Avian pox infection in a free-living crested serpent eagle (Spilornis cheela) in southern Taiwan.

Avian pox viruses (APVs) have been reported to cause infection in diverse avian species worldwide. Herein we report the first case of APV infection in a free-living bird, a subadult crested serpent eagle (Spilornis cheela), in Taiwan. In addition to the typical wart-like lesions distributed on the cere, eyelid, and face, there were also yellowish nodules below the tongue and on the hard palate. Phylogenetic analysis of the 4b core protein gene showed that the APV is very close to that found in white-tailed sea eagle (Haliaeetus albicilla) in Japan recently. Because both cases are located on the same major flyway for migratory birds, the impact of this virus with regard to the wild and migratory raptor species along the East Asian-Australasian Flyway and West Pacific Flyway requires immediate investigation.

Crocalbin: a new calcium-binding protein that is also a binding protein for crotoxin, a neurotoxic phospholipase A2.

Utilizing Marathon-ready cDNA library and a gene-specific primer corresponding to a partial amino acid sequence determined previously, the complete nucleotide sequence for the cDNA of crocalbin, which binds crotoxin (a phospholipase A2) and Ca2+, was obtained by polymerase chain reaction. The open reading frame of the cDNA encodes a novel polypeptide of 315 amino acid residues, including a signal sequence of 19 residues. This protein contains six potential Ca(2+)-binding domains, one N-glycosylation site, and a large amount of acidic amino acid residues. The ability to bind Ca2+ has been ascertained by calcium overlay experiment. Evidenced by sequence similarity in addition, it is concluded that crocalbin is a new member of the reticulocalbin family of calcium-binding proteins.

Purification and partial amino acid sequence of a novel protein of the reticulocalbin family.

Binding proteins in neuronal membranes for a phospholipase A2 with presynaptic neurotoxicity have been purified. Three polypeptides of 87, 65, and 50 K Da were obtained from the synaptic membrane fraction of guinea pig brain utilizing an immobilized crotoxin (a phospholipase A2) column. For large scale purification, porcine brain was used instead, and two polypeptides of 50 and 18 K Da were found. The 65 and 18 K polypeptides may represent hitherto unidentified components of the crotoxin-binding proteins. Partial N-terminal amino acid sequence and a partial sequence for an internal peptide fragment have been determined for the 50 K polypeptide. Search of protein data bank reveals that this polypeptide or protein is a novel member of the reticulocalbin family of calcium-binding proteins.

Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A2 with neurotoxicity.

Crotoxin and taipoxin are both neurotoxic phospholipases A2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca(2+)-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the Na(+)-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Conversion of bovine pancreatic phospholipase A2 at a single site into a competitor of neurotoxic phospholipases A2 by site-directed mutagenesis.

A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A2 variants with neurotoxicity, including crotoxin, by chemical cross-linking experiments (Yen, C.-H., and Tzeng, M.-C. (1991) Biochemistry 30, 11473-11477). The binding of crotoxin to this receptor protein was completely suppressed by sufficient F22Y, a mutated bovine pancreatic phospholipase A2 generated by site-directed mutagenesis of Phe22 of the wild-type enzyme to Tyr. The IC50 of this inhibition was estimated to be 1 microM. In sharp contrast, the wild-type enzyme gave no effect even at 50 microM. This mutation resulted in only minor and localized structural perturbations with little effect on enzymatic activity. Other phospholipase A2 molecules capable of competing with crotoxin for this binding invariably have Tyr at this position. It was concluded that this Tyr residue is an important determinant for the binding of a number of phospholipase A2 variants to the 45-kDa receptor.

Primary structure of an inactive mutant of phospholipase A2 in the venom of Bungarus fasciatus (banded krait).

From the acidic components of Bungarus fasciatus venom, a very small amount (0.16%) of a novel phospholipase A2 was obtained. Both neurotoxicity and enzyme activity were found to be lacking. Amino acid sequence study showed that it has a normal backbone of group I snake venom phospholipase A2 with 118 amino acid residues. The lack of enzyme activity was attributed to its mutation of the indispensable Asp residue to an Ala residue, i.e., the usual His-Asp47 turned out to be His-Ala47. This is the eighth isoform of phospholipase A2 found from the venom of Bungarus fasciatus. Examination of structural homology with three other isoforms revealed 66% similarity at most.

[Identification and typing of Pseudomonas pickettii during an episode of nosocomial outbreak].

