Ubiquitylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome

BAG-1 is a ubiquitin domain protein that links the molecular chaperones Hsc70 and Hsp70 to the proteasome. During proteasomal sorting BAG-1 can cooperate with another co-chaperone, the carboxyl terminus of Hsc70-interacting protein CHIP. CHIP was recently identified as a Hsp70- and Hsp90-associated ubiquitin ligase that labels chaperone-presented proteins with the degradation marker ubiquitin. Here we show that BAG-1 itself is a substrate of the CHIP ubiquitin ligase in vitro and in vivo. CHIP mediates attachment of ubiquitin moieties to BAG-1 in conjunction with ubiquitin-conjugating enzymes of the Ubc4/5 family. Ubiquitylation of BAG-1 is strongly stimulated when a ternary Hsp70.BAG-1.CHIP complex is formed. Complex formation results in the attachment of an atypical polyubiquitin chain to BAG-1, in which the individual ubiquitin moieties are linked through lysine 11. The noncanonical polyubiquitin chain does not induce the degradation of BAG-1, but it stimulates a degradation-independent association of the co-chaperone with the proteasome. Remarkably, this stimulating activity depends on the simultaneous presentation of the integrated ubiquitin-like domain of BAG-1. Our data thus reveal a cooperative recognition of sorting signals at the proteolytic complex. Attachment of polyubiquitin chains to delivery factors may represent a novel mechanism to regulate protein sorting to the proteasome.     

Effects of aversive experience on the behavior within a custom-made plus maze in the short-tailed fruit bat, Carollia perspicillata

Stress exposure evokes a variety of physiological and behavioral responses in an organism, enabling it to cope with stressful situations and changes in the environment. In a previous study, we found that subjecting individuals of Carollia perspicillata to a chronic immobilization stress paradigm resulted in a significant increase of fecal cortisol concentrations. In the present study, we investigated the influence of stress on the behavior of C. perspicillata, by adapting a commonly used behavioral paradigm for characterizing coping styles of animals (i.e., the elevated-plus maze) to bats. Adult bats were subjected 1?h/day to immobilization over a period of 10?days. On the subsequent day, the behavior of each animal was analyzed in a custom-made plus maze, consisting of four arms (two open and two enclosed ones) and designed 3D because of the bats' ability to fly. In this newly invented design, we compared the behaviors of stressed animals and controls. Changes in locomotor and exploratory behavior suggest two divergent adaptive behaviors in C. perspicillata following the chronic stress paradigm, possibly indicating different stress coping styles.     

Effects of cytosolic calcium and limited, possible dual, effects of G protein modulators on guard cell inward potassium channels

The cellular mechanisms that regulate potassium (K⁺) channels in guard cells have been the subject of recent research, as K⁺ channel modulation has been suggested to contribute to stomatal movements. Patch clamp studies have been pursued on guard cell protoplasts of Vicia faba to analyze the effects of physiological cytosolic free Ca²⁺ concentrations, Ca²⁺ buffers and GTP-binding protein modulators on inward-rectifying K⁺ channels. Ca²⁺ inhibition of inward-rectifying K⁺ currents depended strongly on the concentration and effectiveness of the Ca²⁺ buffer used, indicating a large Ca²⁺ buffering capacity and pH increases in guard calls. When the cytosolic Ca²⁺ concentration was buffered to micromolar levels using BAPTA, inward-rectifying K⁺ channels were strongly inhibited. However, when EGTA was used as the Ca²⁺ buffer, much less inhibition was observed, even when pipette solutions contained 1 µM free Ca²⁺. Under the imposed conditions, GTPγS did not significantly inhibit inward-rectifying K⁺ channel currents when cytosolic Ca²⁺ was buffered to low levels or when using EGTA as the Ca²⁺ buffer. Furthermore, GDPβS reduced inward K⁺ currents at low cytosolic Ca²⁺, indicating a novel mode of inward K⁺ channel regulation by G-protein modulators, which is opposite in effect to that from previous reports. On the other hand, when Ca²⁺ was effectively elevated in the cytosol to 1 µM using BAPTA, GTPγS produced an additional inhibition of the inward-rectifying K⁺ channel currents in a population of cells, indicating possible Ca²⁺-dependent action of GTP-binding protein modulators in K⁺ channel inhibition. Assays of stomatal opening show that 90% inhibition of inward K⁺ currents does not prohibit, but slows, stomatal opening and reduces stomatal apertures by only 34% after 2 h light exposure. These data suggest that limited K⁺ channel down-regulation alone may not be rate-limiting, and it is proposed that the concerted action of proton-pump inhibition and additional anion channel activation is likely required for inhibition of stomatal opening. Furthermore, G-protein modulators regulate inward K⁺ channels in a more complex and limited, possibly Ca²⁺-dependent, manner than previously proposed.     

Domain conformational switch of the iron-sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc(1) complex in the past have led to the formulation of the "protonmotive Q-cycle" mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q(P) site with both electrons transferred simultaneously to ISP and cyt b(L) when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc(1) demonstrates that the reduced ISP-ED moves to the c(1)-position to reduce cyt c(1) only after the reduced cyt b(L) is oxidized by cyt b(H). However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q(P) site inhibitors, Pm and Pf, under various redox states of the bc(1) complex, suggest that the electron transfer from heme b(L) to b(H) is the driving force for the releasing of the reduced ISP-ED from the b-position to c(1)-position to reduce cyt c(1).     

