Affinity pulldown of γ‐secretase and associated proteins from human and rat brain

γ‐Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph‐1 and Pen‐2. Little is known about how the substrates are selected by γ‐secretase, but it has been suggested that γ‐secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ‐secretase and regulates the processing of APP‐derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ‐secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ‐secretase inhibitor coupled to biotin via a long linker and a S‐S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC‐MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ‐secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ‐secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ‐secretase complex.     

A cell-based screen for inhibitors of protein folding and degradation

Cancer cells are exposed to external and internal stresses by virtue of their unrestrained growth, hostile microenvironment, and increased mutation rate. These stresses impose a burden on protein folding and degradation pathways and suggest a route for therapeutic intervention in cancer. Proteasome and Hsp90 inhibitors are in clinical trials and a 20S proteasome inhibitor, Velcade, is an approved drug. Other points of intervention in the folding and degradation pathway may therefore be of interest. We describe a simple screen for inhibitors of protein synthesis, folding, and proteasomal degradation pathways in this paper. The molecular chaperone-dependent client v-Src was fused to firefly luciferase and expressed in HCT-116 colorectal tumor cells. Both luciferase and protein tyrosine kinase activity were preserved in cells expressing this fusion construct. Exposing these cells to the Hsp90 inhibitor geldanamycin caused a rapid reduction of luciferase and kinase activities and depletion of detergent-soluble v-Src::luciferase fusion protein. Hsp70 knockdown reduced v-Src::luciferase activity and, when combined with geldanamycin, caused a buildup of v-Src::luciferase and ubiquitinated proteins in a detergent-insoluble fraction. Proteasome inhibitors also decreased luciferase activity and caused a buildup of phosphotyrosine-containing proteins in a detergent-insoluble fraction. Protein synthesis inhibitors also reduced luciferase activity, but had less of an effect on phosphotyrosine levels. In contrast, certain histone deacetylase inhibitors increased luciferase and phosphotyrosine activity. A mass screen led to the identification of Hsp90 inhibitors, ubiquitin pathway inhibitors, inhibitors of Hsp70/Hsp40-mediated refolding, and protein synthesis inhibitors. The largest group of compounds identified in the screen increased luciferase activity, and some of these increase v-Src levels and activity. When used in conjunction with appropriate secondary assays, this screen is a powerful cell-based tool for studying compounds that affect protein synthesis, folding, and degradation.     

Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli

Chemerin is a novel chemokine that binds to the G protein-coupled receptor (GPCR) ChemR23, also known as chemokine-like receptor 1 (CMKLR1). It is secreted as a precursor and executes pro-inflammatory functions when the last six amino acids are removed from its C-terminus by serine proteases. After maturation, Chemerin attracts dendritic cells and macrophages through binding to ChemR23. We report a new method for expression and purification of mature recombinant human Chemerin (rhChemerin) using a prokaryotic system. After being expressed in bacteria, rhChemerin in inclusion bodies was denatured using 6 M guanidine chloride. Soluble rhChemerin was prepared by the protein-specific renaturation solution under defined conditions. It was subsequently purified using ion-exchange columns to more than 95% purity with endotoxin level <1.0 EU/μg. We further demonstrated its biological activities for attracting migration of human dendritic cells and murine macrophages in vitro using established chemotaxis assays.     

Differences in metabolite profile between blood plasma and serum

In metabolomic research, blood plasma and serum have been considered to possess similar compositions and properties. Their perceived equivalence has resulted in researchers choosing arbitrarily between serum and plasma for analysis. Here, routine serum and plasma were prepared and their low-molecular-weight compounds were determined using gas chromatography/time-of-flight mass spectrometry. Principal components analysis was applied to process the acquired data, and marked differences in metabolite profiles were observed between serum and plasma. Of the 72 identified compounds, 36 (50%) discriminate serum from plasma, with 29 and 7 metabolites showing a significantly higher abundance (t test, P < 0.05) in serum and plasma, respectively. Incubation of blood had distinct effects on the analyte peak areas, with the effects being more pronounced for plasma than for serum and more pronounced for a shorter incubation than for a longer incubation. These results highlight the importance in choosing serum or plasma as the analytical sample and in stipulating the incubation time. Because incubation affected the analyte peak areas less in serum than in plasma, we recommend serum as the sample of choice in metabolomic studies.     

Plasmacytoid dendritic cells in antiviral immunity and autoimmunity

Plasmacytoid dendritic cells (pDCs) represent a unique and crucial immune cell population capable of producing large amounts of type I interferons (IFNs) in response to viral infection. The function of pDCs as the professional type I IFN-producing cells is linked to their selective expression of Toll-like receptor 7 (TLR7) and TLR9, which sense viral nucleic acids within the endosomal compartments. Type I IFNs produced by pDCs not only directly inhibit viral replication but also play an essential role in linking the innate and adaptive immune system. The aberrant activation of pDCs by self nucleic acids through TLR signaling and the ongoing production of type I IFNs do occur in some autoimmune diseases. Therefore, pDC may serve as an attractive target for therapeutic manipulations of the immune system to treat viral infectious diseases and autoimmune diseases.     

