Physical mapping of the 5S ribosomal RNA genes on rice chromosome 11

One 5S ribosomal RNA gene (5S rDNA) locus was localized on chromosome 11 of japonica rice by in situ hybridization. The biotinylated DNA probe used was prepared by direct cloning and direct labeling methods, and the locus was localized to the proximal region of the short arm of chromosome 11 (llpl.l) by imaging methods. The distance between the signal site and the centromere is 4.0 arbitrary units, where the total length of the short arm is 43.3 units. The 5S rDNA locus physically identified and mapped in rice was designated as 5SRrn. The position of the 5S rDNA locus reported here differs from that in indica rice; possible reasons for this difference are discussed. DNA sequences of 5S rDNA are also reported.     

Simple and rapid analysis of lamotrigine, a novel antiepileptic, in human serum by high-performance liquid chromatography using a solid-phase extraction technique

A simple and rapid method for the quantitation of concentrations of lamotrigine, a novel antiepileptic, in human serum was developed with high-performance liquid chromatography, using a solid-phase extraction technique. The mobile phase was composed of acetonitrile-10 mM phosphate buffer (pH 3.5) containing 5 mM sodium octanesulphonate (27:73, v/v), and components were detected at 265 nm. Retention times of acetanilide as an internal standard and lamotrigine were 3.4 and 10.3 min, respectively. The coefficients of variation were 3.1–4.5% and 4.4–9.8% for the within-day and between-day precision estimates, respectively. The extraction recovery of lamotrigine added to blank serum was 86–107%. The quantitation limit of lamotrigine was ca.0.2 μg/ml in 100 μl of serum. These results suggest that the method employed in this study is useful for the routine monitoring of sereum concentrations of lamotrigine in epileptic patients.     

Random median sampling to enhance the precision of population estimates

We describe the characteristics of a sampling procedure called random median sampling that was proposed to enhance the precision of population estimates. In performing random median sampling, we first select a sampling item at random from the sampling area. We roughly compare the abundance of individuals in the selected item with that of the adjacent two items in order to identify the item that has median abundance, i.e., the item that has the second largest abundance among the three items. We count the number of individuals of the item having the median abundance. This procedure is repeated n times in the sampling area (i = 1, 2, ..., n). Let m _i be the ith median abundance. The estimates of the mean abundance per sampling item and the variance of estimates are given by Σm _i /n and Σ(m _i –Σm _i /n)²/n(n – 1), respectively. This method is a local application of the median ranked set sampling that was proposed by Muttlak (J Appl Stat Sci 6:245–255, 1997). Random median sampling is effective when the correlation coefficient between adjacent items is small. If the correlation coefficient is close to zero, random median sampling reduces the variance of estimates to 45 or 32% of that in simple random sampling when the distribution follows a normal distribution or a Laplace distribution, respectively. The sample size required to achieve a given precision of estimate decreases accordingly. The effectiveness of random median sampling, however, is small if the correlation coefficient is large. The condition in which random median sampling is superior to simple random sampling is also discussed.     

Chemical and immunological studies on mycobacterial polysaccharides. 1. Purification and properties of polysaccharides from human tubercle bacilli

Defatted human tubercle bacilli, Aoyama B strain, were extracted with 0.1 n NaOH for 24 hr, and the crude polysaccharide fraction was precipitated by the addition of 5 volumes of ethyl alcohol. A yield of 17.8 g of crude polysaccharides was obtained from 800 g of bacilli. The crude polysaccharide was further fractionated into seven fractions by fractional precipitation with ethyl alcohol. Each fraction was purified by successive chromatography on Dowex 50 and diethylaminoethyl cellulose, and by gel filtration on Sephadex G-75 and G-200. Optical rotation and gas chromatographic analyses of purified polysaccharide showed that these polysaccharides contained glucan mannan, arabinomannan, and arabinogalactan. Each polysaccharide was almost completely free from nitrogen, and no tuberculin reaction was produced by 100 mug of each material. Arabinomannan and arabinogalactan showed precipitin reaction, complement fixation, and passive hemagglutination reaction with rabbit antiserum against heat-killed whole bacilli (Aoyama B). In guinea pigs sensitized with Aoyama B bacilli, arabinomannan and arabinogalactan provoked anaphylactic shock when injected intravenously, and Arthus type reaction when injected intracutaneously. With the use of rabbit antiserum, arabinomannan and arabinogalactan showed passive anaphylactic shock, passive cutaneous anaphylaxis, and Prausnitz-Küstner type reactions in guinea pigs. By immunodiffusion analysis, it was shown that the antigenic determinant of arabinomannan was different from that of arabinogalactan.     

