Isolation,purification and partial chemical characterization of a lethal factor from common indian toad (Bufo melanostictus,Schneider) skin extract

Indian toad (Bufo melanostictus, Schneider) skin extract (TSE) is pharmacologically potent and probably contains several bioactive compounds [Das et. al., Indian J Pharmacol, 28 (1996) 72]. A lethal factor was isolated and purified by neutral alumina column chromatography followed by HPLC. Spectroscopic (UV, IR, FAB-MASS) study indicated that the lethal factor (TSE-LF) was a 254 Da long chain compound with carbonyl, hydroxyl and ester as functional groups. LD50 of TSE-LF was found to be 3.5 mg/kg (iv). Biological study showed that TSE-LF possesses hypotensive, cardiotoxic, neurotoxic activity and produced death by apnoea in experimental animal. Cyproheptadine antagonised TSE-LF induced contraction of isolated smooth muscle indicating involvement of histamine/serotonin receptors. TSE-LF induced neurotoxic action on chick biventer cervices was mediated through Ca2+ ion.     

Isolation and sequence of a novel amphibian pancreatic chitinase

An approximately 60-kDa protein with chitinase activity was purified from the pancreas of the toad Bufo japonicus. Its specific activity was 4.5 times higher than that of a commercial bacterial chitinase in fragmenting crab shell chitin, and its optimal pH was approximately 6.0. A cDNA clone encoding a protein consisting of 488 amino acid residues, including part of the peptide sequence determined from the isolated protein, was obtained from a toad pancreas cDNA library. The deduced amino acid sequence indicated that the protein contained regions with high homology to those present in chitinases from different species, with the amino acid residues for the chitinase activity and the chitin-binding ability being completely conserved. We designate the protein as toad pancreatic chitinase (tPCase). Northern blot analysis revealed the mRNA of this enzyme to be expressed exclusively in the pancreas. Toad PCase is the first amphibian chitinase to be identified as well as the first pancreatic chitinase identified in a vertebrate.     

Immunochemical evidence for a transmembrane orientation of both the (Na+, K+)-ATPase subunits

Antibodies were raised against the large catalytic subunit (apparent Mr 96000) and the glycoprotein (apparent Mr 60000) of the sodium- and potassium-dependent adenosine triphosphatase [(Na+, K+)-ATPase] from Bufo marinus. The specificity of each antiserum was assessed by two-dimensional immunoelectrophoresis using toad kidney microsomes or the purified holoenzyme as a source of antigen and by indirect immunoprecipitation of detergent-solubilized (Na+, K+)-ATPase subunits from radioiodinated or biosynthetically labeled kidney holoenzyme, microsomes, or postnuclear supernatant. The anticatalytic subunit serum reacted exclusively with a 96000-dalton protein. The antiserum to the glycoprotein was rendered specific to this subunit by absorption with purified catalytic subunit. The two antisera were agglutinating and lytic in the presence of complement when toad erythrocytes were used as targets, indicating that antigenic determinants of both subunits were exposed on the cell surface. The specific reactivities with surface-exposed antigenic determinants of both subunits could be absorbed with toad red blood cells. Such absorbed antisera still reacted with detergent-treated or untreated kidney microsomes, revealing the presence of cytoplasmic and/or intramembranous antigenic sites. Our immunochemical data demonstrate that the glycoprotein subunit of (Na+, K+)-ATPase spans the lipid bilayer and confirm the transmembrane orientation of the catalytic subunit postulated from functional studies.     

Free fatty acids, diacyl- and triacylglycerols and total phospholipids in vertebrate retina: comparison with brain, choroid and plasma

Abstract— A comparative study of the concentration and fatty acid distribution in diacyl- and triacylgly-cerols. free fatty acids and total phospholipids from rabbit, cattle and toad retina is presented. With respect to the toad, a comparison is made with brain, choroid and plasma lipids. Marked differences in diacylglycerol composition and levels between mammalian and toad retina are found: in the mammal arachidonate predominates (25 per cent), in the toad docosahexaenoate is the main fatty acid (42 per cent). The total phospholipid composition parallels that of the diacylglycerols only in the toad, whereas in the mammalian retina the phospholipids are richer in docosahexaenoate than are the diacylglycerols. It is suggested that there is a relationship between diacylglycerols and phosphoglyceride metabolism in the toad; in the retinas of other species the diacylglycerols may be related to specific phosphatides. In the three species, triacylglycerols show high levels of unsaturation; however, marked differences are found in the distribution of polyunsaturated acyl groups: in the cattle and toad retina docosahexaenoate predominates. whereas in the rabbit a higher proportion of 22:4 is found. Retina free fatty acid pools also show different features in the three species: the cattle retina contains the highest proportion of free 20:4 and 22:6. The triacylglycerol concentration is much higher in the toad choroid than in the retina, although the fatty acid compositions are similar. A possible relationship between these choroid lipids and those of the retina is suggested.     