Early Changes of β-Catenins and Menins in Spinal Cord Dorsal Horn after Peripheral Nerve Injury

Injury to the peripheral nervous system can lead to spontaneous pain, hyperalgesia and allodynia. Previous studies have shown sprouting of Aβ-fibres into lamina II of the spinal cord dorsal horn after nerve injury and the formation of new synapses by these sprouts. β-Catenin and menin as synaptogenic factors are critically involved in synapse formation. However, the roles of β-catenin and menin in neuropathic pain are still unclear. Using Western blot analysis we investigated the changes of β-catenin and menin in the spinal dorsal horn after unilateral spared nerve injury (SNI). We demonstrated an increase in both β-catenin and menin protein levels in the ipsilateral spinal dorsal horn at days 1 and 3 following spared nerve injury (P < 0.05). These increases were associated with changes in paw withdrawal threshold to mechanical stimuli and weight bearing deficit suggestive of pain behavior and spontaneous ongoing pain respectively. However, the injury-associated increases in β-catenins and menins levels returned to control levels at day 14. In conclusion, these results indicate that peripheral nerve injury induces upregulation of β-catenins and menins in the dorsal horn of the spinal cord, which may contribute to the development of chronic neuropathic pain. Antagonists of these molecules may serve as new therapeutic agents.     

Isolation and characterization of lactic acid bacteria strains with ornithine producing capacity from natural sea salt

Two lactic acid bacteria (LAB) having ornithine-producing capacity were isolated from Korean natural sea salt. They were Gram-positive, short rod-type bacteria, and able to grow anaerobically with CO₂ production. The isolates grew well on MRS broth at 30–37°C and a pH of 6.5–8.0. The optimum temperature and pH for growth are 37°C and pH 7.0. The isolates fermented D-ribose, D-galactose, D-lactose, D-maltose, Dcellobiose, D-tagatose, D-trehalose, sucrose, D-melezitose, gentiobiose, D-glucose but not D-melibiose, inositol, and L-sorbose. The 16S rDNA sequences of the two isolates showed 99.5% and 99.6% homology with the Weissella koreensis S5623 16S rDNA (Access no. AY035891). They were accordingly identified and named as Weissella koreensis MS1-3 and Weissella koreensis MS1-14, and produced intracellular ornithine at levels of 72 mg/100 g cell F.W. and 105 mg/100 g cell F.W. and extracellular ornithine at levels of 4.5 mg/100 ml and 4.6 mg/100 ml medium, respectively, by culturing in MRS broth supplemented with 1% arginine. High cell growth was maintained in MRS broth with a NaCl concentration of 0–6%. These results show for the first time that Korean natural sea salts contain lactic acid bacteria Weissella koreensis strains having ornithine producing capacity.     

Prediction of alterations in cardiac functions (ECG data) in the course of the permanent monitoring of cosmonauts starting from selection until return to earth after short space flights

Analysis of alterations in the cardiac activity on the basis of electrocardiographic (ECG) findings in 29 cosmonauts of flight and ground professions aged from 29 to 61 years after 34 short (8–30 days) space flights (SFs) between 1982 and 2006 has been carried out. The ECG data at the stage of clinical selection, clinical-physiological examination (CPE) before a SF, at the stage of the launch of a spacecraft (SC) into orbit and its landing on Earth and at the stage of postflight CPE have been analyzed. The analysis of cardiac activity parameters on the basis of ECG data at different stages of observations has led to the identification of three groups of cosmonauts. There were no significant changes or negative tendencies in the alteration of ECG data in the first group (55.2% of the total number of cosmonauts) during the observation period from selection to the end of the SF. The changes that later became more pronounced during the landing on Earth and were retained during postflight CPE have been found in the second group of cosmonauts (in 34.5% cases) at the time of selection and preflight CPE. Considerable disturbances in cardiac activity that are dangerous for human health have been found in ECGs in the third group (10.3%) during the descent from orbit. The data from the study are the first step in the investigation of possible medical risks for the development and improvement of requirements for the medical selection of crews and the admission of subjects with partial health insufficiency on SFs.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

Rethinking the dispersal of Homo sapiens out of Africa

Current fossil, genetic, and archeological data indicate that Homo sapiens originated in Africa in the late Middle Pleistocene. By the end of the Late Pleistocene, our species was distributed across every continent except Antarctica, setting the foundations for the subsequent demographic and cultural changes of the Holocene. The intervening processes remain intensely debated and a key theme in hominin evolutionary studies. We review archeological, fossil, environmental, and genetic data to evaluate the current state of knowledge on the dispersal of Homo sapiens out of Africa. The emerging picture of the dispersal process suggests dynamic behavioral variability, complex interactions between populations, and an intricate genetic and cultural legacy. This evolutionary and historical complexity challenges simple narratives and suggests that hybrid models and the testing of explicit hypotheses are required to understand the expansion of Homo sapiens into Eurasia.