A silver stain for the detection of nanogram amounts of tRNA following two-dimensional electrophoresis

Three ultrasensitive protein silver-staining methods have been compared with respect to the detection of tRNA in polyacrylamide gels. The method of Sammons (D. W. Sammons, L.D. Adams, and E.E. Nishizawa (1981) Electrophoresis 2, 135-141) has been shown to have remarkable sensitivity, with a detection limit of 0.3 ng tRNA/mm2, allowing the two-dimensional fractionation of submicrogram amounts of bulk tRNA. The application of this technique to developmental and differentiation problems and other areas where the amounts of nonradioactive tRNA available are limited is anticipated.     

Structural analysis and molybdenum-dependent expression of the pAO1-encoded nicotine dehydrogenase genes of Arthrobacter nicotinovorans

The genes of nicotine dehydrogenase (NDH) were identified, cloned and sequenced from the catabolic plasmid pA01 of Arthrobacter nicotinovorans. In immediate proximity to this gene cluster is the beginning of the 6-hydroxy-L-niotine oxidase (6-HLNO) gene. NDH is composed of three subunits (A, B and C) of M_r 30011, 14924 and 87677. It belongs to a family of bacterial hydroxylases with a similar subunit structure; they have molybdopterin dinucleotide, FAD and Fe-S clusters as cofactors. Here the first complete primary structure of a bacterial hydroxylase is provided. Sequence alignments of each of the NDH subunits show similarities to the sequences of eukaryotic xanthine dehydrogenase (XDH) but not to other known molybdenum-containing bacterial enzymes. Based on alignment with XDH it is inferred that the smallest subunit (NDHB) carries an iron-sulphur cluster, that the middle-sized subunit (NDHA) binds FAD, and that the largest NDH subunit (NDHC) corresponds to the molybdopterin-binding domain of XDH. Expression of both the ndh and the 6-hlno genes required the presence of nicotine and molybdenum in the culture medium. Tungsten inhibited enzyme activity but not the synthesis of the enzyme protein. The enzyme was found in A. nicotinovorans cells in a soluble form and in a membrane-associated form. In the presence of tungsten the fraction of membrane-associated NDH increased.     

Twintrons are not unique to the Euglena chloroplast genome: structure and evolution of a plastome cpn60 gene from a cryptomonad

Introns within introns (twintrons) are known only from the Euglena chloroplast genome. Twintrons are group II or III introns, into which another group II or III intron has been transposed. In this paper we describe a non-Euglena twintron structure within a plastid-encoded chaperone gene (cpn60) of the cryptomonad alga Pyrenomonas salina. In addition, the evolutionary relationships between members of the Cpn60 protein family are determined. Our findings permit the inclusion of cryptomonad plastomes in phylogenetic studies of intron evolution and present further evidence for the origin of modern plastids from a cyanobacterial ancestor.This paper is dedicated to Prof. Dr. Peter Sitte on the occasion of his 65th birthday     

Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Phylogenetic utility of the nuclear gene arginine decarboxylase: an example from Brassicaceae

Arginine decarboxylase (ADC) is an important enzyme in the production of putrescine and polyamines in plants. It is encoded by a single or low-copy nuclear gene that lacks introns in sequences studied to date. The rate of Adc amino acid sequence evolution is similar to that of ndhF for the angiosperm family studied. Highly conserved regions provide several target sites for PCR priming and sequencing and aid in nucleotide and amino acid sequence alignment across a range of taxonomic levels, while a variable region provides an increased number of potentially informative characters relative to ndhF for the taxa surveyed. The utility of the Adc gene in plant molecular systematic studies is demonstrated by analysis of its partial nucleotide sequences obtained from 13 representatives of Brassicaceae and 3 outgroup taxa, 2 from the mustard oil clade (order Capparales) and 1 from the related order Malvales. Two copies of the Adc gene, Adc1 and Adc2, are found in all members of the Brassicaceae studied to data except the basal genus Aethionema. The resulting Adc gene tree provides robust phylogenetic data regarding relationships within the complex mustard family, as well as independent support for proposed tribal realignments based on other molecular data sets such as those from chloroplast DNA.      

Development and characterization of a FIA system for selective assay of l-ascorbic acid in food samples

An amperometric detector and an enzymatic reaction were combined for the measurement of l-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA) system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below 5% (5 times repeated, 400 μmol/L conc.), linearity up to 400 μmol/L, limit of detection (LOD) 5 μmol/L, fitting of calibration curve in 25–400 μmol/L range was R ² = 0.99.     

Effects of aerobic exercise therapy and cognitive behavioural therapy on functioning and quality of life in amyotrophic lateral sclerosis: protocol of the FACTS-2-ALS trial

Background

Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain.

Methods

We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles.

Results

Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct.

Conclusions

The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.     

An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.