A C-terminal sequence in the guanine nucleotide exchange factor Sec7 mediates Golgi association and interaction with the Rsp5 ubiquitin ligase

Arf GTPases control vesicle formation from different intracellular membranes and are regulated by Arf guanine nucleotide exchange factors (GEFs). Outside of their conserved catalytic domains, known as Sec7 domains, little is known about Arf GEFs. Rsp5 is a yeast ubiquitin ligase that regulates numerous membrane trafficking events and carries a C2 domain that is specifically required for trans-Golgi network to vacuole transport. In a screen for proteins that interact with the Rsp5 C2 domain we identified Sec7, the GEF that acts on Golgi-associated Arfs. The Rsp5-Sec7 interaction is direct, occurs in vivo, and is conserved among mammalian Rsp5 and Sec7 homologues. A 50-amino acid region near the Sec7 C terminus is required for Rsp5 binding and for normal Sec7 localization. Binding of Sec7 to Rsp5 is dependent on the presence of the phosphoinositide 3-kinase Vps34, suggesting that phosphatidylinositol 3-phosphate (PI(3)P) plays a role in regulating this interaction. Overexpression of Sec7 significantly suppresses the growth and sorting defects of an rsp5 C2 domain point mutant. These observations identify a new functional region within the Sec7/BIG family of Arf GEFs that is required for trans-Golgi network localization.     

The influence of visual stimulation on the behaviour of cats housed in a rescue shelter

This study explored the influence of 5 types of visual stimulation (1 control condition [no visual stimulation] and 4 experimental conditions [blank television screen; and, televised images depicting humans, inanimate movement, animate movement]) on the behaviour of 125 cats housed in a rescue shelter. Twenty-five cats were randomly assigned to one of the five conditions of visual stimulation for 3 h a day for 3 days. Each cat's behaviour was recorded every 5 min throughout each day of exposure to the visual stimuli. Cats spent relatively little of the total observation time (6.10%) looking at the television monitors. Animals exposed to the programmes depicting animate and inanimate forms of movement spent significantly more of their time looking at the monitors than those exposed to the moving images of humans or the blank screen. The amount of attention that the cats directed towards the television monitors decreased significantly across their 3 h of daily presentation, suggesting habituation. Certain components of the cats’ behaviour were influenced by visual stimulation. Animals in the animate movement condition spent significantly less time sleeping, and displayed a non-significant trend to spend more time resting, and in the exercise area of their pens, than those in the other conditions of visual stimulation. Overall, the results from this study suggest that visual stimulation in the form of two-dimensional video-tape sequences, notably that combining elements of prey items and linear movement, may hold some enrichment potential for domestic cats housed in rescue shelters. Such animals, however, may not benefit from this type of enrichment to the same degree as species with more well-developed visual systems, such as primates.     

Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.     

Trans-channel interactions in batrachotoxin-modified rat skeletal muscle sodium channels: kinetic analysis of mutual inhibition between mu-conotoxin GIIIA derivatives and amine blockers

R13X derivatives of μ-conotoxin GIIIA bind externally to single sodium channels and block current incompletely with mean “blocked” durations of several seconds. We studied interactions between two classes of blockers (μ-conotoxins and amines) by steady state, kinetic analysis of block of BTX-modified Na channels in planar bilayers. The amines cause all-or-none block at a site internal to the selectivity filter. TPrA and DEA block single Na channels with very different kinetics. TPrA induces discrete, all-or-none, blocked events (mean blocked durations, ∼100 ms), whereas DEA produces a concentration-dependent reduction of the apparent single channel amplitude (“fast” block). These distinct modes of action allow simultaneous evaluation of block by TPrA and DEA, showing a classical, competitive interaction between them. The apparent affinity of TPrA decreases with increasing [DEA], based on a decrease in the association rate for TPrA. When an R13X μ-conotoxin derivative and one of the amines are applied simultaneously on opposite sides of the membrane, a mutually inhibitory interaction is observed. Dissociation constants, at +50 mV, for TPrA (∼4 mM) and DEA (∼30 mM) increase by ∼20%-50% when R13E (nominal net charge, +4) or R13Q (+5) is bound. Analysis of the slow blocking kinetics for the two toxin derivatives showed comparable decreases in affinity of the μ-conotoxins in the presence of an amine. Although this mutual inhibition seems to be qualitatively consistent with an electrostatic interaction across the selectivity filter, quantitative considerations raise questions about the mechanistic details of the interaction.     

