Synthesis of phenanthroindolizidine alkaloids and evaluation of their antitumor activities and toxicities

We previously reported that phenanthroindolizidine alkaloid 3 and its derivatives had markedly potent in vitro cytotoxicity. However, they had low in vivo antitumor activities and high in vivo toxicities, which was a serious problem. To address this problem, new phenanthroindolizidine derivatives were synthesized and their antitumor activities and toxicities were evaluated. This study describes the relationship between the chemical structures, antitumor activities, and toxicities of these phenanthroindolizidine derivatives. Based on its properties, compound 8 was found to be the most suitable potential antitumor agent.     

First crystal structures of Na+,K+-ATPase: new light on the oldest ion pump

Na(+),K(+)-adenosine triphosphatase (NKA) is the first P-type ion translocating adenosine triphosphatase (ATPase) ever identified, and the significance of this class of proteins was highlighted by the 1997 Nobel Prize in Chemistry awarded to Jens C. Skou for the discovery in 1957. More than half a century passed between the initial identification and the publication of a high-resolution crystal structure of NKA. Although the new crystal structures provided many surprises and insights, structural biology on this system remains challenging, as NKA is a very difficult protein to crystallize. Here we explain the reasons behind the challenges, introduce a mechanism that governs the function, and summarize current knowledge of NKA structure in comparison with another member of the P-type ATPase family, Ca(2+)-ATPase.     

Prediction of N-myristoylation modification of proteins by SVM

Attachment of a myristoyl group to NH(2)-terminus of a nascent protein among protein post-translational modification (PTM) is called myristoylation. The myristate moiety of proteins plays an important role for their biological functions, such as regulation of membrane binding (HIV-1 Gag) and enzyme activity (AMPK). Several predictors based on protein sequences alone are hitherto proposed. However, they produce a great number of false positive and false negative predictions; or they cannot be used for general purpose (i.e., taxon-specific); or threshold values of the decision rule of predictors need to be selected with cautiousness. Here, we present novel and taxon-free predictors based on protein primary structure. To identify myristoylated proteins accurately, we employ a widely used machinelearning algorithm, support vector machine (SVM). A series of SVM predictors are developed in the present study where various scales representing physicochemical and biological properties of amino acids (from the AAindex database) are used for numerical transformation of protein sequences. Of the predictors, the top ten achieve accuracies of >98% (the average value is 98.34%), and also the area under the ROC curve (AUC) values of >0.98. Compared with those of previous studies, the prediction accuracies are improved by about 3 to 4%.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Correlations among brain gray matter volumes, age, gender, and hemisphere in healthy individuals

To determine the relationship between age and gray matter structure and how interactions between gender and hemisphere impact this relationship, we examined correlations between global or regional gray matter volume and age, including interactions of gender and hemisphere, using a general linear model with voxel-based and region-of-interest analyses. Brain magnetic resonance images were collected from 1460 healthy individuals aged 20-69 years; the images were linearly normalized and segmented and restored to native space for analysis of global gray matter volume. Linearly normalized images were then non-linearly normalized and smoothed for analysis of regional gray matter volume. Analysis of global gray matter volume revealed a significant negative correlation between gray matter ratio (gray matter volume divided by intracranial volume) and age in both genders, and a significant interaction effect of age × gender on the gray matter ratio. In analyzing regional gray matter volume, the gray matter volume of all regions showed significant main effects of age, and most regions, with the exception of several including the inferior parietal lobule, showed a significant age × gender interaction. Additionally, the inferior temporal gyrus showed a significant age × gender × hemisphere interaction. No regional volumes showed significant age × hemisphere interactions. Our study may contribute to clarifying the mechanism(s) of normal brain aging in each brain region.     

Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells

Background

Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined.

Methodology/Principal Findings

Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin.

Conclusions/Significance

RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.     

Immunohistochemical localization of phosphatidylcholine in rat mandibular condylar surface and lower joint cavity by cryotechniques

The immunolocalization of phospholipids has not yet been clearly demonstrated in temporomandibular joints (TMJs). We have examined the distribution of one of phospholipids, phosphatidyl-choline (PC), in the rat mandibular condylar surface and lower joint cavity. Some fresh resected TMJs with their disks attached were immediately plunged into isopentane-propane cryogen (-193 degrees C). Cryostat sections were cut, mounted on NH3+-coated slides, and fixed with paraformaldehyde (PF). Cryosections were first immunostained with anti-mouse PC antibody (JE-1). Subsequently, they were labeled with immunogold particles following silver enhancing for light microscopic analyses. Some cryosections were subjected to double immunofluoresecence labeling with anti-fibronectin antibody or hyaluronic acid-binding protein in combination with the anti-PC antibody. As an immunocontrol, other cryosections were pretreated with phospholipase A2 before such immunofluorescence labeling. We have confirmed the presence of PC in the lower joint cavity of rat TMJs as well as on the mandibular condylar surface layer, which was colocalized with hyaluronic acid and fibronectin respectively. However, by treatment with phospholipase A2, such immunolabeling for PC was clearly decreased, showing that the PC is a component in the rat in vivo TMJ. These findings suggest that PC, hyaluronic acid and fibronectin may interact each other in the TMJ articular surface areas to play a functional role for lubrication in TMJ.     

Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3 A resolution (Rwork of 17.8% and Rfree of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (alpha2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle.     

Down-regulation of osteoprotegerin production in bone marrow macrophages by macrophage colony-stimulating factor

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) induce the differentiation of bone marrow macrophages (BMMs) into osteoclasts. To delineate mechanisms involved, the effect of M-CSF on the production of osteoprotegerin (OPG), decoy receptor of RANKL, in BMMs was investigated. Mouse bone marrow cells were cultured with M-CSF for 4 days and adherent cells formed were used as BMMs. BMMs were cultured with or without M-CSF, and analyzed for expression of OPG and receptor activator of NF-kappaB (RANK; receptor for RANKL) mRNAs by real-time polymerase chain reaction and secretion of OPG by enzyme-linked immunosorbent assay. BMMs expressed macrophage markers, CD115 (c-fms), Mac-1 and F4/80, and showed phagocytotic activity. In addition, BMMs expressed OPG mRNA and secreted OPG into medium. M-CSF inhibited both the OPG mRNA expression and the OPG secretion dose-dependently and reversibly. The expression of RANK mRNA was not significantly affected by M-CSF. The results showed that M-CSF suppresses the OPG production in BMMs, which may increase the sensitivity of BMMs to RANKL.     

A chimeric human-cat fusion protein blocks cat-induced allergy

Animal allergens are an important cause of asthma and allergic rhinitis. We designed and tested a chimeric human-cat fusion protein composed of a truncated human IgG Fcgamma1 and the major cat allergen Fel d1, as a proof of concept for a new approach to allergy immunotherapy. This Fcgamma-Fel d1 protein induced dose-dependent inhibition of Fel d1-driven IgE-mediated histamine release from cat-allergic donors' basophils and sensitized human cord blood-derived mast cells. Such inhibition was associated with altered Syk and ERK signaling. The Fcgamma-Fel d1 protein also blocked in vivo reactivity in FcepsilonRIalpha transgenic mice passively sensitized with human IgE antibody to cat and in Balb/c mice actively sensitized against Fel d1. The Fcgamma-Fel d1 protein alone did not induce mediator release. Chimeric human Fcgamma-allergen fusion proteins may provide a new therapeutic platform for the immune-based therapy of allergic disease.