The association of assistive mobility devices and social participation in people with spinal cord injuries.

OBJECTIVE: We assumed that assistive technology in mobility devices (that is, wheelchairs with external power and driving modified vehicle (MV) with or without driving on wheelchair) may facilitate social participation for wheelchairs users who have spinal cord injuries (SCIs). This study examined the relationship between mobility devices and social participation in this population. METHODS: We included 2986 individuals who had received initial rehabilitation at one of 18 regional centers of the Model Spinal Cord Injury System in the United States, had been interviewed between 2004 and 2010, and were wheelchair users (use a wheelchair > or = 40 h per week and cannot ambulate 150 feet at home). We performed secondary panel-data analysis using a mixed-effect model on data from 3498 follow-up interviews. Participation (measured by the Craig Handicap Assessment and Reporting Technique-Short Form (CHART-SF) and employment status) and the use of wheelchair and MV were recorded. RESULTS: Among the participants, 33% drove an MV, and 44% used an external-powered wheelchair. The use of an MV was positively related to employment and CHART-SF score, regardless of driving directly or driving with a wheelchair. People who drove an MV were found to have approximately two more business associates to contact to once a month and ∼2 additional days out of home per week compared with those without an MV. No significant association was shown between the type of wheelchair used and participation. CONCLUSION: The use of an MV was found to be positively associated with social participation in an SCI population.

Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A(2) from the venoms of rattlesnakes and other pit vipers.

Basic phospholipase A(2) homologs with Lys49 substitution at the essential Ca(2+)-binding site are present in the venom of pit vipers under many genera. However, they have not been found in rattlesnake venoms before. We have now screened for this protein in the venom of rattlesnakes and other less studied pit vipers. By gel filtration chromatography and RP-HPLC, Lys49-phospholipase-like proteins were purified from the venoms of two rattlers, Crotalus atrox and Crotalus m. molossus, and five nonrattlers, Porthidium nummifer, Porthidium godmani, Bothriechis schlegelii, Trimeresurus puniceus, and Trimeresurus albolabris. Their N-terminal amino acid sequences were shown to be characteristic for this phospholipase subfamily. The purified basic proteins from rattlesnakes caused myonecrosis and edema in experimental animals. We have also cloned the cDNAs and solved the complete sequences of four novel Lys49-phospholipases from the venom glands of C. atrox, P. godmani, B. schlegelii, and Deinagkistrodon acutus (hundred-pace). Phylogenetic analyses based on the amino acid sequences of 28 Lys49-phospholipases separate the pitviper of the New World from those of the Old World, and the arboreal Asiatic species from the terrestrial Asiatic species. The implications of the phylogeny tree to the systematics of pit vipers, and structure-function relationship of the Lys49-phospholipases are discussed.

Differential expression and geographic variation of the venom phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus.

To investigate the geographic variations in venoms of two medically important pitvipers, we have purified and characterized the phospholipases A2 (PLA2s) from the pooled venoms of Calloselasma rhodostoma from Malaysia, Thailand, Indonesia, and Vietnam, as well as the individual venom of Trimeresurus mucrosquamatus collected from both North and South Taiwan. Enzymatic and pharmacological activities of the purified PLA2s were also investigated. The complete amino acid sequences of the purified PLA2s were determined by sequencing the corresponding cDNAs from the venom gland and shown to be consistent with their molecular weight data and the N-terminal sequences. All the geographic venom samples of C. rhodostoma contain a major noncatalytic basic PLA2-homolog and two or three acidic PLA2s in different proportions. These acidic PLA2s contain Glu6-substitutions and show distinct inhibiting specificities toward the platelets from human and rabbit. We also found that the T. mucrosquamatus venoms from North Taiwan but not those from South Taiwan contain an Arg6-PLA2 designated as TmPL-III. Its amino acid sequence is reported for the first time. This enzyme is structurally almost identical to the low- or nonexpressed Arg6-PLA2 from C. rhodostoma venom gland, and thus appears to be a regressing venom component in both of the Asian pitvipers.

Serine protease isoforms of Deinagkistrodon acutus venom: cloning, sequencing and phylogenetic analysis.

The major coagulating fibrinogenase of Deinagkistrdon acutus venom, designated acutobin, was purified by anion-exchange chromatography, gel filtration and reverse-phase HPLC. Approximately 80% of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Extensive screening of the venom gland cDNA species after amplification by PCR resulted in the isolation of four distinct cDNA clones encoding acutobin and three other serine proteases, designated Dav-PA, Dav-KN and Dav-X. The complete amino acid sequences of these enzymes were deduced from the cDNA sequences. The amino-acid sequence of acutobin contains a single chain of 236 residues including four potential N-glycosylation sites. The purified acutobin (40 kDa) contains approx. 30% carbohydrate by weight, which could be partly removed by N-glycanase. The phylogenetic tree of the complete amino acid sequences of 40 serine proteases from 18 species of Crotalinae shows functional clusters reflecting parallel evolution of the three major venom enzyme subtypes: coagulating enzymes, kininogenases and plasminogen activators. The possible structural elements responsible for the functional specificity of each subtype are discussed.

