A comprehensive expression analysis of the Arabidopsis proline-rich extensin-like receptor kinase gene family using bioinformatic and experimental approaches

The Arabidopsis proline-rich extensin-like receptor kinase (PERK) family consists of 15 predicted receptor kinases. A comprehensive expression analysis was undertaken to identify overlapping and unique expression patterns within this family relative to their phylogeny. Three different approaches were used to study AtPERK gene family expression, and included analyses of the EST, MPSS and NASCArrays databases as well as experimental RNA blot analyses. Some of the AtPERK members were identified as tissue-specific genes while others were more broadly expressed. While in some cases there was a good association between these different expression patterns and the position of the AtPERK members in the kinase phylogeny, in other cases divergence of expression patterns was seen. The PERK expression data identified by the bioinformatics and experimental approaches were found generally to show similar trends and supported the use of data from large-scale expression studies for obtaining preliminary expression data. Thus, the bioinformatics survey for ESTs and microarrays is a powerful comprehensive approach for obtaining a genome-wide view of genes in a multigene family.     

Peroxidative aggregation of alpha-synuclein requires tyrosines

Alpha-synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of alpha-synuclein in vitro, the specific amino acid types of alpha-synuclein involved in this type of aggregation have not been identified. We show, using human cytochrome c plus H(2)O(2) as the source oxidative stress, that the tyrosines of alpha-synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.     

V(D)J recombination frequencies can be profoundly affected by changes in the spacer sequence

Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two V(H)S107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is approximately 9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.     

Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase

Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI(2) synthase) with increasing concentrations of peroxynitrite (PN) up to 250 microm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 microm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 microm PN, PGI(2) synthase was about half-maximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-Asn-Tyr(3-nitro)-COOH corresponding to positions 427-430 of PGI(2) synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI(2) synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide.     

Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.     

Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.     

Evaluation of four methods for detecting the beta-lactamase activity in Neisseria gonorrhoeae isolated in Cuba

Four methods (chromogenic, acidimetric, inhibition, and iodometric) for demonstration of the beta-lactamase production by 70 isolates of Neisseria gonorrhoeae, were evaluated in Cuba. There was 100% correlation between all beta-lactamase methods and the standardized penicillin dilution susceptibility test for penicillinase-non-producing N. gonorrhoeae. For penicillinase-producing N. gonorrhoeae strains, there was a perfect correlation between the chromogenic method and penicillin susceptibility testing, but one and two strains failed to give a positive result for beta-lactamase with the inhibition/acidimetric and the iodometric methods, respectively. There was a high concordance between the chromogenic method, considered as gold standard and the rest of penicillinase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. The four methods evaluated were accurate, reproducible, easily readable, economical, and ease to use for screening primary isolates of N. gonorrhoeae in Cuba. We recommended the use of the inhibition method, when testing the penicillinase activity in gonococcal isolates in provincial and municipal reference laboratories.     

Substituted 4-[4-(dimethylamino)styryl]pyridinium salt as a fluorescent probe for cell microviscosity

In aqueous solution, 4-[4-(dimethylamino)styryl]pyridine (DMASP) derivatives displayed dual fluorescence, in which excitation at either 469 or 360 nm produced an emission band near 600 nm. Increasing the viscosity of the environment intensified the fluorescence emission obtained at the longer wavelength of excitation, whereas the emission at the lower wavelength of excitation showed little change in intensity. Thus, using the ratio of the 600 nm emission obtained by exciting at 469 nm to that obtained with 360 nm excitation, it is possible to obtain a value related to the local viscosity that does not depend on the system parameters. The fluorescence emission of the dye in aqueous solution, as well as in living cells, is well suited for use with visible fluorescence spectroscopy. The N-carboxymethyl butyl ester DMASP derivative (1) was found to be irreversibly loaded into living smooth muscle cells, presumably because it is hydrolyzed by cellular esterases, transforming it into a membrane-impermeable fluorescent carboxylate DMASP derivative. (2) After calibrating 2 against glycerol/water and sucrose/water mixtures of known viscosity, the fluorescence ratio generated from cultured smooth muscle cells in dual-excitation mode gave an average intracellular viscosity of 4.5 cP. This value corresponds to those reported in the literature.     

Effects of cold on CO2 exchange in winter rape leaves

A viable wheat hybrid intermediate of the same height as the parents was obtained by crossing the female parent of tall variety NP4 with the male parent of the dwarf variety HD2160. Seeds of the hybrid and its parents were germinated and their growth pattern as well as the activities of peroxidase, indolyl-3-acetic acid oxidase and amylase in extracts made from them were studied at the early seedling stages i.e. up to 96 h.
A positive correlation existed between the length of the axis at the early seedling stage and at mature plant height as far as the parental varieties are concerned but no such correlation was observed with the hybrid. Growth of the hybrid seedlings was less than of its parents. Light appeared to stimulate the longitudinal growth of the axis to different extents in the parents and hybrid. Higher activities of peroxidase, indolyl-3-acetic acid oxidase and amylase were observed in the hybrid as compared to both of its parents. Lethal wheat hybrid also exhibits increased activities of amylase, indolyl-3-acetic acid oxidase and peroxidase. Therefore, it appears that seedling growth and enzyme activities at the seedling stage are not always correlated with hybrid vigour.