Direct effect of complement factor C5a on the contractile state of isolated smooth muscle cells

The complement-(C) derived factor C5a has long been recognized as a potent contractile agonist in smooth muscle (1,2); however, controversy remains as to whether the effects of this anaphylatoxin are direct or secondary to the release of histamine (3) and/or other mediators (4-8) from nonmuscle cells within the tissue. To resolve this controversy, we have assessed the contractile effects of purified human C5a and C5a des Arg in a homogeneous preparation of enzymatically dispersed smooth muscle cells derived from the stomach of the toad, Bufo marinus. This preparation, which is insensitive to histamine at concentrations as high as 10(-4) M, responds normally to a variety of electrical (9), mechanical (10), and pharmacologic (11, 12) stimuli. These smooth muscle cells also respond to purified human anaphylatoxin; exposure to the cells to purified human C5a or C5a des Arg produce contractions of the smooth muscle cells that are accompanied by increased Ca2+ influx. The contractile response was unaffected by antagonists to histamine or acetylcholine but was reduced by 30% by pretreatment with the leukotriene antagonist FPL55712. A direct contractile effect of C5a on amphibian smooth muscle cells is suggested.     

Studies on the erythropoietic effect of plasma from anemic toads both with and without testis.

Injection of plasma from experimentally induced anemic toad with intact testis increases erythropoiesis in starved toads evidenced by the increase of red blood cell, hemoglobin and hematocrit, whereas the plasma of castrated and phenylhydrazine-HCl treated anemic toad failed to do so. It can be suggested that the erythropoiesis stimulating factor (ESF) is produced from the testis of toad and the production of this factor (ESF) was found to increase during anemia in an attempt to correct the anemic condition.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

从蟾酥中纯化一种内源性钠泵抑制因子

内源性Ｎａ+ Ｋ+ ＡＴＰ酶 (钠泵 )抑制因子是新发现的一种由肾上腺或下丘脑分泌并贮存的具有生理和病理意义的一种生物活性物质 ,在高血压的发生和发展中可能是重要的因素之一 .内源性钠泵抑制因子与高血压发病关系的研究近年来报道较多 ,已成为该领域国际研究的热点 .Ｈａｍｌｙｎ和Ｈａｕｐｅｒｔ研究组从人的血液及牛的下丘脑中纯化出一种与哇巴因相似的明显抑制钠泵活性的哇巴因样物质(ｏｕａｂａｉｎ ｌｉｋｅｃｏｍｐｏｕｎｄ ,ＯＬＣ) [1 2 ] .Ｓｃｈｏｎｅｒ研究组由牛肾上腺中纯化得到一种分子量为 6 0 0 (ＵＶ 2 5 0ｎｍ)的前…     

Synthesis and characterization of methylbromoamiloride,a potential biochemical probe of epithelial Na+ channels

Summary We report the synthesis of a radioactive, methylated analog of bromoamiloride which inhibits the amiloride-sensitive, epithelial Na⁺ channel reversibly and with high affinity. This synthesis was achieved by methylation of a nitrogen in the acylguanidinium moiety with tritiated methyliodide of high specific activity. This methylated bromoamiloride molecule (CH₃BrA) was purified by both thin layer and high performance liquid chromatography. Proton nuclear magnetic resonance and mass spectroscopy techniques were used to determine the structure of this analog. This compound inhibited both short-circuit current ofin vitro frog skin and²²Na⁺ influx into apical plasma membrane vesicles made from cultured toad kidney cells (line A6) with the same or lower apparent inhibitory dissociation constant as bromoamiloride. Irradiation with ultraviolet light rendered this inhibition irreversible in both A6 vesicles and frog skin. Preparation of radioactive CH₃BrA yielded specific activities in excess of 1 Ci/mmol. We suggest that this compound will be useful in the isolation and purification of this ubiquitous Na⁺ channel.     