From January to April 1989, Pseudomonas pickettii was isolated from clinical specimens of 24 hospitalized patients at the National Taiwan University Hospital in Taipei. The source of the organism was the 0.9% NaCl solution prepared by the hospital pharmacy. A total of 39 isolates of P. pickettii were collected, including 28 from clinical specimens and 11 from 0.9% saline and distilled water during the outbreak. These microorganisms were studied by using four methods, namely, conventional biochemical method, Vitek Auto-Microbic System (Vitek AMS), gas-liquid chromatographic analysis of cellular fatty acids composition and determination of the minimum inhibitory concentrations of 10 different antimicrobial agents. By conventional biochemical method, 16 isolates were typed as biovar 1 and 23 strains were biovar 3. Strains of both biovars were recovered from clinical specimens and 0.9% saline. Vitek AMS was able to identify P. pickettii correctly, but the result of biotyping was not satisfactory. Analysis of cellular fatty acids could rapidly identify P. pickettii to the species level, but could not distinguish the different biovars. By determination of the MICs, the antibiogram could be classified into 9 patterns. Of 16 isolates of P. pickettii biovar 1, 7 (44%) belonged to pattern I, and 9 (56%) pattern II. Strains of both patterns were found in cultures of clinical specimens and 0.9% saline. Of 23 isolates of P. pickettii biovar 3, 11 (48%) belonged to pattern III, 4 (17%) pattern IV and 8 (35%) pattern V to IX. Pattern III and pattern IV were seen in isolates from clinical specimens and 0.9% saline, while pattern V to IX were only seen in isolates from clinical specimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a new binding protein for crotoxin and other neurotoxic phospholipase A2s on brain synaptic membranes.

Crotoxin and other neurotoxic phospholipase A2s exert neurotoxicity by acting primarily at the presynaptic level. Strong binding of crotoxin and several others to synaptic membranes has been demonstrated previously. In this study we used simple chemical cross-linking techniques to identify the neuronal membrane molecules involved in the binding of these toxins. After 125I-crotoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate, disuccinimidyl dithiobis(propionate) or ethylene glycol bis(succinimidyl succinate) resulted in the formation of a predominant radioactive conjugate of approximately 60 kDa, which was different from the conjugate formed by photoaffinity labeling technique in a previous report. The membrane component in the conjugate was shown to be a single-chain protein of approximately 45 kDa. In subfractions of synaptosomes, this binding protein was mostly found in the synaptic membrane fraction and was not present in the mitochondrial fraction. Plasma membranes from several nonneural tissues also did not contain this binding protein. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of around 1 x 10(-8) M. Mojave toxin and some other phospholipase A2s were also highly inhibitory to this conjugation, and notexin and others were less effective, while beta-bungarotoxin and pancreatic PLA2 were totally ineffective. We concluded that a new protein of 45 kDa specifically present in neuronal membranes is another major molecule responsible for the binding of crotoxin and other phospholipase A2s.

Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling.

Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.

Taipoxin-binding protein on synaptic membranes: identification by affinity labeling.

Affinity labeling techniques were used to identify the neuronal membrane molecules involved in the binding of taipoxin, a neurotoxic protein with phospholipase A2 activity. After [125I]taipoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate resulted in the formation of a predominant radioactive conjugate of 60,000 Da. Notexin and some other PLA2s are weakly inhibitory to this conjugation, while beta-bungarotoxin and some others are not inhibitory. The 60K conjugate was not detected when plasma membranes from several nonneuronal tissues were used. We concluded that a 45,000 Da protein specifically present in neuronal membranes is (a subunit of) the major molecule responsible for taipoxin binding.

Unusual amino acid sequence of fasciatoxin, a weak reversibly acting neurotoxin in the venom of the banded krait, Bungarus fasciatus.

A weak reversibly acting neurotoxin, fasciatoxin, was found in the venom of Bungarus fasciatus. The sequencing was completed by manual and automated Edman analyses of the reduced and carboxymethylated protein and of the peptides obtained from enzyme digestions. It is composed of 63 amino acid residues with four disulphide bonds and a unique sequence at the C-terminal end. According to the criteria set by Ryden, Gabel & Eaker [(1973) Int. J. Pept. Protein Res. 5, 261-273], fasciatoxin lacks all of the five functionally invariant residues of neurotoxins. The hydropathy index indicates that fasciatoxin is devoid of a strong hydrophilicity domain for binding to the receptor site. Structural comparison with some typical neurotoxins also reveals the uniqueness of fasciatoxin in that the extent of similarity is only about 30%.

beta-Bungarotoxin antagonizes the effect of alpha-latrotoxin from black widow spider venom on the neuromuscular junction.

Sustained contraction of the chick biventer cervicis nerve-muscle preparations evoked by alpha-latrotoxin was antagonized quickly by beta-bungarotoxin. This effect of beta-bungarotoxin was dependent on its phospholipase A2 activity. In contrast, pancreatic phospholipase A2 was ineffective even at a much higher dose. It is concluded that alpha-latrotoxin needs intact presynaptic membrane to exert its effect.

Use of chick biventer cervicis muscle in the bioassay of alpha-latrotoxin from black widow spider venom.

alpha-Latrotoxin purified from black widow spider venom caused a sustained contraction of the chick biventer cervicis muscle. Muscle response to exogenous acetylcholine was not impaired. The time to reach half maximal contracture height was reproducible with small variations and can be used to quantitate the activity of alpha-LTX preparations.