Involvement of protein phsophatase-1 in cytoskeletal organization of cultured endothelial cells

The phosphorylation and dephosphorylation of cytoskeletal proteins regulate the shape of eukaryotic cells. To elucidate the role of serine/threonine protein phosphatases (PP) in this process, we studied the effect of calyculin A (CLA), a potent and specific inhibitor of protein phosphatases 1 (PP-1) and 2A (PP-2A) on the cytoskeletal structure of cultured human umbilical vien endothelial cells (HUVECs). The addition of CLA (5 min) caused marked alterations in cell morphology, such as cell constriction and bleb formation. Microtubules and F-actin were reorganized, becoming markedly condensed around the nucleus. Although the fluorescence intensity of phosphoamino acids was not significantly different to immunocytochemistry between cells with and without CLA, polypeptides of 135, 140, 158, and 175 kDa were specifically phosphorylated on serine and/or threonine residues. There was no significant effect on tyrosine residues. The effects of CLA on cytoskeletal changes and protein phosphorylation were almost completely inhibited by the non-selective kinase inhibitor, K-252a. The effect of CLA on cell morphology was at least 100 times more potent than that of okadaic acid, consistent with the inhibitory potency against PP-1. The catalytic subunit of PP-1 was also identified in HUVECs by Western blotting with its monoclonal antibody. These results suggest that PP-1 is closely involved in sustaining the normal structure of the cytoskeleton. © 1995 Wiley-Liss, Inc.     

Life history of amphibians and gravity.

Anurans hold a unique position in vertebrate phylogeny, as they made the major transition from water to land. Through evolution they have acquired fundamental mechanisms to adapt to terrestrial gravity. Such mechanisms are now shared among other terrestrial vertebrates derived from ancestral amphibians. Space research, using amphibians as a model animal, is significant based on the following aspects: (1) Anuran amphibians show drastic changes in their living niche during their metamorphosis. Environments for tadpoles and for terrestrial life of frogs are quite different in terms of gravity and its associated factors. (2) Certain tadpoles, such as Rhacophorus viridis amamiensis, have a transparent abdominal wall. Thus visceral organs and their motion can be observed in these animals in non-invasive manner through their transparent abdominal skin. This feature enables biologists to evaluate the physiological state of these amphibians and study the autonomic control of visceral organs. It is also feasible for space biologists to examine how such autonomic regulation could be altered by microgravity and exposure to the space environment.     

Purification and Characterization of Streptomyces Sialidases

Some strains of Streptomyces produce sialidases. Two sialidases were purified over 1,000-fold from a culture filtrate of two Streptomyces species. They had the same properties in molecular weight, behavior to ions and other reagents, and substrate specificity. They showed very small differences in kinetic properties, pH optima, and heat stability. These Streptomyces sialidases differed markedly from Clostridium perfringens sialidase in molecular weight, p-chloromercuribenzoate sensitivity, and substrate specificity. Approximate molecular weights of the sialidases from Streptomyces and C. perfringens were 32,000 and 57,000, respectively. p-Chloromercuribenzoate (10(-3) M) caused complete inhibition of C. perfringens sialidase but not of Streptomyces sialidases.     

Schistosoma japonicum: Immunopathology of nephritis in Macaca fascicularis

Macaca monkeys experimentally infected with Schistosoma japonicum developed a chronic progressive kidney lesion characterized by an increase of mesangial matrix, local glomerular hypercellularity, and local thickening of glomerular basement membrane. Immunofluorescence studies revealed the localization of IgG, IgM, IgA, and IgE immunoglobulins mostly in the mesangial area of the glomeruli accompanied by the deposition of Schistosoma antigens. By electron microscopy, in addition to the local thickening of the glomerular basement membrane, dense homogeneous deposits and those with moth-eaten appearance were detected in the mesangial matrix. These findings suggest that worms in the bloodstream continuously release antigenic materials that stimulate host's antibody response belonging to various immunoglobulin classes including IgE. The produced antibodies and antigens would form immune complexes that deposited in the glomeruli. The increased vascular permeability caused by antigen-IgE antibody interaction may play an important role in the deposition of immune complexes and in the rapid development of kidney injury.     

Variation in Natural Abundance of 15N among Plant Parts and in 15N/14N Fractionation during N2 Fixation in the Legume-Rhizobia Symbiotic System

