Effect of heparin chain length on the interaction with tissue factor pathway inhibitor (TFPI)

Tissue factor pathway inhibitor (TFPI) is a heparin-binding protein involved in the extrinsic blood coagulation system. In order to elucidate the minimal size of heparin chain required for the interaction with TFPI, we prepared a series of heparin-derived oligosaccharides with tailored chain length ranged from disaccharide to eicosasaccharide after the successive treatments of heparin, including partial N-desulphation, deaminative cleavage with nitrous acid and gel-filtration. Affinity chromatography study of each oligosaccharide fraction using TFPI as the ligand indicated that increasing the degree of polymerisation causes increased affinity, and that a remarkable change in the affinity occurs between the decamers and dodecamers. Measurement of factor Xa inhibitory activity of TFPI in the presence of each oligosaccharide fraction indicated that the fractions shorter than dodecamers only slightly enhanced the TFPI activity for factor Xa inhibition, while the fractions larger than octadecamers had an effect comparable to full-length heparin. These were compatible to the results from the kinetic analyses of the interaction between TFPI and heparin-derived oligosaccharide with an evanescent wave-based biosensor system, IAsys, using a TFPI C-terminal peptide as the ligand.     

Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; comparison of gene expression between the blood-brain and -retinal barriers

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selectively expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3'-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR.     

Critical role of PICT-1, a tumor suppressor candidate, in phosphatidylinositol 3,4,5-trisphosphate signals and tumorigenic transformation

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulates diverse cellular functions by dephosphorylating the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). Recent study revealed that PICT-1/GLTSCR2 bound to and stabilized PTEN protein in cells, implicating its roles in PTEN-governed PIP(3) signals. In this study, we demonstrate that RNA interference-mediated knockdown of PICT-1 in HeLa cells down-regulated endogenous PTEN and resulted in the activation of PIP(3) downstream effectors, such as protein kinase B/Akt. Furthermore, the PICT-1 knockdown promoted HeLa cell proliferation; however the proliferation of PTEN-null cells was not altered by the PICT-1 knockdown, suggesting its dependency on PTEN status. In addition, apoptosis of HeLa cells induced by staurosporine or serum-depletion was alleviated by the PICT-1 knockdown in the similar PTEN-dependent manner. Most strikingly, the PICT-1 knockdown in HeLa and NIH3T3 cells promoted anchorage-independent growth, a hallmark of tumorigenic transformation. Furthermore, PICT-1 was aberrantly expressed in 18 (41%) of 44 human neuroblastoma specimens, and the PICT-1 loss was associated with reduced PTEN protein expression in spite of the existence of PTEN mRNA. Collectively, these results suggest that PICT-1 plays a role in PIP(3) signals through controlling PTEN protein stability and the impairment in the PICT-1-PTEN regulatory unit may become a causative factor in human tumor(s).     

Protection of glutathione S-transferase from bilirubin inhibition

Inhibition of the enzyme activity of glutathione S-transferase (GST) by a physiological concentration of bilirubin was studied using various substrates. When rat liver cytosol was used as an unfractionated GST, its GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene was decreased to one-half by bilirubin, while the activity toward 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, or 1,2-epoxy-(p-nitrophenoxy)propane and also the non-selenium dependent GSH-peroxidase activity toward cumene hydroperoxide (CHPx activity) were hardly affected under the same conditions. In contrast, bilirubin inhibited each of the purified GST isozymes and no remarkable difference in bilirubin inhibition was observed with any of the substrates tested. From the chromatographic analysis of the cytosol incubated with [3H]bilirubin, it was found that a major part of the added bilirubin binds to subunit 1 (Ya) of GST isozyme, leaving not only the conjugation activity derived from 3-4 type GST but also the CHPx activity of subunit 2 (Yc) quantitatively intact. The bilirubin inhibition of both the conjugation activity of GST 3-4 and the CHPx activity of GST 2-2 was prevented almost completely by addition of a 3-fold molar excess of GST 1-1. From these results, it was assumed that the enzyme activities of both 3-4 type GSTs and subunit 2 (Yc) were protected from the inhibitory action of bilirubin by the scavenger effect of subunit 1 (Ya).     

