Antibodies to Asp-Asp-Glu-Asp can inhibit transport of nuclear proteins into the nucleus

The signal sequence of simian virus 40 (SV40) large T-antigen for translocation into the nucleus is composed of positively charged amino acids Lys-Lys-Lys-Arg-Lys. Rabbit antibodies to a synthetic peptide containing the negatively charged amino acid sequence Asp-Asp-Asp-Glu-Asp were obtained. Indirect immunofluorescence of the antigens recognized by the antibody was punctate at the nuclear rim or the nuclear surface, depending on the plane of focus. The antibody blocked transport of nuclear proteins into the nucleus. The antigens recognized by the antibody were predominantly localized to the nuclear pores.     

Expressions of tumor necrosis factor-α, its receptor I,II and receptor-associated factor 2 in the porcine corpus luteum during the estrous cycle and early pregnancy

We examined the gene and protein levels of tumor necrosis factor (TNF)-α, its receptors (types I and II, designated TNF-RI and TNF-RII, respectively), TNF receptor-associated factor 2 (TRAF2) and morphological features in the porcine corpus luteum (CL), on Days 13 and 17 (Day 0 = the last day of estrus) of the estrous cycle or of early pregnancy. Gene expression levels of TNF-α, TNF-RI, TNF-RII and TRAF2 were unaffected by the day or reproductive status. TNF-α concentration was significantly higher in the CL on Day 17 of pregnancy than on Day 13 of pregnancy and on day 17 of the estrous cycle. The TNF-RI protein level was significantly higher in the CL on Days 13 and 17 of pregnancy than those of the estrous cycle, significantly increasing on Day 17 compared with those on Day 13 in pregnancy. In relation to TNF-RII protein levels, although there were no change during pregnancy, there was a tendency (P?=?0.0524) to up-regulate as pregnancy proceeded. In estrous cycle, TNF-RII protein levels decreased significantly as luteolysis proceeded. TRAF2 protein level was significantly higher in the CL on Days 13 and 17 of pregnancy than during estrous. There were few apoptotic bodies in the CL between Days 13 and 17 of pregnancy than during esrous. There were few apoptotic bodies in the CL between Days 13 and 17 of pregnancy. The number of apoptotic bodies was much greater than the CL on Day 17 of the estrous than those of pregnancy. Thus, the TNF-α and TNF-RI and TNF-RII pathways including the TRAF2 protein, known to control of cell differentiation, tissue renewal and apoptosis, might participate in maintaining the porcine CL during early pregnancy.     

Age validation and growth variability of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis> off the Pacific coast of northern Japan

ABSTRACT:   This study examined age and growth of Japanese flounder Paralichthys olivaceus off the Pacific coast of northern Japan, and determined whether the growth patterns of male and female fish in northern (40–41°N) and southern (37–38°15'N) waters differ. In total 8095 specimens were collected between January 1999 and December 2005. Zonation consisting of opaque and translucent bands on the otolith was evident. Within each opaque band a thin and clear check (ring mark) was observed in all specimens examined. Monthly change in the frequency of appearance of a ring mark on the outer margin of the otolith indicates that ring marks form between July and August. The von Bertalanffy growth model showed a sexual dimorphism in growth, as females grew faster and reached a larger size than males. The growth patterns obtained by tracking the observed total length for monthly collections showed a rapid increase in total length between August and October. Spatial variation in the growth pattern of male and female fish between northern and southern waters was evident, as southern fish were significantly larger than northern counterparts during 1.25–3.00 years post hatch.     

Sorption Behavior of Arsenate by Mg-Bearing Minerals at Hyperalkaline Condition: Implications for Oxyanions Sequestration During the Use and Disposal of Alkaline Wastes

The utilization and disposal of alkaline waste materials such as slag and coal fly ash as cement aggregates and raw materials in cement manufacturing can pose environmental and health hazards because these waste materials usually contain elevated concentration of toxic elements. This study examined the possibility of controlling the pore water chemistry of these waste materials in order to induce the secondary mineral formation of Mg-bearing minerals as major sorbing solids for oxyanions during the utilization and disposal of alkaline wastes. The formation of Mg-bearing minerals was examined at ambient temperature and alkaline pH conditions in the Mg?CSi?CAl system. The interaction of Mg-bearing minerals with oxyanions using arsenate as an analog was examined during and after mineral formation. The results revealed that the generated Mg-bearing mineral phases were smectite and brucite in Mg?CSi system and hydrotalcite and serpentine in Mg?CSi?CAl system. Moreover, hydrotalcite, serpentine, brucite, and smectite phases formed under low Si ratio showed high sorption capacity for arsenate, but only high Al content hydrotalcite and serpentine showed substantial irreversible fraction of sorbed arsenate. Hence, the generation of these kinds of hydrotalcite and serpentine phases as scavengers for oxyanions must be considered during the utilization and disposal of alkaline wastes.     

