PMab-219: A monoclonal antibody for the immunohistochemical analysis of horse podoplanin

Monoclonal antibodies (mAbs) against human, mouse, rat, rabbit, dog, cat, and bovine podoplanin (PDPN), a lymphatic endothelial cell marker, have been established in our previous studies. However, mAbs against horse PDPN (horPDPN), which are useful for immunohistochemical analysis, remain to be developed. In the present study, mice were immunized with horPDPN-overexpressing Chinese hamster ovary (CHO)-K1 cells (CHO/horPDPN), and hybridomas producing mAbs against horPDPN were screened using flow cytometry. One of the mAbs, PMab-219 (IgG_2a, kappa), specifically detected CHO/horPDPN cells via flow cytometry and recognized horPDPN protein using Western blotting. Furthermore, PMab-219 strongly stained CHO/horPDPN via immunohistochemistry. These findings suggest that PMab-219 is useful for investigating the function of horPDPN.     

A Novel Highly Potent Autotaxin/ENPP2 Inhibitor Produces Prolonged Decreases in Plasma Lysophosphatidic Acid Formation In Vivo and Regulates Urethral Tension

Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted enzyme that has lysophospholipase D activity, which converts lysophosphatidylcholine to bioactive lysophosphatidic acid. Lysophosphatidic acid activates at least six G-protein coupled recpetors, which promote cell proliferation, survival, migration and muscle contraction. These physiological effects become dysfunctional in the pathology of cancer, fibrosis, and pain. To date, several autotaxin/ENPP2 inhibitors have been reported; however, none were able to completely and continuously inhibit autotaxin/ENPP2 in vivo. In this study, we report the discovery of a highly potent autotaxin/ENPP2 inhibitor, ONO-8430506, which decreased plasma lysophosphatidic acid formation.The IC₅₀ values of ONO-8540506 for lysophospholipase D activity were 6.4–19 nM for recombinant autotaxin/ENPP2 proteins and 4.7–11.6 nM for plasma from various animal species. Plasma lysophosphatidic acid formation during 1-h incubation was almost completely inhibited by the addition of >300 nM of the compound to human plasma. In addition, when administered orally to rats at a dose of 30 mg/kg, the compound demonstrated good pharmacokinetics in rats and persistently inhibited plasma lysophosphatidic acid formation even at 24 h after administration.Smooth muscle contraction is a known to be promoted by lysophosphatidic acid. In this study, we showed that dosing rats with ONO-8430506 decreased intraurethral pressure accompanied by urethral relaxation. These findings demonstrate the potential of this autotaxin/ENPP2 inhibitor for the treatment of various diseases caused by lysophosphatidic acid, including urethral obstructive disease such as benign prostatic hyperplasia.     

Performance of hydrophobic chromatography in purification of alpha-amylase

Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors.     

Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Acquired Thermotolerance and Temperature-Induced Protein Accumulation in the Extremely Thermophilic Bacterium Rhodothermus obamensis

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95°C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.     

Immunoperoxidase Staining for Estrogen and Progesterone Receptors in Archival Formalin Fixed, Paraffin Embedded Breast Carcinomas after Microwave Antigen Retrieval

Immunoperoxidase staining was performed for estrogen and progesterone receptors in 93 cases of primary breast carcinoma. Breast tumor samples were fixed in formalin and embedded in paraffin. Antigen retrieval was performed by microwave heating in citrate buffer, pH 6.0, using precisely defined and reproducible conditions. The cases studied included material from the current year and from paraffin blocks retrieved from archival storage dating back to 1981. In all cases, estrogen and progesterone receptor values determined by biochemical assay were available for comparison with the immunohistochemical results. We found 94% agreement of results between the two methods.     

Exposure of Leaves of Cucumis sativus L. to Low Temperatures in the Light Causes Uncoupling of Thylakoids II. Non-Destructive Measurements with Intact Leaves

To examine the effects of chilling of leaves of cucumber (Cucumissativus L.) in moderate light on the coupling state of thylakoidsin situ, changes in fluorescence, changes in light scatteringand flash-induced changes in absorbance at 518 nm were examinedin intact leaves. After chilling of leaves at 5?C in the lightfor 5 h, the non-photochemical quenching of fluorescence, ameasure of energisation of thylakoids, was largely suppressed.The treatment also caused a suppression of light-induced changesin the light scattering by leaves, which depends on the formationof a pH gradient across thylakoid membranes. When thylakoidswere prepared by very gentle methods from the leaves chilledin the light, through a step of preparation of intact chloro-plasts,the transport of electrons from H₂O to ferricyanide was uncoupled,being insensitive to an uncoupler, methylamine. These data provide consistent evidence that the thylakoids areuncoupled in situ by the chilling of leaves in the light and,as a consequence of the uncoupling, the energisation of themembranes is suppressed. However, the decay of the flash-inducedchange in absorbance at 518 nm in leaves was not markedly acceleratedby the treatment. The thylakoids isolated from leaves chilledin the light, which were in the uncoupled state, also did notshow a rapid decay, unless an efficient uncoupler such as gramicidinwas added. These results suggest that even a considerable uncouplingof thylakoids, brought about by chilling of leaves in the light,is not sufficient to cause a marked acceleration of the decayof the flash-induced change in absorbance at 518 nm. Therefore,analysis at 518 nm is not always a sensitive method for assessingthe coupling state of thylakoids. (Received July 1, 1991; Accepted October 4, 1991)     

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²⁺

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.     

Selection of High Ubiquinone 10-Producing Strain of Tobacco Cultured Cells by Cell Cloning Technique

A novel enzyme, which was named N^α-benzyloxycarbonyl amino acid urethane hydrolase, was purified from a cell-free extract of Streptococcus faecalis R ATCC 8043, using N^α-benzyloxycarbonyl glycine as substrate. The enzyme was purified 1300-fold with an activity yield of 8%. The purified enzyme was homogeneous by disc electrophoresis. The molecular weight of the native enzyme is about 220,000 by gel filtration, and a molecular weight of 32,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate. The isoelectric point was 4.48. The enzyme was inhibited by p-chloromercuribenzoate. The presence of divalent cations (i.e., Co²⁺ or Zn²⁺) is essential for its activity.