Tricorn protease in bacteria: characterization of the enzyme from Streptomyces coelicolor

Tricorn protease is believed to act downstream of the proteasome, or of other ATP-dependent proteases, cleaving the oligopeptides (mostly 6 to 12 residues) released by them into small peptides (2 to 4 residues), before an array of aminopeptidases finally converts them into free amino acids. Hitherto, the occurrence of Tricorn protease seemed to be limited to some archaea, but genes encoding Tricorn homologs have now been found in several bacterial genomes. Among them is Streptomyces coelicolor A3(2), which has, in fact, two Tricorn-like genes, ScC77.16c and ScE87.19. The proteins encoded by them (TRI-ScC77 and TRI-ScE87) are very similar in their PDZ and TSP domains, but rather divergent in their beta-propeller domains. We have expressed one of them, TRI-ScC77, in E. coil and characterized the recombinant protein structurally and functionally. TRI-ScC77 forms a homohexameric complex of approximately 700 kDa, both in E. coil and in S. coelicolor, with enzymatic properties very similar to the complex from the archaeon Thermoplasma acidophilum. The fact that Tricorn-like proteins exist not only in thermoacidophiles, but also in bacteria inhabiting radically different environments, rules out the possibility that Tricorn protease is an adaptive element that helps to meet the challenges of an extreme habitat.     

Epidermal transmittance of leaves of Vicia faba for UV radiation as determined by two different methods

Leaves of Vicia faba were collected from the field and the greenhouse and transmittance of epidermal peels from adaxial and abaxial sides was determined in the wavelength range from 250 to 800 nm using a spectrophotometer equipped for the measurement of turbid samples. From the same leaves, epidermal transmittance was estimated by a recently developed fluorometric method. Both methods gave highly correlated results with a slope of the regression line between both methods close to 1 and an intercept close to 0. Transmittances at around 310 nm as low as 3% were detected in the adaxial epidermis of field-grown leaves, while transmittance could be as high as 70% in the abaxial epidermis of greenhouse-grown leaves. There was a strong correlation between UV-A (ca. 366 nm) and UV-B (ca. 310 nm) transmittance detected by both methods which could be explained by the pigment composition in methanolic extracts where flavonols accounted for 90% of the absorption at 310 nm in the extract, while hydroxycinnamic acid derivatives which absorb only at the shorter wavelength constituted about 5%. It is concluded that the fluorescence method which allows rapid measurements on intact leaves can provide a quantitative estimate of epidermal transmittance for UV-B (280–320 nm) and UV-A (320–400 nm) radiation.This revised version was published online in October 2005 with corrections to the Cover Date.     

Genetic analysis of resistance to Puccinia striiformis f.sp. tritici in cv. Aflak at adult plant stage

In order to investigate heritability and gene action for yellow rust resistance in wheat, a resistance yellow rust cultivar Aflak was crossed to susceptible cultivar Avocet‘s’. Parents, F₁, F₂ and F₃ generations were cultured according to randomised complete block design with two replications in the research station of Gharakhil, Iran. Parents and other generations were inoculated with 70E0A+ race. Traits including severity and infection type were recorded and then coefficient of infection was calculated. For this trait, generations mean and variance analysis were performed and results showed that there were significant differences among generations for coefficient of infection. Results showed that in addition to additive and dominance effects, at least one kind of epistasis interaction (additive × additive) control this trait. Although additive and dominance effects control this trait, but with attention to generations variance analysis, the results showed that additive variance had important role to control this trait.     

Force spectroscopy of substrate molecules en route to the proteasome's active sites

We used an atomic force microscope to study the mechanism underlying the translocation of substrate molecules inside the proteasome. Our specific experimental setup allowed us to measure interaction forces between the 20S proteasome and its substrates. The substrate (β-casein) was covalently bound either via a thiol-Au bond or by a PEG-based binding procedure to the atomic force microscope cantilever tip and offered as bait to proteasomes from Methanosarcina mazei. The proteasomes were immobilized densely in an upright orientation on mica, which made their upper pores accessible for substrates to enter. Besides performing conventional single-molecule force spectroscopy experiments, we developed a three-step procedure that allows the detection of specific proteasome-substrate single-molecule events without tip-sample contact. Using the active 20S wild type and an inactive active-site mutant, as well as two casein mutants bound with opposite termini to the microscope tip, we detected no directional preference of the proteasome-substrate interactions. By comparing the distribution of the measured forces for the proteasome-substrate interactions, were observed that a significant proportion of interaction events occurred at higher forces for the active versus the inactive proteasome. These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome.     

Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography

The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.     

A mutation in CHN-1/CHIP suppresses muscle degeneration in Caenorhabditis elegans

Duchenne muscular dystrophy (DMD) is one of the most severe X-linked, inherited diseases of childhood, characterized by progressive muscle wasting and weakness as the consequence of mutations in the dystrophin gene. The protein encoded by dystrophin is a huge cytosolic protein that links the intracellular F-actin filaments to the members of the dystrophin-glycoprotein-complex (DGC). Dystrophin deficiency results in the absence or reduction of complex components that are degraded through an unknown pathway. We show here that muscle degeneration in a Caenorhabditis elegans DMD model is efficiently reduced by downregulation of chn-1, encoding the homologue of the human E3/E4 ubiquitylation enzyme CHIP. A deletion mutant of chn-1 delays the cell death of body-wall muscle cells and improves the motility of animals carrying mutations in dystrophin and MyoD. Elimination of chn-1 function in the musculature, but not in the nervous system, is sufficient for this effect, and can be phenocopied by proteasome inhibitor treatment. This suggests a critical role of CHIP/CHN-1-mediated ubiquitylation in the control of muscle wasting and degeneration and identifies a potential new drug target for the treatment of this disease.     

Impact of Early Life Adversity on Reward Processing in Young Adults: EEG-fMRI Results from a Prospective Study over 25 Years

Several lines of evidence have implicated the mesolimbic dopamine reward pathway in altered brain function resulting from exposure to early adversity. The present study examined the impact of early life adversity on different stages of neuronal reward processing later in life and their association with a related behavioral phenotype, i.e. attention deficit/hyperactivity disorder (ADHD). 162 healthy young adults (mean age = 24.4 years; 58% female) from an epidemiological cohort study followed since birth participated in a simultaneous EEG-fMRI study using a monetary incentive delay task. Early life adversity according to an early family adversity index (EFA) and lifetime ADHD symptoms were assessed using standardized parent interviews conducted at the offspring''s age of 3 months and between 2 and 15 years, respectively. fMRI region-of-interest analysis revealed a significant effect of EFA during reward anticipation in reward-related areas (i.e. ventral striatum, putamen, thalamus), indicating decreased activation when EFA increased. EEG analysis demonstrated a similar effect for the contingent negative variation (CNV), with the CNV decreasing with the level of EFA. In contrast, during reward delivery, activation of the bilateral insula, right pallidum and bilateral putamen increased with EFA. There was a significant association of lifetime ADHD symptoms with lower activation in the left ventral striatum during reward anticipation and higher activation in the right insula during reward delivery. The present findings indicate a differential long-term impact of early life adversity on reward processing, implicating hyporesponsiveness during reward anticipation and hyperresponsiveness when receiving a reward. Moreover, a similar activation pattern related to lifetime ADHD suggests that the impact of early life stress on ADHD may possibly be mediated by a dysfunctional reward pathway.