Chemical constituents of Brazilian Propolis from the state of Bahia and their growth inhibitory activities against cancer cells

A novel 2-phenoxychromone (1) and five known flavones (2-6) were isolated from northeastern Brazilian propolis in the state of Bahia. The chemical structures of these six compounds were determined by spectroscopic investigations and single-crystal X-ray analysis. The isolated compounds showed growth-inhibitory activities, in varying degrees, against human tumor cell lines. This is the first report on the discovery of a novel 2-phenoxychromone from propolis.     

The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture

Human herpes virus 6 (HHV-6) is a common human pathogen that is most often detected in hematopoietic cells. Although human cells harboring chromosomally integrated HHV-6 can be generated in vitro, the availability of such cell lines originating from in vivo tissues is limited. In this study, chromosomally integrated HHV-6B has been identified in a human vascular endothelial cell line, HUV-EC-C (IFO50271), derived from normal umbilical cord tissue. Sequence analysis revealed that the viral genome was similar to the HHV-6B HST strain. FISH analysis using a HHV-6 DNA probe showed one signal in each cell, detected at the distal end of the long arm of chromosome 9. This was consistent with a digital PCR assay, validating one copy of the viral DNA. Because exposure of HUV-EC-C to chemicals did not cause viral reactivation, long term cell culture of HUV-EC-C was carried out to assess the stability of viral integration. The growth rate was altered depending on passage numbers, and morphology also changed during culture. SNP microarray profiles showed some differences between low and high passages, implying that the HUV-EC-C genome had changed during culture. However, no detectable change was observed in chromosome 9, where HHV-6B integration and the viral copy number remained unchanged. Our results suggest that integrated HHV-6B is stable in HUV-EC-C despite genome instability.     

DM-20, a proteolipid apoprotein, is an encephalitogen of acute and relapsing autoimmune encephalomyelitis in mice

Experimental allergic encephalomyelitis (EAE) was successfully induced in BALB/c mice with DM-20, a protein component of proteolipid apoprotein. DM-20 was separated by ion exchange column chromatography with CM-Trisacryl from proteolipid apoprotein obtained from bovine spinal cords. Its purity was ascertained by SDS-polyacrylamide gel electrophoresis, a dot immunobinding procedure, and amino acid analysis. Nine of 15 animals with a single injection of 100 micrograms of DM-20 and five of seven animals with a booster injection developed hind leg paralysis or axial rotatory movement 16 to 27 days after sensitization (mean 21.3 days). Five of the 14 animals relapsed 2 to 6 wk after the first attack. Histological examination revealed inflammatory lesion, with significant demyelination in the central nervous system. Antibody levels to DM-20 were not related to the clinical signs. Five of 11 BALB/c nude mice reconstituted with T cells developed similar clinical and pathologic signs. This DM-20-induced EAE in mice may provide a valuable model because it is similar to multiple sclerosis and because it can be induced in inbred mice in which immune mechanisms can be easily studied.     

Two activated forms of protease-sensitive protein kinase isolated from rat liver plasma membrane

Rat liver plasma membrane contains a protease-activated kinase which corresponds to protein kinase C. When the solubilized enzyme was digested with trypsin in the absence of NaCl, a partially activated form with an approximate molecular weight of 8 X 10(4) was produced. However, another active form of molecular weight of 5 X 10(4) was obtained when the enzyme was digested in the presence of NaCl. The larger molecular weight form was converted to the smaller form by tryptic digestion in the presence of NaCl. These results suggest the existence of two protease-activated forms of protein kinase C.     

High-level expression of human proapolipoprotein A-I in Escherichia coli using expression plasmids containing tandemly polymerized proapolipoprotein A-I structural genes

Summary A gene coding human proapolipoprotein A-I (proapo A-I) was inserted into a plasmid with a consensus ribosome binding sequence in Escherichia coli. One to three copies of this gene were tandemly inserted to construct proapo A-I expression plasmids, pMTpAI, pMT(pAI)₂ and pMT(pAI)₃, respectively. The cells harbouring pMT(pAI)₃, could produce proapo A-I at a level of 49 g/ml A₆₀₀ and up to approx. 48% of the total cellular protein. The product was soluble in E. coli, and formed protein-lipid complexes with dimyristoyl phosphatidyl choline, which formed stacked disc structures. Analyses of the rec proapo A-I formed in the bacteria was identical to human proapo A-I except for methionine at the N-terminus.