The Erv family of sulfhydryl oxidases

The Erv flavoenzymes contain a compact module that catalyzes the pairing of cysteine thiols into disulfide bonds. High-resolution structures of plant, animal, and fungal Erv enzymes that function in different contexts and intracellular compartments have been determined. Structural features can be correlated with biochemical properties, revealing how core sulfhydryl oxidase activity has been tailored to various functional niches. The introduction of disulfides into cysteine-containing substrates by Erv sulfhydryl oxidases is compared with the mechanisms used by NADPH-driven disulfide reductases and thioredoxin-like oxidoreductases to reduce and transfer disulfides, respectively.     

Phenology and Stem Diameter Increment Seasonality in a Costa Rican Wet Tropical Forest

The relationship between phenology and tree stem diameter increment is largely unexplored in tropical species, especially in wet tropical forests. To explore links between these phenomena, we measured stem diameter increment and phenology of ten canopy tree species from a range of functional types in the Atlantic lowlands of Costa Rica to test for seasonal and interannual patterns. We measured stem diameter increment using band dendrometers and visually assessed leaf and reproductive phenology monthly from 1997 to 2000. We categorized the species into groups based on patterns of leaf exchange and reproduction. Species were either deciduous with synchronous or asynchronous leaf drop, or evergreen with continuous or seasonal leaf flushing. Flowering occurred supra-annually, annually, or continuously. Of the ten species studied, four species, Cecropia insignis, Dipteryx panamensis, Lecythis ampla, and Simarouba amara , had consistent seasonal stem diameter increment patterns in both years. Dipteryx panamensis and L. ampla were deciduous with synchronized leaf drop . Cecropia insignis was evergreen and produced new leaves continuously. Simarouba amara , also evergreen, exchanged leaves over a brief period once a year. We tested whether stem diameter increment was correlated to phenology using logistic regression. Leaflessness significantly explained patterns in stem diameter increment but reproductive phenology did not. Deciduous trees were 2.6–9.3 times more likely to grow less than average the month following leaffall than in months when trees had full crowns.     

Vegetation and fire history of a Chinese site in southern tropical Xishuangbanna derived from phytolith and charcoal records from Holocene sediments

Aim The aims of this paper are to reconstruct the vegetation and fire history over the past 2000 years in a well‐preserved rain‐forest area, to understand interactions between climate, fire, and vegetation, and to predict how rain forest responds to global warming and increased intensity of human activity. Location Xishuangbanna, south‐west China, 21–22° N, 101–102° E. Methods Phytolith (plant opal silica bodies) morphotypes, assemblages, and indices were used to reconstruct palaeovegetation and palaeoclimate changes in detail. Micro‐charcoal particles found in phytolith slides, together with burnt phytoliths and highly weathered bulliform cells, were employed to reconstruct a record of past fire occurrence. A survey of field sediments, lithology, and ¹⁴C dating were also employed. Results Phytoliths were divided into 11 groups and classified into 33 well‐described morphotypes according to their shape under light microscopy and their presumed anatomical origins and ecological significance. The phytolith assemblages were divided into six significant zones that reveal a complete history of vegetation changes corresponding to climate variation and fire occurrence. Phytolith assemblages and indices show that the palaeoclimate in the study area is characterized by the alternation of warm–wet and cool–dry conditions. Phytolith and charcoal records reveal that 12 fire episodes occurred. Comparison of burnt phytoliths with an aridity index (Iph) shows that fire episodes have a strong relationship with drought events. Main conclusions Our results indicate that fire occurrence in the tropical rain forest of Xishuangbanna is predominantly under the control of natural climate variability (drought events). Nearly every fire episode is coupled with a climatic event and has triggered vegetation composition changes marked by a pronounced expansion of grasses. This indicates that drought interacts with fire to exert a strong influence on the ecological dynamics of the rain forest. However, the impact of human activity in recent centuries is also significant. Our results are important for understanding the interactions between climate, fire, and vegetation, and for predicting how rain forest responds to global warming and increased human activity.     

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers

To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spin-labeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.