Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [3H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D2-dopamine receptors.     

Tendon‐to‐bone attachment: From development to maturity

The attachment between tendon and bone occurs across a complex transitional tissue that minimizes stress concentrations and allows for load transfer between muscles and skeleton. This unique tissue cannot be reconstructed following injury, leading to high incidence of recurrent failure and stressing the need for new clinical approaches. This review describes the current understanding of the development and function of the attachment site between tendon and bone. The embryonic attachment unit, namely, the tip of the tendon and the bone eminence into which it is inserted, was recently shown to develop modularly from a unique population of Sox9‐ and Scx‐positive cells, which are distinct from tendon fibroblasts and chondrocytes. The fate and differentiation of these cells is regulated by transforming growth factor beta and bone morphogenetic protein signaling, respectively. Muscle loads are then necessary for the tissue to mature and mineralize. Mineralization of the attachment unit, which occurs postnatally at most sites, is largely controlled by an Indian hedgehog/parathyroid hormone‐related protein feedback loop. A number of fundamental questions regarding the development of this remarkable attachment system require further study. These relate to the signaling mechanism that facilitates the formation of an interface with a gradient of cellular and extracellular phenotypes, as well as to the interactions between tendon and bone at the point of attachment. Birth Defects Research (Part C) 102:101–112, 2014. © 2014 Wiley Periodicals, Inc.     

Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis

Autophagy is a major catabolic process responsible for the delivery of proteins and organelles to the lysosome/vacuole for degradation. Malfunction of this pathway has been implicated in numerous pathological conditions. Different organelles have been found to contribute to the formation of autophagosomes, but the exact mechanism mediating this process remains obscure. Here, we show that lipid droplets (LDs) are important for the regulation of starvation-induced autophagy. Deletion of Dga1 and Lro1 enzymes responsible for triacylglycerol (TAG) synthesis, or of Are1 and Are2 enzymes responsible for the synthesis of steryl esters (STE), results in the inhibition of autophagy. Moreover, we identified the STE hydrolase Yeh1 and the TAG lipase Ayr1 as well as the lipase/hydrolase Ldh1 as essential for autophagy. Finally, we provide evidence that the ER-LD contact-site proteins Ice2 and Ldb16 regulate autophagy. Our study thus highlights the importance of lipid droplet dynamics for the autophagic process under nitrogen starvation.     

A Rab-GAP TBC domain protein binds hepatitis C virus NS5A and mediates viral replication

Hepatitis C virus (HCV) is an important cause of liver disease worldwide. Current therapies are inadequate for most patients. Using a two-hybrid screen, we isolated a novel cellular binding partner interacting with the N terminus of HCV nonstructural protein NS5A. This partner contains a TBC Rab-GAP (GTPase-activating protein) homology domain found in all known Rab-activating proteins. As the first described interaction between such a Rab-GAP and a viral protein, this finding suggests a new mechanism whereby viruses may subvert host cell machinery for mediating the endocytosis, trafficking, and sorting of their own proteins. Moreover, depleting the expression of this partner severely impairs HCV RNA replication with no obvious effect on cell viability. These results suggest that pharmacologic disruption of this NS5A-interacting partner can be contemplated as a potential new antiviral strategy against a pathogen affecting nearly 3% of the world's population.     

Oxidation as a post-translational modification that regulates autophagy

The toxicity associated with accumulation of reactive oxygen species (ROS) has led to the evolution of various defense strategies to overcome oxidative stress, including autophagy. This pathway is involved in the removal and degradation of damaged mitochondria and oxidized proteins. At low levels, however, ROS act as signal transducers in various intracellular pathways. In a recent study we described the role of ROS as signaling molecules in starvation-induced autophagy. We showed that starvation stimulates formation of ROS, specifically H(2)O(2), in the mitochondria. Furthermore, we identified the cysteine protease HsAtg4 as a direct target for oxidation by H(2)O(2), and specified a cysteine residue located near the HsAtg4 catalytic site as critical for this regulation. Here we focus on Atg4, the target of regulation, and discuss possible mechanisms for the regulation of this enzyme in the autophagic process.     

Autophagy mediates nonselective RNA degradation in starving yeast

During nitrogen starvation, a nonselective bulk degradation of cytosolic proteins and organelles including ribosomes, termed macro‐autophagy (hereafter termed autophagy), is induced. The precise mechanism of RNA degradation by this pathway has not been yet elucidated. In this issue of the The EMBO Journal, Huang et al characterize an autophagy‐dependent RNA catabolism in yeast and identify the enzymes responsible for the degradation process.     

Muscle force regulates bone shaping for optimal load-bearing capacity during embryogenesis

The vertebrate skeleton consists of over 200 individual bones, each with its own unique shape, size and function. We study the role of intrauterine muscle-induced mechanical loads in determining the three-dimensional morphology of developing bones. Analysis of the force-generating capacity of intrauterine muscles in mice revealed that developing bones are subjected to significant and progressively increasing mechanical challenges. To evaluate the effect of intrauterine loads on bone morphogenesis and the contribution of the emerging shape to the ability of bones to withstand these loads, we monitored structural and mineral changes during development. Using daily micro-CT scans of appendicular long bones we identify a developmental program, which we term preferential bone growth, that determines the specific circumferential shape of each bone by employing asymmetric mineral deposition and transient cortical thickening. Finite element analysis demonstrates that the resulting bone structure has optimal load-bearing capacity. To test the hypothesis that muscle forces regulate preferential bone growth in utero, we examine this process in a mouse strain (mdg) that lacks muscle contractions. In the absence of mechanical loads, the stereotypical circumferential outline of each bone is lost, leading to the development of mechanically inferior bones. This study identifies muscle force regulation of preferential bone growth as the module that shapes the circumferential outline of bones and, consequently, optimizes their load-bearing capacity during development. Our findings invoke a common mechanism that permits the formation of different circumferential outlines in different bones.     

Fruit cuticle lipid composition and fruit post-harvest water loss in an advanced backcross generation of pepper (Capsicum sp.)

To understand the role of fruit cuticle lipid composition in fruit water loss, an advanced backcross population, the BC(2)F(2) , was created between the Capsicum annuum (PI1154) and the Capsicum chinense (USDA162), which have high and low post-harvest water loss rates, respectively. Besides dramatic differences in fruit water loss, preliminary studies also revealed that these parents exhibited significant differences in both the amount and composition of their fruit cuticle. Cuticle analysis of the BC(2)F(2) fruit revealed that although water loss rate was not strongly associated with the total surface wax amount, there were significant correlations between water loss rate and cuticle composition. We found a positive correlation between water loss rate and the amount of total triterpenoid plus sterol compounds, and negative correlations between water loss and the alkane to triterpenoid plus sterol ratio. We also report negative correlations between water loss rate and the proportion of both alkanes and aliphatics to total surface wax amount. For the first time, we report significant correlations between water loss and cutin monomer composition. We found positive associations of water loss rate with the total cutin, total C(16) monomers and 16-dihydroxy hexadecanoic acid. Our results support the hypothesis that simple straight-chain aliphatic cuticle constituents form more impermeable cuticular barriers than more complex isoprenoid-based compounds. These results shed new light on the biochemical basis for cuticle involvement in fruit water loss.     

Modulation of N-ethylmaleimide-sensitive factor activity upon amino acid deprivation

Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply.