Heterogeneity of intermediate filament proteins from rabbit spinal cord

Large amounts of a neurofilament-enriched fraction may be prepared from spinal cord homogenates by a simple, three-step procedure. This involves flotation of filament-containing axon fragments, extraction with Triton X-100, and washing by sedimentation through a sucrose density gradient. The material obtained by this procedure includes both large mats of individual 10-nm filaments and tightly packed bundles of filaments. SDS-gel electrophoresis of these fractions indicates that the fractions are formed of four polypeptides: the three which are generally considered to form neurofilaments (P200, P150, and P68) and another, with a molecular weight of about 50,000 daltons (P50), which is thought to be derived from fibrous astrocytes. Analysis of these filament fractions on two-dimensional gels indicates heterogeneity among each of the different molecular weight classes. The largest polypeptide of neurofilaments, P200, focuses at several spots in the pH gradient. P68 and P150 are more acidic: each appears as a pair of overlapping spots. P50 resolves into a complex of spots of about the same molecular weight but with different isoelectric points. Heterogeneity is not unique to these filament polypeptides but appears to be a characteristic of all fibrous proteins of the nervous system.     

Heavy and light forms of some aminoacyl-tRNA synthetases in fraction X,microsomes and cytosol of rabbit liver

Summary Aminoacyl-tRNA synthetase activity for alanine, glutamic acid, lysine and phenylalanine was studied in the three subcellular fractions of rabbit liver: fraction X, microsomes and cytosol. From 60 to 80% of the enzyme activities were found in fraction X and microsomes. Fraction X was especially rich in the synthetase activities. By means of gel chromatography, heavy (over 10⁶ daltons) and light (below 480 × 10³ daltons) forms of lysyl- and phenylalanyl- but only light ones of alanyl- and glutamyl-tRNA synthetase activities were found in all the subcellular fractions studied. It is concluded that in higher organisms (mammals) all aminoacyl-tRNA synthetases, at least in part, are associated with cell structural constituents.Abbreviations ALA, GLU, LYS, PHE alanyl-, glutamyl-, lysyl-, phenylalanyl-tRNA synthetase - PMSF phenylmethylsulfonyl fluoride - BSA bovine serum albumin     

Enhanced nucleotide analysis enables the quantification of deoxynucleotides in plants and algae revealing connections between nucleoside and deoxynucleoside metabolism

Detecting and quantifying low-abundance (deoxy)ribonucleotides and (deoxy)ribonucleosides in plants remains difficult; this is a major roadblock for the investigation of plant nucleotide (NT) metabolism. Here, we present a method that overcomes this limitation, allowing the detection of all deoxy- and ribonucleotides as well as the corresponding nucleosides from the same plant sample. The method is characterized by high sensitivity and robustness enabling the reproducible detection and absolute quantification of these metabolites even if they are of low abundance. Employing the new method, we analyzed Arabidopsis thaliana null mutants of CYTIDINE DEAMINASE, GUANOSINE DEAMINASE, and NUCLEOSIDE HYDROLASE 1, demonstrating that the deoxyribonucleotide (dNT) metabolism is intricately interwoven with the catabolism of ribonucleosides (rNs). In addition, we discovered a function of rN catabolic enzymes in the degradation of deoxyribonucleosides in vivo. We also determined the concentrations of dNTs in several mono- and dicotyledonous plants, a bryophyte, and three algae, revealing a correlation of GC to AT dNT ratios with genomic GC contents. This suggests a link between the genome and the metabolome previously discussed but not experimentally addressed. Together, these findings demonstrate the potential of this new method to provide insight into plant NT metabolism.

A new method for the quantification of (deoxy)ribonucleotides and nucleosides enables an in-depth analysis of the nucleotide metabolism in plants.     

Metal-enhanced fluorescence of tryptophan residues in proteins: Application toward label-free bioassays

The detection of submonolayers of proteins based on native fluorescence is a potentially valuable approach for label-free detection. We have examined the possibility of using silver nanostructures to increase the emission of tryptophan residues in proteins. Fluorescence spectra, intensities, and lifetimes of multilayers and submonolayers of proteins deposited on the surfaces of silver island films were measured. Increased fluorescence intensities from two- to three-fold and similar decreases in lifetimes were observed in the presence of the silver nanoparticles compared with the proteins on the surface of the bare quartz. The observed spectral effects of silver nanoparticles on tryptophan fluorescence indicates the possibility for the design of analytical tools for the detection of proteins without traditional labeling by extrinsic fluorophores.     

Prevalence of Prototheca spp. on dairy farms in Poland – a cross-country study

The Prototheca algae have recently emerged as an important cause of bovine mastitis globally. Here, we present results of a first large-scale, cross-country survey on the prevalence of Prototheca spp. in dairy cows, and their environment in Poland. A total of 1211 samples were collected and microbiologically analysed. Included within this number were milk (n = 638), body swabs (n = 374) and environmental samples (n = 199), originating from 400 dairy cows and their surroundings, on 16 dairy farms, based in all major provinces of the country. Prototheca spp. were the third, after Streptococcus and Staphylococcus spp., most common mastitis pathogens. The overall prevalence of protothecal mastitis was 8.3% (33/400), with the majority (75.8%) of cases having a subclinical course, and all but one attributable to P. zopfii genotype 2. Prototheca spp. were cultured from body swabs of both healthy and mastitic cows, yet the isolation rate among the latter was conspicuously lower (12.3% vs. 17.8%). Forty-two (21.2%) environmental samples yielded growth of Prototheca spp. However, no clear association between Prototheca mastitis in dairy cows and the algal isolation from the herd environment was found. Nor was there any association between the environmental recovery of the algae and farm management practices.     

t-RNA-nucleotidyltransferase activity in Lupinus luteus seeds

t-RNA-nucleotidyltransferase activity was detected in Lupinus luteus seed.The enzyme was partly purified, and some of its properties are described.