1H, 13C, and 15N resonance assignments of human phosphohistidine phosphatase 1 (PHPT1)

Phosphohistidine phosphatase 1 (PHPT1) is the first protein histidine phosphatase identified in vertebrates. The NMR assignments of human PHPT1 are essential for solution structure determination and NMR study of the protein interactions of PHPT1 with its potential substrates.     

Transducing the Hedgehog signal across the plasma membrane

Cell signaling mediated by the Hedgehog (Hh) family of secreted proteins is essential for metazoan development and its malfunction causes congenital disorders and cancer. The seven-transmembrane protein Smoothened (Smo) transduces the Hh signal across the plasma membrane in both vertebrates and invertebrates but the underlying mechanisms remain ill defined. In Drosophila, Hh induces phosphorylation of Smo at multiple sites by PKA and CK1, leading to its cell surface accumulation and activation. Recently, we have obtained evidence that Hh-induced phosphorylation promotes Smo activity by inducing a conformational switch and dimerization of its carboxy-terminal cytoplasmic tail (C-tail). Furthermore, we provided evidence that a similar mechanism regulates mammalian Smo. We discuss how Smo conformational change regulates the intracellular signaling complex and how Smo transduces the graded Hh signaling activities through different conformational states.     

Development of a RP-HPLC method for screening potentially counterfeit anti-diabetic drugs

Pharmaceutical counterfeiting is becoming a serious problem in the world, especially in developing countries including China. Herein an isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for screening counterfeit medicines and adulterated dietary supplement products. The developed method could be employed to separate and determine simultaneously six anti-diabetic drugs (glipizide, gliclazide, glibenclamide, glimepiride, gliquidone, repaglinide) on an isocratic solvent system using an Alltima C18 column (5 microm, 150 mmx4.6 mm) with an isocratic mobile phase of methanol-phosphate buffer (pH 3.0; 0.01 mol/L) (70:30, v/v), at a flow rate of 1.0 mL/min and at a wavelength of 230 nm. The proposed method was successfully applied to the analysis of medicinal and dietary supplement samples purchased from the local market in China.     

Membrane subproteomic analysis of Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been known for a long time to prevent tuberculosis (TB) worldwide since 1921. Nonetheless, we know little about BCG membrane proteome. In the present study, we utilized alkaline incubation and Triton X-114-based methods to enrich BCG membrane proteins and subsequently digested them using proteolytic enzyme. The recovered peptides were further separated by 2-D LC and identified by ESI-MS/MS. As a result, total 474 proteins were identified, including 78 integral membrane proteins (IMPs). Notably, 18 BCG IMPs were described for the first time in mycobacterium. Further analysis of the 78 IMPs indicated that the theoretical molecular mass distribution of them ranged from 8.06 to 167.86 kDa and pI scores ranged from 4.40 to 11.60. Functional classification revealed that a large proportion of the identified IMPs (67.9%, 53 out of 78) were involved in cell wall and cell processes functional group. In conclusion, here we reported a comprehensive profile of the BCG membrane subproteome. The present investigation may allow the identification of some valuable vaccine and drug target candidates and thus provide basement for future designing of preventive, diagnostic, and therapeutic strategies against TB.     

Characterization and fine mapping of <Emphasis Type="Italic">RppQ</Emphasis>, a resistance gene to southern corn rust in maize

Southern corn rust (SCR) is a fungal disease caused by Puccinia polysora Underw, which can infect maize and may result in substantial yield losses in maize production. The maize inbred line Qi319 carries the SCR resistance gene RppQ. In order to identify molecular markers linked to the RppQ gene, several techniques were utilized including random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), and amplified fragment length polymorphism (AFLP). In addition, sequence characterized amplified region (SCAR) techniques combined with bulked segregant analysis (BSA) were used. Seven RAPD markers, eight SSR markers, and sixty-three AFLP primer combinations amplified polymorphisms between two parents and two bulk populations. A large F₂ population was used for genetic analysis and for fine mapping of the RppQ gene region. One AFLP polymorphic band, M-CAA/E-AGC₃₂₄, was converted to a SCAR marker, MA7, which was mapped to a position 0.46 cM from RppQ. Finally, the RppQ gene was mapped between the SCAR marker MA7 and the AFLP marker M-CCG/E-AGA₁₅₇ with distances of 0.46 and 1.71 cM, respectively.     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