Sixteen legumes were grown in N-free media so that N was suppliedentirely by symbiotic N₂ fixation. The plant tissues were analyzedfor natural ¹⁵N abundance (expressed as ¹⁵N per mil relativeto air N₂) with a ratio mass spectrometer. The nodules of desmodium,centro, siratro, soybean and winged bean showed high enrichmentin ¹⁵N (+9), while red clover showed slight enrichment (+2).The nodules of 9 other forage legumes (Townsville stylo, whiteclover, alsike clover, common vetch, Chinese milk vetch, senna,alfalfa, ladino clover, and hairy vetch) showed little enrichmentin ¹⁵N. In all the legumes investigated, particularly in the ureide-transportingplants such as desmodium, centro, siratro, soybean, winged beanand field bean, the ¹⁵N value of the shoots was negative (–3.2).The ¹⁵N value of the shoots in winged bean and field bean variedby about 1 depending on the Rhizobium strains used. The isotopicmass balance of 13 legumes indicated that isotopic fractionationoccurs during N₂ fixation by the legume-rhizobia symbiosis witha preference for ¹⁴N over ¹⁵N, resulting in a ¹⁵N value of –0.2to –2 in the whole plant. The results indicate that ¹⁵N/¹⁴N isotopic discrimination witha preference for the lighter atom may occur in both N₂ fixationand export of fixed N from nodules. ¹Present address: Department of Soils and Fertilizers, NationalAgriculture Research Center, Kannondai, Tsukuba, Ibaraki 305,Japan. (Received October 8, 1985; Accepted April 7, 1986)     

Effects of zotepine, chlorpromazine and haloperidol on D-1, D-2, D-3 and D-4 subtypes of dopamine receptors in rat striatal and bovine caudate nucleus membranes

Inhibitory effects of zotepine (Zot) on D-1, D-2, D-3 and D-4 subtypes of dopamine (DA) receptors were investigated in crude synaptic membranes of rat striatum and bovine caudate nucleus and compared to those of chlorpromazine (CPZ) and haloperidol (HAL). From the IC₅₀-values of Zot, CPZ and HAL, the K-values of each drug are estimated as follows: 34.4, 152 and 244 nM (D-1, ³H-labeled cis-flupenthixol binding (1.0 nM) to rat membranes); 37.4, 7.1 and 2.4 nM (D-2, [³H]spiperone (Spi) binding (0.5 nM) to rat membranes in the presence of 0.1 μM ketanserin); 73.1, 15.2 and 22.4 nM (D-3, ³H-labeled N-propylapomorphine (NPA) binding (0.29 nM) to bovine membranes in the presence of 0.1 μM Spi); 9.5, 65.3 and 3.1 nM (D-4, [³H]NPA binding (0.29 nM) bovine membranes in the presence of 25 nM DA), respectively. Zot binds with higher affinity to D-4 but lower affinity to D-3 than to other subtypes. It is also presumed that Zot binds to D-1 with high affinity and D-2 and D-3 with low affinity compared to CPZ and HAL.     

Identification of 5-Hydroxy Eicosatetraenoic Acid in Cerebrospinal Fluid After Subarachnoid Hemorrhage

Abstract: To detect and identify lipid peroxides in the CFS following subarachnoid hemorrhage (SAH), CSF samples were obtained sequentially from 10 patients who developed typical vasospasm and were analyzed by HPLC and gas chromatography-mass spectrometry. One of the peaks appearing on the 7th day after SAH was identified as 5-hydroxy eicosatetraenoic acid. On HPLC, an identical peak was detected in samples from other SAH patients. The results gave unequivocal evidence that peroxides of arachidonic acid are present in the CSF following SAH, and a correlation between them and the occurrence of vasospasm seemed likely. The hypothesis that lipid peroxides are involved in the genesis of vasospasm deserves further investigation.     

Allelic polymorphism of murine IgE controlled by the seventh immunoglobulin heavy chain allotype locus

Two alloantisera against hybridoma-derived IgE detected allotypic determinants expressed on the murine s chain. An antiserum raised in BALB/c mice against monoclonal IgE of C57BL/6 origin reacted exclusively with IgE of strains having Igh-1^b (IgG_2a) allotype. The second antiserum, C57BL/6 anti-BALB/c monoclonal IgE, reacted with IgE of strains having Igh-1^a, Igh-1^d, Igh-1^e and Igh-1^j allotypes. The genetic studies of (BALB/c x C57BL/6)F₁ and backcross F₂ animals indicated that the locus controlling the IgE allotype is linked to the Igh-1 locus. This was further confirmed by the possession of respective IgE allotypes by Igh-C congenic mice, BALB/c and BAB-14, C3H.SW/Hz and CWB/Hz. Thus, the allotype detected on the chain is controlled by the seventh murine immunoglobulin allotype locus, and should be designated as the Igh-7 allotype.Abbreviations used in this paper PCA passive cutaneous anaphylaxis - RID radioimmunodiffusion - i.p. intraperitoneally - EA egg albumin - Igh-C immunoglobulin heavy chain constant region locus - DNP 2,4-dinitrophenyl - PBS phosphate-buffered saline - NMS normal mouse serum - KLH keyhole limpet hemocyanin Visiting investigator supported by the Scientific and Humanistic Development Council from the Central University of Venezuela, currently at the following address: Consejo de Desarrollo Cientifico y Universidad Central de Venezuela, Av. Principal Urb. La Floresta Ota., Silenia Caracas, Venezuela.     

Characterization of the Carbohydrate Chain on the Substance P Receptor in the Rat Brain Cortex: Effect of Lectins on [3H]Substance P Binding

The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of alpha-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system.     

Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.