A new polypeptide toxin from the nematocyst venom of an Okinawan sea anemone Phyllodiscus semoni (Japanese name "unbachi-isoginchaku")

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD50, 50 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50, 80 ng/ml).     

Involvement of the Calcium-sensing Receptor in Human Taste Perception

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca²⁺, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.     

PET Analysis of Dopaminergic Neurodegeneration in Relation to Immobility in the MPTP-Treated Common Marmoset,a Model for Parkinson’s Disease

Background

Positron Emission Tomography (PET) measurement was applied to the brain of the common marmoset, a small primate species, treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The marmoset shows prominent Parkinson’s disease (PD) signs due to dopaminergic neural degeneration. Recently, the transgenic marmoset (TG) carrying human PD genes is developing. For phenotypic evaluations of TG, non-invasive PET measurement is considered to be substantially significant. As a reference control for TG, the brain of the MPTP-marmoset as an established and valid model was scanned by PET. Behavioral analysis was also performed by recording locomotion of the MPTP-marmoset, as an objective measure of PD signs.

Methodology/Principal Findings

Marmosets received several MPTP regimens (single MPTP regimen: 2 mg/kg, s.c., per day for 3 consecutive days) were used for PET measurement and behavioral observation. To measure immobility as a central PD sign, locomotion of marmosets in their individual living cages were recorded daily by infrared sensors. Daily locomotion counts decreased drastically after MPTP regimens and remained diminished for several months or more. PET scan of the brain, using [¹¹C]PE2I as a ligand of the dopamine (DA) transporter, was performed once several months after the last MPTP regimen. The mean binding potential (BP_ND) in the striatum (putamen and caudate) of the MPTP-marmoset group was significantly lower than that of the MPTP-free control group (n = 5 for each group). In the MPTP-marmosets, the decrease of BP_ND in the striatum closely correlated with the decrease in locomotion counts (r = 0.98 in putamen and 0.91 in caudate).

Conclusion/Significance

The present characterization of neural degeneration using non-invasive PET imaging and of behavioral manifestation in the MPTP marmoset mimics typical PD characteristics and can be useful in evaluating the phenotype of TG marmosets being developed.     

Changes in the properties and composition of zona pellucida of pigs during fertilization in vitro.

Several hundred fertilized pig eggs were prepared by an in vitro fertilization (IVF) technique in which follicular phase ovarian eggs were matured in vitro to metaphase II before incubation with capacitated epididymal spermatozoa for 12 h at 39 degrees C. Parthenogenetic eggs were also prepared by stimulation of the mature eggs with an electric pulse. The zonae were solubilized with 0.2% pronase/phosphate-buffered saline (PBS) or lactic acid/PBS. The time taken for solubilization was 30-40% shorter than for unfertilized eggs, indicating that zona hardening was induced during fertilization. At the same time, the sperm receptor activity of the zona was reduced. Electrophoretic analyses of zona glycoproteins from the ovarian, mature and fertilized eggs revealed that the amount of 90 kDa proteins decreased substantially during fertilization. This fraction could barely be detected in the zonae from parthenogenetic eggs. However, modification with a fluorescent probe showed that the general architecture of the zona remained unchanged during fertilization. These results suggest that the minor 90 kDa proteins are specifically degraded by the protease(s) released from the oocyte at fertilization, thereby leading to the block to polyspermy.     

AHK5 histidine kinase regulates root elongation through an ETR1-dependent abscisic acid and ethylene signaling pathway in Arabidopsis thaliana

The Arabidopsis thaliana genome encodes a small family of histidine (His) protein kinases, some of which have redundant functions as ethylene receptors, whereas others serve as cytokinin receptors. The most poorly characterized of these is authentic histidine kinase 5 (AHK5; also known as cytokinin-independent 2, CKI2). Here we characterize three independent ahk5 mutants, and show that they have a common phenotype. Our results suggest that AHK5 His-kinase acts as a negative regulator in the signaling pathway in which ethylene and ABA inhibit the root elongation through ETR1 (an ethylene receptor).     

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.