Possible involvement of cerebroside sulfate in opiate receptor binding.

Cerebroside sulfate (CS) appears to fulfill most of the structural requirements of a hypothetical opiate receptor. It possesses many of the properties that are thought to be necessary for the identification of an "opiate receptor," exhibiting high affinity and stereoselective binding to a number of narcotic drugs. Although these properties are insufficient to establish identity of the receptor, it is highly significant that the affinity of this binding can be correlated with the analgetic potency of these drugs in both man and rodents. CS is an endogenous component of brain tissue, and a partially purified opiate receptor from mouse brain has been found to be CS. Other experiments indicate that reduced availability of brain CS decreases the analgetic effects of morphine and this is accompanied by a reduction in number of binding sites, suggesting that the interaction of opiates with CS observed in vitro may also have importance in vivo. CS was also found to be a component of the opiate receptor after marking with 125I-labeled diazosulfanilic acid. The possibility that CS or the SO4-2 group of this lipid may be the "anionic site" of the opiate receptor should be considered.     

Comparative proteome analysis using amine-reactive isobaric tagging reagents coupled with 2D LC/MS/MS in 3T3-L1 adipocytes following hypoxia or normoxia

Hypoxia during the expansion of adipocytes is known to contribute both to the secretion of multiple inflammation-related adipokines as well as to obesity. We therefore investigated the nature of protein changes occurring in adipocytes during hypoxia by observation of the intracellular proteins that are expressed in 3T3-L1 adipocytes. Lysates were utilized for quantitative proteome analysis using isobaric tags for relative and absolute quantitation (iTRAQ) combined with peptide separation by multi-dimensional liquid chromatography. Antioxidants and elongation factors, as well as glycolytic enzymes were increased in hypoxic adipocytes. These changes were supported by similar changes suggested by real-time PCR. The proteins showing changes are all potential targets for revering the mechanism behind the phenomenon of induction of obese adipocytes by hypoxia. This study can therefore aid in defining the proteomic changes that occur in adipocytes in response to oxygen stress, and can further characterize adipocyte metabolism and adaptation to low oxygen conditions.     

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed.     

Proteomic profiling of lymphedema development in mouse model

The lymphatic vascular system plays an important role in tissue fluid homeostasis. Lymphedema is a chronic, progressive, and incurable condition that leads to lymphatic fluid retention; it may be primary (heritable) or secondary (acquired) in nature. Although there is a growing understanding of lymphedema, methods for the prevention and treatment of lymphedema are still limited. In this study, we investigated differential protein expressions in sham‐operated and lymphedema‐operated mice for 3 days, using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry analysis. Male improved methodology for culturing noninbred (ICR) mice developed lymphedema in the right hindlimb. Twenty functional proteins were found to be differentially expressed between lymphedema induced‐right leg tissue and normal left leg tissue. Out of these proteins, the protein levels of apolipoprotein A‐1 preprotein, alpha‐actinin‐3, mCG21744, parkinson disease, serum amyloid P‐component precursor, annexin A8, mKIAA0098 protein, and fibrinogen beta chain precursor were differentially upregulated in the lymphedema mice compared with the sham‐operated group. Western blotting analysis was used to validate the proteomics results. Our results showing differential up‐regulation of serum amyloid P‐component precursor, parkinson disease, and apolipoprotein A‐1 preprotein in lymphedema model over sham‐operated model suggest important insights into pathophysiological target for lymphedema. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of abscisic acid application on root isoflavonoid concentration and nodulation of wild-type and nodulation-mutant soybean plants

Isoflavonoids (daidzein, genistein, and coumestrol) are involved in induction of nod genes in Bradyrhizobium japonicum and may be involved in nodule development as well. Abscisic acid (ABA) may also impact nodulation since ABA is reportedly involved in isoflavonoid synthesis. The current study was conducted to evaluate whether ABA plays a role in differential nodulation of a hypernodulated soybean (Glycine max L. Merr.) mutant and the Williams parent. Exogenous ABA application resulted in a decrease in nodule number and weight in both lines. Isoflavonoid concentrations were also markedly decreased in response to ABA application in both inoculated and noninoculated soybean roots. The inoculation treatment itself resulted in a marked increase in isoflavonoid concentrations of NOD1-3, regardless of ABA levels, while only slight increases occurred in Williams. The nodule numbers of both soybean lines across several ABA concentration treatments were highly correlated with the concentration of all three isoflavonoids. However, differences in internal levels of ABA between lines were not detected when grown in the absence of external ABA additions. It is concluded that differential nodule expression between the wild type and the hypernodulated mutant is not likely due to differential ABA synthesis.     

Spontaneous Reperfusion after In Situ Thromboembolic Stroke in Mice

Injection of thrombin into the middle cerebral artery (MCA) of mice has been proposed as a new model of thromboembolic stroke. The present study used sequential multiparametric Magnetic Resonance Imaging (MRI), including Magnetic Resonance Angiography (MRA), Diffusion-Weighted Imaging (DWI) and Perfusion-Weighted Imaging (PWI), to document MCA occlusion, PWI-DWI mismatch, and lesion development. In the first experiment, complete MCA occlusion and reproducible hypoperfusion were obtained in 85% of animals during the first hour after stroke onset. In the second experiment, 80% of animals showed partial to complete reperfusion during a three-hour follow-up. Spontaneous reperfusion thus contributed to the variability in ischemic volume in this model. The study confirmed the value of the model for evaluating new thrombolytic treatments, but calls for extended MRI follow-up at the acute stage in therapeutic studies.