QAlign: quality-based multiple alignments with dynamic phylogenetic analysis

Integrating different alignment strategies, a layout editor and tools deriving phylogenetic trees in a 'multiple alignment environment' helps to investigate and enhance results of multiple sequence alignment by hand. QAlign combines algorithms for fast progressive and accurate simultaneous multiple alignment with a versatile editor and a dynamic phylogenetic analysis in a convenient graphical user interface.     

Inflammatory cells in rat skeletal muscle are elevated after electrically stimulated contractions.

We determined the effect of muscle contractions resulting from high-frequency electrical stimulation (HFES) on inflammatory cells in rat tibialis anterior (TA), plantaris (Pln), and soleus (Sol) muscles at 6, 24, and 72 h post-HFES. A minimum of four and a maximum of seven rats were analyzed at each time point. HFES, applied to the sciatic nerve, caused the Sol and Pln to contract concentrically and the TA to contract eccentrically. Neutrophils were higher (P < 0.05) at 6 and 24 h after HFES in the Sol, Pln, and TA muscles relative to control muscles. ED1(+) macrophages in the Pln were elevated at 6 and 24 h after HFES and were also elevated in the Sol and TA after HFES relative to controls. ED2(+) macrophages in the Sol and TA were elevated at 24 and 72 h after HFES, respectively, and were also elevated in the Pln after HFES relative to controls. In contrast to the TA muscles, the Pln and Sol muscles showed no gross histological abnormalities. Collectively, these data indicate that both eccentric and concentric contractions can increase inflammatory cells in muscle, regardless of whether overt histological signs of injury are apparent.     

Evidence for plasmid like DNA in a filamentous fungus, the ascomycete Podospora anserina

Summary The existece of plasmid like DNA was demonstrated in senescent mycelia of Podospora anserina (strain s) by biophysical and electronmicroscopic methods. According to their contour length of about 1.4 and 2.7 m respectively the molecular weight for the monomer is in the range of 3·10⁶.This work was supported by the Deutsche Forschungsgemeinschaft. The sojourn of P.A.L. for six months in Bochum was made possible by a Senior U.S. Scientist Award of the Alexander von Humboldt Stiftung (Bonn).     

Phosphorylation of p70S6k correlates with increased skeletal muscle mass following resistance exercise

High-resistance exercise training results in an increase inmuscle wet mass and protein content. To begin to address the acute changes following a single bout of high-resistance exercise, a newmodel has been developed. Training rats twice a week for 6 wk resultedin 13.9 and 14.4% hypertrophy in the extensor digitorum longus (EDL)and tibialis anterior (TA) muscles, respectively. Polysome profilesafter high-resistance lengthening contractions suggest that the rate ofinitiation is increased. The activity of the 70-kDa S6 protein kinase(p70^S6k), a regulator oftranslation initiation, is also increased following high-resistancelengthening contractions (TA, 363 ± 29%; EDL, 353 ± 39%).Furthermore, the increase inp70^S6k activity 6 h after exercisecorrelates with the percent change in muscle mass after 6 wk oftraining (r = 0.998). The tightcorrelation between the activation ofp70^S6k and the long-term increasein muscle mass suggests thatp70^S6k phosphorylation may be agood marker for the phenotypic changes that characterize musclehypertrophy and may play a role in load-induced skeletal muscle growth.

     

Semiquantitative analysis of ECM molecules in the different cartilage layers in early and advanced osteoarthritis of the knee joint

The study was conducted to examine the expression of collagen type I and II in the different cartilage layers in relation to other ECM molecules during the progression of early osteoarthritic degeneration in human articular cartilage (AC). Quantitative real-time (RT)-PCR and colorimetrical techniques were used for calibration of Photoshop-based image analysis in detecting such lesions. Immunohistochemistry and histology were performed with 40 cartilage tissue samples showing mild (ICRS grade 1b) respectively moderate/advanced (ICRS grade 3a or 3b) (20 each) osteoarthritis compared with 15 healthy biopsies. Furthermore, we quantified our results on the gene expression of collagen type I and II and aggrecan with the help of real-time (RT)-PCR. Proteoglycan content was measured colorimetrically. The digitized images of histology and immunohistochemistry stains were analyzed with Photoshop software. T-test and Spearman correlation analysis were used for statistical analysis. In the earliest stages of AC deterioration the loss of collagen type II was associated with the appearance of collagen type I, shown by increasing amounts of collagen type I mRNA. During subsequent stages, a progressive loss of structural integrity was associated with increasing deposition of collagen type I as part of a natural healing response. A decrease of collagen type II is visible especially in the upper fibrillated area of the advanced osteoarthritic samples, which then leads to an overall decrease. Analysis of proteoglycan showed losses of the overall content and a loss of the classical zonal formation. Correlation analysis of the proteoglycan Photoshop measurements with the RT-PCR revealed strong correlation for Safranin O and collagen type I, medium for collagen type II, alcian blue and glycoprotein but weak correlation with PCR aggrecan results. Photoshop based image analysis might become a valuable supplement for well known histopathological grading systems of lesioned articular cartilage. The evidence of collagen type I production early in the OA disease process coupled with the ability of chondrocytes to up-regulate collagen type II production suggests that therapeutic agents that suppress collagen type I production and increase collagen type II production may enable chondrocytes to generate a more effective repair response.     

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.