Seasonal changes and controlling factors of gross N transformation in an evergreen plantation forest in central Japan

We conducted a year-round measurement of gross N transformation rates using the ¹⁵N dilution method, and analyzed seasonal changes and the mechanisms regulating gross N transformation in the Kiryu Experimental Forest in central Japan. While soil microbial biomass C (SMB-C) decreased from the dormant to growing seasons at the organic (O) horizon, no significant trend was observed in SMB-N. This resulted in SMB-C/N being high in the dormant season and low in the growing season, and suggests that the microbial composition changed seasonally. No clear seasonal trend was found in gross NH₄ ⁺ production rates at either the O or surface mineral soil horizons. In contrast, the NH₄ ⁺ consumption rate varied seasonally, with high values in January and April during the dormant season and low values in July and October during the growing season. There was no clear trend in seasonal fluctuation of net NH₄ ⁺ production rates. Gross NH₄ ⁺ production and gross NH₄ ⁺ consumption rates were 10 times greater than the gross nitrification rate. Almost all of the produced NH₄ ⁺ was immobilized, indicating that N tightly cycles at this study site. Considered together with results of the gross N transformation rates, the dominance of high SMB-C/N microbes might stimulate immobilization in the dormant season. At this study site, the change in microbial composition likely influences gross N transformation through immobilization efficiency.     

Expression of Membrane Complement Regulatory Proteins Crry and CD55 in Normal Rats

Some anticancer therapeutic antibodies are designed to act through complement-dependent cytotoxicity (CDC). It has been reported that there are many membrane complement regulatory proteins (mCRPs) that inhibit CDC. In the present study, we examined the expression of two mCRPs, the complement receptor 1-related gene/protein Y (Crry) and the decay-accelerating factor CD55, in three normal rats by immunohistochemistry. Crry and CD55 were detected widely in rat organs and tissues. Crry was found mainly in the urinary, digestive, respiratory, immunohematopoietic, circulatory and neuroendocrine systems. CD55 was found in the urinary, digestive and neuroendocrine systems. However, the two molecules were expressed in separate cells within the same organ. These results suggest that the distribution of mCRPs is related to the strict regulation of CDC activation in these organs and tissues and that the two molecules have a nonoverlapping expression pattern, a fact indicating specific roles in CDC regulation.     

Molecular cloning and sequence analysis of cDNAs encoding for Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus actins

The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of these genes confirmed the high similarity among actins from ticks in comparison to other species.     

Canine tyrosine hydroxylase (TH) gene and dopamine beta -hydroxylase (DBH) gene: their sequences, genetic polymorphisms, and diversities among five different dog breeds

Dopamine and noradrenaline are catecholamine neurotransmitters that are produced by biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta -hydroxylase (DBH). As a first step to elucidate the genetic background of canine behavioral traits, we selected these genes as targets and sequenced these canine genes, and found that both were highly homologous with those of human beings. Then brain cDNAs derived from ten unrelated Beagles were used to search for polymorphisms in these genes. Four single nucleotide polymorphisms (SNPs) (C97T, G168A, G180A and C264T), one of which (C97T) will cause amino acid substitution in the TH gene, and two SNPs (C789A and A1819G), both of which will cause amino acid substitutions in the DBH gene were identified. The allelic frequencies among five dog breeds (47 Golden Retrievers, 41 Labrador Retrievers, 40 Malteses, 26 Miniature Schnauzers, and 39 Shibas) were examined and found to have significant variation between them with regards to all these SNPs, except for C97T in the TH gene and A1819G in the DBH gene. The polymorphisms of C97T and A1819G were found only in the Shiba. The present results suggest that the polymorphisms of the genes encoding catecholamine biosynthetic enzymes may become important markers for examining the genetic background of behavioral characteristics in dogs.     

A chilling-insensitive stage in germination of a low-temperature-adapted radish, rat’s tail radish (Raphanus sativus L.) cv. “Pakki-hood”

The rat’s tail radish (Raphanus sativus cv. “Pakki-hood”), cultivated in the northern part of Thailand, does not require low temperature for flower-bud initiation and shows high germinability even at low temperature. To analyze its characteristic temperature response, seedlings were subjected to a 3 day-chilling treatment at 0°C after a series of incubations for germination at 26°C which ranged from 3 h to 4 days. The chilling treatment after an initial stage (3 h to less than 2 days) of incubation at 26°C did not significantly affect the hypocotyl regrowth, but the same treatment after a more than 2-day incubation at 26°C reduced subsequent hypocotyl growth of plants. 1-aminocyclopropane-1-carboxylic acid (ACC)-dependent ethylene-forming activity was also reduced by the chilling treatment after 2–4-day incubation at 26°C. Electrolyte leakage from tissues of seedlings in the chilling treatment was increased in proportion to the length of the incubation at 26°C, indicating severe damage of chilling to plasma membranes after a longer incubation. We found a transition of chilling-sensitivity at 2 days in the incubation period for germinating seeds of Pakki-hood. Seedlings of Pakki-hood chilled earlier than the transition are relatively unaffected in their subsequent growth, but the seedlings become sensitive to chilling after the transition.     

Characterization of specific antibodies and the establishment of sandwich ELISA and ELISPOT systems for swine IL-4

We produced four monoclonal antibodies (mAb) and two polyclonal antibodies using the purified cytokine expressed in bacteria and characterized them. Specific binding of each of the mAb and polyclonal antibodies to recombinant swine IL-4 (rSwIL-4) purified from Escherichia coli and baculovirus was demonstrated in an indirect ELISA and/or in western blotting. We established a sandwich enzyme-linked immunosorbent assay (ELISA) for measuring concentration of SwIL-4 in biological samples and established an enzyme-linked immunospot (ELISPOT) assay for detecting IL-4-secreting cells using a mAb and a polyclonal IgG from goat. The detection limit of the sandwich ELISA for SwIL-4 was 78 pg/ml. Using sandwich ELISA, SwIL-4 was detected in the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Mycoplasma hyopneumoniae and could quantitate in supernatants of mitogen-stimulated PBMC culture. The ELISPOT system is useful for the detection of IL-4 producing cells in swine PBMC culture.