Use of an epitope-tagged cDNA library to isolate cDNAs encoding proteins with nuclear localization potential

We have combined epitope tagging with an expression cDNA library in order to isolate cDNAs encoding nuclear proteins. This system allows us to detect proteins expressed from the cDNA library by using antibodies against the epitope tag. As a tag, we used the 85-aa N-terminal peptide of the SV40 T antigen which lacks the nuclear localization signal (NLS). A strong expression vector, pEF204 [Kim et al., Gene 91 (1990) 217–223], was modified into an epitope-tagging vector, pTkim, by putting the tag-coding region and a cDNA cloning site immediately after its promoter. From cDNA libraries constructed using pTkim, we isolated eight cDNA clones whose tagged proteins were localized within the nuclei. From partial sequence analysis, two cDNAs were shown to code for the ribosomal (r-) proteins, simian L44 and human L21, and the others were shown to be new. Furthermore, six cDNAs including those encoding the r-proteins could direct a non-karyophilic T antigen [Fischer-Fantuzzi et al. Virology 153 (1986) 87–95] into nuclei, showing that they have NLSs. These results indicate that this system is useful for isolating new cDNAs which code for nuclear proteins.     

Lipid composition and cholesterol esterification in serum lipoprotein fraction of the horse, Equus caballus.

1. Changes in lipid components of lipoproteins during incubation of horse serum at 37 degrees C were investigated. In non-incubated serum, cholesterol and lecithin existed predominantly in alpha-lipoprotein or in high-density lipoprotein (HDL). Lysolecithin was mainly associated with the fraction with density above 1.21. 2. When serum was separated into alpha- and beta-lipoproteins by the heparin precipitation method after 1 hr incubation, the decrease in alpha-lipoprotein free cholesterol and lecithin was about four times that in beta-lipoprotein counterparts. 3. When serum lipoproteins were separated by ultracentrifugation, the decrease in each lipoprotein free cholesterol was closely paralleled with that in lecithin. 4. HDL appeared to be a preferential substrate for the lecithin: cholesterol acyltransferase reaction. 5. Disc electrophoretic patterns indicated significant differences in the composition of horse serum lipoproteins from those of human and rat.     

Effects of alpha-tocopherol and tocotrienols on blood pressure and linoleic acid metabolism in the spontaneously hypertensive rat (SHR).

Both alpha-tocopherol and a 1:1.7 mixture of alpha-tocopherol and tocotrienols at a 0.2% dietary level significantly depressed the age-related increase in the systolic blood pressure of spontaneously hypertensive rats (SHRs) after 3 weeks of feeding. The aortic production of prostacyclin was increased 1.5 times both by alpha-tocopherol and a tocotrienol mixture, suggesting a possible relevance to their hypotensive effect. These vitamins did not influence the delta 6- and delta 5-desaturase activities of liver microsomes, but fatty acid profiles of the liver phospholipids predicted a reduction of linoleic acid desaturation. These effects were in general more clear with tocotrienols than with alpha-tocopherol. Platelet aggregation by 5 microM ADP remained uninfluenced. Thus, tocotrienols may have effects on various lipid parameters somewhat different from those of alpha-tocopherol.     

Ecology and seasonal distribution of Vibrio parahaemolyticus in aquatic environments of a temperate region

Abstract An environmental survey was done to study the ecology and distribution of Vibrio parahaemolyticus in 5 selected stations in Okayama Prefecture, which included fresh, brackish, and marine aquatic environments. Water and plankton samples were collected monthly for quantitative and qualitative analyses during the period October, 1987 to October, 1989 for V. parahaemolyticus . The pathogen was not detected from fresh water environments. A seasonality of the organism was observed in brackish and marine environments where average salinity ranged between 0.39 and 1.28%.Plankton samples yielded higher densities of V. parahaemolyticus compared with water samples. By applying several enrichment techniques, the pathogen was detected quite frequently during the winter months in the environments with temperatures ranging between 10 and 14°C. The identification following conventional tests, by the API 20E system and by serological methods reveal that the API 20E system is satisfactory to identify V. parahaemolyticus and further confirms that the serological method could be a simpler and more rapid procedure for V. parahaemolyticus identification.     

Adjustment errors of feedback guided human forearm movements elicited by random position signal sequences

The effects of visual and auditory relevant feedback on human forearm movements elicited by random position signal sequences were examined. We observed the adjustment error sequences when the arm moved briskly and accurately to the target position. As the results, the mean adjustment errors for each speed (clock interval) of random position signal sequence are different in the left-right target positions, and also different in the flexion-extension movements. In faster speed (1s), the adjustment errors are approximately flat as compared with those in other speeds, on the other hand, in moderate speed (2s) or slower speed (3s), these indicate a upward tendency from left to right positions, and also indicate a upward tendency from flexion to extension movements. However, in the reaction times, the moving times, and the adjusting times, there are no significant differences for the left-right target positions and for the flexion-extension movements.     

Cloning of the Promoter Regions of Mouse TGF-{beta} Receptor Genes by Inverse PCR with Highly Overlapped Primers

In order to isolate promoters of mouse TGF-ß receptorgenes, we used inverse PCR with highly overlapped primers correspondingto the 5' sequence of the receptor cDNAs. Nested primer setsonly covered a 30- to 40-base region of the sequences. HinfI-digestedand self-ligated mouse genomic DNA was used as a PCR template.Only one band for each receptor was seen after PCR. The amplifiedDNA fragments could direct luciferase production when the luciferasecoding sequence was ligated after the fragments. The sequenceof the fragment which correspond to the type II receptor showedpartial homology with the promoter region of the human TGF-ßtype II receptor. Thus, the inverse PCR with highly overlappedprimers could be an easy way to isolate the promoter regionsof many genes.     

Anti-inflammatory activity of a globular adiponectin function on RAW 264 cells stimulated by lipopolysaccharide from Aggregatibacter actinomycetemcomitans

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-κB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IκB kinase α/β and the degradation of IκB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-α and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.