data from tens of thousands of array element per field, was successfully performed without the need for laser scanning. The performance of the MB Biochip Reader? for DNA microarray imaging was similar to the commercially available photomultiplier tube (PMT)-based microarray scanner, ScanArray Lite. The system potentially could be applied toward real-time analysis in many other fluorescent techniques in addition to real-time DNA microarray analysis.     

Characterization of the mouse gene for the U-box-type ubiquitin ligase UFD2a

UFD2a is a mammalian homolog of Saccharomyces cerevisiae Ufd2, originally described as an E4 ubiquitination factor. UFD2a belongs to the U-box family of ubiquitin ligases (E3s) and likely functions as both an E3 and E4. We have isolated and characterized the mouse gene (Ube4b) for UFD2a. A full-length (approximately 5700 bp) Ube4b cDNA was isolated and the corresponding gene spans >100 kb, comprising 27 exons. Luciferase reporter gene analysis of the 5(') flanking region of Ube4b revealed that nucleotides -1018 to -943 (relative to the translation initiation site) possess promoter activity. This functional sequence contains two putative Sp1 binding sites but not a TATA box. Immunoblot and immunohistochemical analyses revealed that UFD2a is expressed predominantly in the neuronal tissues. We also show that UFD2a interacts with VCP (a AAA-family ATPase) that is thought to mediate protein folding. These data implicate UFD2a in the degradation of neuronal proteins by the ubiquitin-proteasome pathway.