Phytane and neoclerodane diterpenes from the aerial parts of Inula nervosa Wall.

Three new aliphatic diterpenes (1–3), together with three known neoclerodane-type diterpenes (4–6) were isolated from the aerial parts of Inula nervosa Wall. The structures of 1-3 were elucidated on the basis of 1D and 2D spectroscopic analysis. Additionally, phytane-type and neoclerodane-type diterpenes have not been reported in any species of the genus Inula yet. The phytane-type and neoclerodane-type diterpenes obtained from I. nervosa Wall. suggest this plant maybe have remote genetic relations with other Inula species.     

Progressive Kidney Degeneration in Mice Lacking Tensin

Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, −/− mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell– matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell–matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.     

Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency

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Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.     

Sensitive and High Resolution Localization and Tracking of Membrane Proteins in Live Cells with BRET

Peripheral and integral membrane proteins can be located in several different subcellular compartments, and it is often necessary to determine the location of such proteins or to track their movement in living cells. Image‐based colocalization of labeled membrane proteins and compartment markers is frequently used for this purpose, but this method is limited in terms of throughput and resolution. Here we show that bioluminescence resonance energy transfer (BRET) between membrane proteins of interest and compartment‐targeted BRET partners can report subcellular location and movement of membrane proteins in live cells. The sensitivity of the method is sufficient to localize a few hundred protein copies per cell. The spatial resolution can be sufficient to determine membrane topology, and the temporal resolution is sufficient to track changes that occur in less than 1 second. BRET requires little user intervention, and is thus amenable to large‐scale experimental designs with standard instruments.     

The neurotoxicity of iron,copper and cobalt in Parkinson’s disease through ROS-mediated mechanisms

Parkinson’s disease (PD) is the second most common neurodegenerative disease with gradual loss of dopaminergic neurons. Despite extensive research in the past decades, the etiology of PD remains elusive. Nevertheless, multiple lines of evidence suggest that oxidative stress is one of the common causes in the pathogenesis of PD. It has also been suggested that heavy metal-associated oxidative stress may be implicated in the etiology and pathogenesis of PD. Here we review the roles of redox metals, including iron, copper and cobalt, in PD. Iron is a highly reactive element and deregulation of iron homeostasis is accompanied by concomitant oxidation processes in PD. Copper is a key metal in cell division process, and it has been shown to have an important role in neurodegenerative diseases such as PD. Cobalt induces the generation of reactive oxygen species (ROS) and DNA damage in brain tissues.     

Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos. Structure, taxonomic and biosynthetic implications

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin-layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly monounsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia. The permanganate-periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen-4-oic and docosen-6-oic acids. Both types of mycolate esters yielded the same set of long-chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds. After hydrogenation of the double bond located in the alkyl-branched chain, the two types of mycolates had the same mobility on thin-layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates. Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.     

Doxorubicin‐induced cardiotoxicity is maturation dependent due to the shift from topoisomerase IIα to IIβ in human stem cell derived cardiomyocytes

Doxorubicin (DOX) is widely used to treat various cancers affecting adults and children; however, its clinical application is limited by its cardiotoxicity. Previous studies have shown that children are more susceptible to the cardiotoxic effects of DOX than adults, which may be related to different maturity levels of cardiomyocyte, but the underlying mechanisms are not fully understood. Moreover, researchers investigating DOX‐induced cardiotoxicity caused by human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) have shown that dexrazoxane, the recognized cardioprotective drug for treating DOX‐induced cardiotoxicity, does not alleviate the toxicity of DOX on hiPSC‐CMs cultured for 30 days. We have suggested that this may be ascribed to the immaturity of the 30 days hiPSC‐CMs. In this study, we investigated the mechanisms of DOX induced cardiotoxicity in cardiomyocytes of different maturity. We selected 30‐day‐old and 60‐day‐old hiPSC‐CMs (day 30 and day 60 groups), which we term ‘immature’ and ‘relatively mature’ hiPSC‐CMs, respectively. The day 30 CMs were found to be more susceptible to DOX than the day 60 CMs. DOX leads to more ROS (reactive oxygen species) production in the day 60 CMs than in the relatively immature group due to increased mitochondria number. Moreover, the day 60 CMs mainly expressed topoisomerase IIβ presented less severe DNA damage, whereas the day 30 CMs dominantly expressed topoisomerase IIα exhibited much more severe DNA damage. These results suggest that immature cardiomyocytes are more sensitive to DOX as a result of a higher concentration of topoisomerase IIα, which leads to more DNA damage.