Purification and characterization of RNA polymerase I from a higher plant.

RNA polymerase I was purified from chromatin isolated from auxin-treated soybean hypocotyl. Purification was achieved by using Agarose A-1.5m gel filtration, DEAE-cellulose, CM-sephadex, and phosphocellulose chromatography, and sucrose density gradient centrifugation. With denatured calf thymus DNA as template, the enzyme has a high specific activity (200-300 nmol/mg/30 min at 28 degrees C) which is comparable to other RNA polymerase I enzymes purified from animals and yeast. While the gel profiles indicate that purification to homogeneity (greater than 90%) may not have been achieved, the enzyme appears to be composed of possibly 7 subunits, several of which are similar to the subunits of yeast RNA polymerase I. The putative subunits and molar ratios are 183 000 (1), 136 000 (1), 50 000 (0.5), 46 000 (0.5), 40 000 (0.5), 33 000 (0.2), and 28 000 (2). The purified enzyme strongly prefers a completely denatured template such as poly(dC).     

Clinical manifestations of taeniasis in Taiwan aborigines.

From 1974 to 1989, a total of 24,500 aborigines at 67 villages in ten mountainous districts/towns in Taiwan were examined for the Taiwan Taenia infection and 12% were found to be infected. In order to define the clinical manifestations of taeniasis caused by the Taiwan Taenia, 1661 aborigines in ten mountainous districts were surveyed. The overall clinical rate was 76%. The clinical rate was highest among Atayal aborigines (81%), followed by Bunun (66%) and Yami (61%) aborgines and lowest among Ami aborigines (40%). Among 1153 infected people, 10% had passed gravid segments in the faeces for less than 1 year, 24% for 1-3 years, 17% for 4-5 years, 23% for 6-10 years, 16% for 11-20 years, 7% for 21-30 years, and 3% over 30 years. Twenty-six occurrences of gastrointestinal and neurological symptoms were reported by 1258 infected persons. Passing proglottides in the faeces (95%) was the most frequent sign, followed by pruritis ani (77%), nausea (46%), abdominal pain (45%), dizziness (42%), increased appetite (30%), headache (26%), etc.     

Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Site-directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase: effect on activity and pH dependence.

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate-dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem. 248, 63-71] that the activity is pH-dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side-chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH-dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.     

Neurofascin: a novel chick cell-surface glycoprotein involved in neurite-neurite interactions

We have identified neurofascin, a novel chick cell-surface glycoprotein involved in neurite-neurite interactions. Neurofascin is defined by its reactivity with monoclonal antibody (MAb) F6, which detects two polypeptides (160 and 185 kd) in immunotransfers of brain plasma membrane proteins. Immunoaffinity chromatography using immobilized MAb F6 yields major molecular mass bands at 185, 160, 135-110, and 92 kd. Fingerprint analyses show that these polypeptides are related. Neurofascin is expressed primarily in fiber-rich areas of embryonic cerebellum, spinal cord, and retina. Fab fragments of polyclonal antibodies to neurofascin interfere with the outgrowth of retinal and sympathetic axons in two different in vitro bioassays. Neurofascin is immunologically distinct from other known neurite-associated surface glycoproteins.     

In vitro propagation of Alstroemeria ‘Alsaan’

Plantlets were regenerated from Alstroemeria Alsaan rhizome tips cultured in vitro on solid and liquid media based on Murashige and Skoog salt formulation. The quality of the cultures was superior when intact rather than longitudinally sliced rhizome tips were used as explants and when a temperature of 8°C rather than 22°C was used at the initiation stage. More roots were produced on rhizome tips containing a rhizome apical meristem than on rhizome sections lacking such a meristem. Most (90%) of the rooted plantlets were successfully acclimatized and developed into true-to-type flowering plants.     

An electron microscopic study of retinal degeneration in Sprague-Dawley rats

Retinal degeneration in untreated, female Sprague-Dawley rats was studied by electron microscopy and horseradish peroxidase tracer technique. The degeneration appeared to have started at a very young age. The severity of the defect varied from a decrease of photoreceptor nuclei to total loss of receptor cells and the pigment epithelium. In mild degeneration some regions of the retinal pigment epithelium became bilayered and the basal plasma membrane became flattened or formed elaborate infoldings. Breaks in Bruch's membrane occurred in severe degeneration. Degeneration of the pigment epithelium allowed permeation of tracer material from the choroid into the retina.     

Expression of the precore region of an avian hepatitis B virus is not required for viral replication.

The core-antigen-coding region of all hepadnaviruses is preceded by a short, in-phase open reading frame termed precore whose expression can give rise to core-antigen-related polypeptides. To explore the functional significance of precore expression in vivo, we introduced a frameshift mutation into this region of the duck hepatitis B virus (DHBV) genome and examined the phenotype of this mutant DNA by intrahepatic inoculation into newborn ducklings. Animals receiving mutant DNA developed DHBV infection, as judged by the presence in hepatocytes of characteristic viral replicative intermediates; molecular cloning and DNA sequencing confirmed that the original mutation was present in the progeny genomes. Infection could be efficiently transmitted to susceptible ducklings by percutaneous inoculation with serum from mutant-infected animals, indicating that infectious progeny virus was generated. These findings indicate that expression of the precore region of DHBV is not essential for genomic replication, core particle morphogenesis, or intrahepatic viral spread.     

Sequence and translation of the murine coronavirus 5''-end genomic RNA reveals the N-terminal structure of the putative RNA polymerase.

A 28-kilodalton protein has been suggested to be the amino-terminal protein cleavage product of the putative coronavirus RNA polymerase (gene A) (M.R. Denison and S. Perlman, Virology 157:565-568, 1987). To elucidate the structure and mechanism of synthesis of this protein, the nucleotide sequence of the 5' 2.0 kilobases of the coronavirus mouse hepatitis virus strain JHM genome was determined. This sequence contains a single, long open reading frame and predicts a highly basic amino-terminal region. Cell-free translation of RNAs transcribed in vitro from DNAs containing gene A sequences in pT7 vectors yielded proteins initiated from the 5'-most optimal initiation codon at position 215 from the 5' end of the genome. The sequence preceding this initiation codon predicts the presence of a stable hairpin loop structure. The presence of an RNA secondary structure at the 5' end of the RNA genome is supported by the observation that gene A sequences were more efficiently translated in vitro when upstream noncoding sequences were removed. By comparing the translation products of virion genomic RNA and in vitro transcribed RNAs, we established that our clones encompassing the 5'-end mouse hepatitis virus genomic RNA encode the 28-kilodalton N-terminal cleavage product of the gene A protein. Possible cleavage sites for this protein are proposed.