Comparison of xanthine: NAD+ oxidoreductase from liver of toad Bufo viridis and other vertebrates

1. Xanthine oxidoreductase was isolated from toad Bufo viridis (a mainly ureotelic amphibian species) and partially purified. The enzyme occurred as a stable xanthine: NAD+ oxidoreductase (EC 1.1.1.204), unconvertible to the oxidase form. 2. Some properties of the enzyme resembled those of xanthine oxidoreductase from an ammonotelic fish, Cyprinus carpio, and the ureotelic rat, but in other aspects it was similar to this enzyme from an uricotelic snake, Natrix natrix. 3. Inhibition of the toad enzyme by NADH at high non-physiological concentrations rules out a modulation of its oxypurine-hydroxylating activity by in vivo changes in the NADH/NAD+ ratio. Therefore, toad xanthine oxidoreductase plays no regulatory role in the purine nucleotide metabolism.     

Isolation and identification of the 3-hydroxy-5-hydroxymethyl-pyridinium compound as a novel advanced glycation end product on glyceraldehyde-related Maillard reaction

Glyceraldehyde (200 mM) and N alpha-acetyllysine (100 mM) were incubated in 0.2 M sodium phosphate buffer (pH 7.4) at 37 degrees C for a week. A major compound, glyceraldehyde-related Maillard reaction product, was purified from the reaction mixture using reverse phase (ODS)-HPLC. It was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named as GLAP (Glyceraldehyde derived Pyridinium compound), using NMR and MS analyses. It was suggested that GLAP as a novel advanced glycation end product (AGE) is one of the key compounds in the glyceraldehyde-related Maillard reaction.     

Proteins of rod outer segments of toad retina: binding with calmodulin and with GTP

Proteins of purified rod outer segments from toad retina were analysed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The binding of proteins with calmodulin and with guanosine triphosphate was studied by electroblotting the proteins resolved by electrophoresis onto nitrocellulose sheets and by incubating the blots with labelled ligands. The results indicate that rod outer segments from toad retina contain nine proteins which bind to calmodulin and one protein, different from transducin, that binds to guanosine triphosphate.     

The role of mitochondria-rich cells in the chloride current conductance across toad skin

In early studies of salt transport across frog and toad skin, it was assumed that chloride movement is extracellular. However, later studies suggested that chloride movement is largely transcellular. Chloride transport across toad skin is greatly diminished in skins of salt-acclimated toads (Bufo viridis) and was correlated with the number of mitochondria-rich (m.r.) cells in the epithelium. The activated chloride conductance could be recovered upon in vitro incubation with theophylline. It was found that the short-circuit current (Isc) and the chloride conductance (Gcl) in toad skin could be separated experimentally by selective use of synthetic oxytocin (Syntocinon) or theophylline, and by substituting impermeable anions for chloride. With the use of the vibrating probe we demonstrated directly that chloride-dependent peak currents are localized only over m.r. cells, under hyperpolarized (V = -100 mV) conditions. It is concluded that the m.r. cells form the principal site for passive chloride movement across amphibian skin. This cellular pathway is regulated through a cyclic AMP-mediated process. It is suggested that the spatial separation of the sodium and chloride channels is essential to maintain the granulosum cells which are engaged in sodium transport hyperpolarized, and thus providing the driving force for the sodium entry into the cells.     

Free radicals in mercury-resistant bacteria indicate a novel metabolic pathway

A mercury resistant-soil bacterium P.10.15, identified as a close relative of Pseudomonas veronii, was shown to accumulate a specific compound in the stationary phase of growth. This compound is converted to a long-lived free radical under oxidizing conditions, as registered by its EPR signal at room temperature. The compound was purified by ion-exchange and gel-filtration chromatography and identified by mass spectroscopy, 2D NMR, and EPR as a trisaccharide beta-D-GlcpNOH,CH3-(1-->6)-alpha-D-Glcp-(1-->1)-alpha-D-Glcp, or, in other words, as 6-O-(2-deoxy-2-[N-methyl]hydroxylamino-beta-D- glucopyranosyl)-alpha-alpha-trehalose, previously discovered in Micrococcus luteus (lysodeikticus) and named lysodektose. The compound is suggested to be a novel intermediate of a previously unknown basic metabolic pathway of trehalose transformation in bacteria, a potential target for antibacterial drug development.     

