Production and characterization of long-acting recombinant human albumin–EPO fusion protein expressed in CHO cell

A long-lasting recombinant human albumin-linker-erythropoietin (EPO) is a human albumin gene fused to the N-terminal of EPO with a (GGSGG)_n-repeated linker inserted between albumin and EPO. Albumin–EPO fusion genes were co-transfected with the dhfr gene. Albumin–EPO fusion protein has three kinds of sub-types (IALE, AD2LE, AD1LE). Albumin–EPO fusion protein was quantified with human EPO ELISA. The in vitro efficacy of albumin–EPO fusion protein was estimated using F-36E cell, and in vivo efficacy of albumin–EPO fusion protein was estimated using normocythemic mice (B6D2F1). We also determined the in vivo half-life in a Sprague–Dawley rat. A PLA program analysis result demonstrated that the albumin–EPO fusion protein IALE is about 7.8-fold more potent than rHuEPO in increasing the hematocrit of normal mice.     

Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A·A base pairing and a putative structure of the coralyne-induced homo-adenine duplex

Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson–Crick DNA. Hud’s laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine–adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the six most favorable duplex structures. The two most stable structures had trans glycosidic bonds, but distinct pairing geometries, i.e. either Watson–Crick Hoogsteen (transWH) or Watson–Crick Watson–Crick (transWW) with stability of transWH > transWW. To narrow down the possibilities, 7-deaza adenine base substitutions (dA→7) were engineered into homo-(dA) sequences. These substitutions significantly reduced the thermal stability of the coralyne-induced homo-(dA) structure. These experiments strongly suggest the involvement of N7 in the coralyne-induced A·A base pairs. Moreover, due to the differential effect on melting as a function of the location of the dA→7 mutations, these results are consistent with the N1–N7 base pairing of the transWH pairs. Together, the simulation and base substitution experiments predict that the coralyne-induced homo-(dA) duplex structure adopts the transWH geometry.     

Proteomic analysis revealed a strong association of a high level of alpha1-antitrypsin in gastric juice with gastric cancer

To investigate the pathology of gastric disorders, we compared the proteomic patterns of gastric juice from patients with various gastric disorders. In healthy subjects pepsin A, pepsin B and gastric lipase were the major proteins detected by two-dimensional gel electrophoresis. These digestive enzymes were not detected in 60% of gastric cancer cases (18 out of 30 analyzed cases). Interestingly, an extraordinary amount of alpha(1)-antitrypsin was observed in these cases. In contrast to gastric cancer cases, alpha(1)-antitrypsin was detected in only 5% of patients (three out of 56) with chronic atrophic gastritis, and the detection frequency went up as the disease developed (one of four intestinal metaplasia cases, two of seven tubular adenoma cases, a single examined case of hyperplastic polyp and 60% of gastric cancer). Zymography showed that a 60 kDa protease strongly associated with alpha(1)-antitrypsin and mass spectrometric analysis revealed that the gastric alpha(1)-antitrypsin was a protease-cleaved form. Our data suggest that alpha(1)-antitrypsin and 60 kDa protease may serve as good diagnostic and prognostic markers for conditions associated with gastric cancer.     

Effects of transferring in vitro-cultured rabbit embryos to recipient oviducts on mucin coat deposition, implantation and development

A mucin coat is deposited on rabbit embryos during passage through the oviduct; rabbit blastocysts cultured from the 1-cell stage in vitro have no mucin coat. When cultured blastocysts are transferred to recipients, the lack of mucin coat might account in part for subsequent failure of pregnancy. We have investigated the possibility that mucin coat deposition is induced following transfer of in vitro 72 h-cultured blastocysts to oviducts of asynchronous or synchronous recipients. One-cell embryos were collected by flushing oviducts 19-20 h post-coitus and were cultured in vitro for 72 h until they reached the blastocyst stage. The blastocysts were transferred to the oviducts of recipients that were synchronized either with the donors (synchronous) or 1 day later than the donors (asynchronous). They were recovered after 24-48 h and the mucin coat thickness and embryo degeneration rate were measured. The degeneration rate of blastocysts recovered from uteri of synchronous recipients was higher than that from asynchronous recipients (72.2% vs 40.0%). The mucin coats around embryos recovered from oviducts of asynchronous recipients after 48 h were thicker than those from synchronous recipients. More asynchronous recipients were pregnant and gave birth to more pups than synchronous recipients. These results indicate that the oviducts of asynchronous recipients secreted more mucin around the transferred embryos, causing higher rates of implantation of the in vitro-cultured blastocysts.     

Tilt aftereffects generated by bilaterally symmetrical patterns

Tilt aftereffects were generated by bilaterally symmetrical dot patterns. Both expansion and contraction effects, similar in size and magnitude to effects usually reported with luminance contours, were observed after adaptation to symmetrical patterns tilted 15 deg or 75 deg respectively from a vertically oriented test. Large effects were found when both adapting and test stimuli were symmetrical patterns while smaller effects were found when the adapting stimulus was symmetrical and test stimulus was a grating. A third experiment, which manipulated the number of dots near the axis line, confirmed the above findings; expansion and contraction effects were observed again. The results of these experiments suggest that the neural mechanism underlying the perception of luminance contours may be linked to the mechanism for the detection of symmetry.     

<Emphasis Type="Italic">Pedobacter yonginense</Emphasis> sp. nov., isolated from a mesotrophic artificial Lake in Korea

A non-motile red-pigmented bacterium, designated strain HMD1002^T, was isolated from an artificial lake located on the campus of Hankuk University of Foreign Studies, South Korea. The major fatty acids were iso-C_15:0 (29.6%), Summed Feature 3 (comprising C_16:1 ω7c and/or iso-C_15:0 2-OH; 17.5%) and iso-C_17:0 3-OH (12.5%). The major isoprenoid quinone was menaquinone-7 (MK-7). The DNA G+C content was 41.0 mol%. A phylogenetic tree based on 16S rRNA gene sequences showed that strain HMD1002^T formed a lineage in the genus Pedobacer and was closely related to Pedobacer terrae (96.3%) and Pedobacer suwonensis (95.8%) in sequence similarity. On the basis of the evidence presented in this study, strain HMD1002^T represents a novel species of the genus Pedobacter, for which the name Pedobacter yonginense sp.nov. is proposed. The type strain is HMD1002^T (=KCTC 22721^T = CEC^T 7544^T).     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).