Identification of novel proteins associated with both alpha-synuclein and DJ-1

The molecular mechanisms leading to neurodegeneration in Parkinson disease (PD) remain elusive, although many lines of evidence have indicated that alpha-synuclein and DJ-1, two critical proteins in PD pathogenesis, interact with each other functionally. The investigation on whether alpha-synuclein directly interacts with DJ-1 has been controversial. In the current study, we analyzed proteins associated with alpha-synuclein and/or DJ-1 with a robust proteomics technique called stable isotope labeling by amino acids in cell culture (SILAC) in dopaminergic MES cells exposed to rotenone versus controls. We identified 324 and 306 proteins in the alpha-synuclein- and DJ-1-associated protein complexes, respectively. Among alpha-synuclein-associated proteins, 141 proteins displayed significant changes in the relative abundance (increase or decrease) after rotenone treatment; among DJ-1-associated proteins, 119 proteins displayed significant changes in the relative abundance after rotenone treatment. Although no direct interaction was observed between alpha-synuclein and DJ-1, whether analyzed by affinity purification followed by mass spectrometry or subsequent direct co-immunoprecipitation, 144 proteins were seen in association with both alpha-synuclein and DJ-1. Of those, 114 proteins displayed significant changes in the relative abundance in the complexes associated with alpha-synuclein, DJ-1, or both after rotenone treatment. A subset of these proteins (mortalin, nucleolin, grp94, calnexin, and clathrin) was further validated for their association with both alpha-synuclein and DJ-1 using confocal microscopy, Western blot, and/or immunoprecipitation. Thus, we not only confirmed that there was no direct interaction between alpha-synuclein and DJ-1 but also, for the first time, report these five novel proteins to be associating with both alpha-synuclein and DJ-1. Further characterization of these docking proteins will likely shed more light on the mechanisms by which DJ-1 modulates the function of alpha-synuclein, and vice versa, in the setting of PD.     

Halothane binding proteome in human brain cortex

Inhaled anesthetics bind specifically to a wide variety of proteins in the brain. This set of proteins must include those that contribute to the physiological and behavioral phenotypes of anesthesia and the related side effects. To identify the anesthetic-binding targets and functional pathways associated with these targets in human brain, halothane photolabeling and two-dimensional (2D) gel electrophoresis were used. Both membrane and soluble proteins from human temporal cortex were prepared. More than 300 membrane and 400 soluble protein spots were detected on the stained blots, of which 23 membrane and 34 soluble proteins were labeled by halothane and identified by mass spectroscopy. Their functional classification reveals five groups, including carbohydrate metabolism, protein folding, oxidative phosphorylation, nucleoside triphosphatase, and dimer/kinase activity with different correlative stringency. When network analysis of the interaction between these protein molecules is used, the weighted interaction accentuates the cellular protein components important in cell growth and proliferation, cell cycle and cell death, and cell-cell signaling and interactions, although no pathway was specific. This study provides evidence for multiple anesthetic binding targets and suggests potential pathways involved in their actions.     

Nanoparticle-mediated drug delivery and gene therapy

Biomedical application of nanotechnology is a rapidly developing area that raises new prospect in the improvement of diagnosis and treatment of human diseases. The ability to incorporate drugs or genes into a functionalized nanoparticle demonstrates a new era in pharmacotherapy for delivering drugs or genes selectively to tissues or cells. It is envisioned that the transfer of nanoengineering capability into disease therapy will provide constant and concentrated drug delivery to targeted tissues, minimizing systemic side effects and toxicity. We have in this article highlighted the recent state of the art in nanomedicine, focusing particularly on the achievement of nanotechnology in nanoscale drug and gene delivery in vitro and in vivo. In addition, a specific emphasis has been placed on the use of nanotechnology to improve controlled drug release and sustainable drug delivery in solid tumors and on new drug therapies for age-related neurodegenerative disorders.     

Associations between daytime and nighttime plasma orexin A levels and cognitive function in patients with obstructive sleep apnea

Sleep and Biological Rhythms - The relationship between plasma orexin A (OXA) levels and cognitive function in patients with obstructive sleep apnea (OSA) remains unclear. This study aimed to...