Aldosterone stimulates Na+ transport without affecting citrate synthase activity in cultured cells

Aldosterone increases citrate synthase activity in toad urinary bladder and mammalian kidney. It has been suggested that this action is important to aldosterone stimulation of Na+ transport, and it has been used as a marker of those epithelia which are stimulated by aldosterone. We describe three continuous lines of cultured cells derived from toad urinary bladder and toad kidney in which aldosterone increases active Na+ transport but does not increase the activity of citrate synthase. Therefore, in cultured cells at least, citrate synthase is not a critical enzyme for, or a suitable marker of, aldosterone stimulation of Na+ transport.     

Characterization of a major secretory protein in the cane toad (Bufo marinus) choroid plexus as an amphibian lipocalin-type prostaglandin D synthase

Here we report the enzymatic and ligand-binding properties of a major secretory protein in the choroid plexus of cane toad, Bufo marinus, whose protein is homologous with lipocalin-type prostaglandin (PG) D synthase (L-PGDS) and is recombinantly expressed in Xenopus A6 cells and Escherichia coli. The toad protein bound all-trans retinal, bile pigment, and thyroid hormones with high affinities (K(d)=0.17 to 2.00 microM). The toad protein also catalysed the L-PGDS activity, which was accelerated in the presence of GSH or DTT, similar to the mammalian enzyme. The K(m) value for PGH(2) (17 microM) of the toad protein was almost the same as that of rat L-PGDS (14 microM), whereas the turnover number (6 min(-1)) was approximately 28 fold lower than that of rat L-PGDS. Site-directed mutagenesis based on a modeled structure of the toad protein revealed that Cys(59) and Thr(61) residues were crucial for the PGDS activity. The quadruple Gly(39)Ser/Ala(75)Ser/Ser(140)Thr/Phe(142)Tyr mutant of the toad protein, resembling mouse L-PGDS, showed a 1.6 fold increase in the turnover number and a shift in the optimum pH for the PGDS activity from 9.0 to 8.5. Our results suggest that the toad protein is a prototype of L-PGDS with a highly functional ligand-binding pocket and yet with a primitive catalytic pocket.     

Purification and Characterization of Nk‐3FTx: A Three Finger Toxin from the Venom of North East Indian Monocled Cobra

Snake venom three finger toxins (3FTxs) are a non‐enzymatic family of venom proteins abundantly found in elapids. We have purified a 7579.5 ± 0.591 Da 3FTx named as Nk‐3FTx from the venom of Naja kaouthia of North East India origin. The primary structure was determined by a combination of N‐terminal sequencing and electrospray ionization  liquid chromatography‐mass spectrometry/mass spectrometry. Biochemical and biological characterization reveal that it is nontoxic to human cell lines and exhibit mild anticoagulant activity when tested on citrated human plasma. Nk‐3FTx was found to affect the compound action potential (CAP) and nerve conduction velocity of isolated toad sciatic nerve. This is the first report of a non‐conventional 3FTx from Naja kaouthia venom that reduces CAP for its neurotoxic effect. Further studies can be carried out to understand the mechanism of action and to explore its potential therapeutic application.     

黄河上游环境污染对花背蟾蜍抗氧化酶活性及丙二醛含量的影响

选择黄河上游污染程度较严重的兰州地区和相对无污染的刘家峡地区作为研究样点,比较分析了两地花背蟾蜍（Bufo raddei）肝脏和肾脏中抗氧化酶SOD、CAT和GPx活性及丙二醛（MDA）含量。与刘家峡地区相比,兰州地区花背蟾蜍肝脏的SOD活性升高,CAT和GPx活性极显著降低,肾脏GPx活性显著增高,肝脏和肾脏MDA含量明显高于刘家峡。结果表明,黄河上游环境污染使得花背蟾蜍体内氧化胁迫加重,导致其组织脂质过氧化程度升高。     

Electrotonic transmission between primary afferent fibers and the motor neurons of the isolated spinal cord of various representative amphibians

Studies have been made of monosynaptic excitatory postsynaptic potentials elicited by stimulation of the posterior root in motoneurones of the isolated spinal cord of the frog Rana ridibunda, toad Bufo bufo, and clawed toad Xenopus laevis. In all the amphibians studied, the early component of monosynaptic EPSP was not blocked in Ca-free medium containing 2 mM Mn2+. It is suggested that electrical coupling in anuran amphibians reflects certain stage of evolution of the synaptic transmission in vertebrates.