Phospholipases A2 from Callosellasma rhodostoma venom gland cloning and sequencing of 10 of the cDNAs, three-dimensional modelling and chemical modification of the major isozyme.

Callosellasma rhodostoma (Malayan pitviper) is a monotypic Asian pitviper of medical importance. Three acidic phospholipases A2 (PLA2s) and one basic PLA2-homolog were purified from its venom while 10 cDNAs encoding distinct PLA2s were cloned from venom glands of a Thailand specimen of this species. Complete amino-acid sequences of the purified PLA2s were successfully deduced from their cDNA sequences. Among the six un-translated PLA2 cDNAs, two apparently result from recombination of its Lys49-PLA2 gene with its Asp49-PLA2 genes. The acidic PLA2s inhibit platelet-aggregation, while the noncatalytic PLA2-homolog induces local edema. This basic PLA2-homolog contains both Asp49 and other, unusual substitutions unique for the venom Lys49-PLA2 subtype (e.g. Leu5, Trp6, Asn28 and Arg34). Three-dimensional modelling of the basic protein revealed a heparin-binding region, and an abnormal calcium-binding pocket, which may explain its low catalytic activity. Oxidation of up to six of its Met residues or coinjection with heparin reduced its edema-inducing activity but methylation of its active site His48 did not. The distinct Arg/Lys-rich and Met-rich region at positions 10-36 of the PLA2 homolog presumably are involved in its heparin-binding and the cell membrane-interference leading to edema and myotoxicity.

Glycoprotein Ib-binding protein from the venom of Deinagkistrodon acutus--cDNA sequence, functional characterization, and three-dimensional modeling.

Agkicetin-C, a potent glycoprotein Ib antagonist from the venom of the Chinese pit viper, Deinagkistrodon acutus, has been purified and characterized (5). It is a disulfide-linked heterodimer containing subunits of 132 and of 123 amino acid residues. Herein, the complete amino acid sequences were resolved by cloning and nucleotide sequencing of the cDNAs. The sequences of its subunits are homologous to those of other snake venom proteins of the C-type (Ca2+-dependent) lectin superfamily. A three-dimensional model of agkicetin-C was constructed based on the crystal structure of habu coagulation factor IX/X-binding protein. By careful alignment of all the related sequences available and comparing the 3D-model of agkicetin-C with structures of other homologous proteins of different functions, some variable residues of agkicetin-C were identified, which possibly are responsible for the specificity of this distinct subtype of the C-type lectin-like venom proteins.

Purification, cloning and sequence analyses for pro-metalloprotease-disintegrin variants from Deinagkistrodon acutus venom and subclassification of the small venom metalloproteases.

Acidic and basic hemorrhagic metalloproteases were purified from the venom of Deinagkistrodon acutus (from Fujian Province, China) using gel filtration and anion exchange on FPLC and reversed-phase HPLC. Their hemorrhagic activities and N-terminal sequences were characterized. Extensive screening of the venom gland cDNA after PCR amplification resulted in the identification and sequencing of a total of seven cDNA clones encoding the multidomain precursors of six acidic and one alkaline low molecular mass metalloproteases. Two of the precursors contain a processable disintegrin domain. Disintegrins of 5 kDa were also purified from the venom. The partial amino-acid sequences and molecular masses determined by electrospray ionization mass spectrometry of the purified proteins specifically match those deduced from two of the cDNA sequences. Moreover, phylogenetic analyses based on 30 complete sequences of low molecular mass venom metalloproteases revealed that they may be classified into three functional subtypes: acidic hemorrhagins, basic and moderate hemorrhagins, and nonhemorrhagic enzymes. Subtype-specific amino-acid substitutions in the C-terminal regions of the enzymes were highlighted to explore the structure-activity relationships of the enzymes.

Purification and characterization of the venom phospholipases A2 from Asian monotypic crotalinae snakes.

Phospholipases A2 were purified from the venoms of Asian monotypic crotalinae snakes including Callosellasma, Hypnale, Deinagkistrodon, and Tropidolaemus by a combination of gel filtration and reversed-phase chromatographic methods. One to four isoforms of the enzyme were found in each of the venoms. The venom enzymes were subjected to N-terminal sequencing up to the 30th amino acids, and their molecular weights were analyzed by electrospray-ionization mass spectrometry. Homologous antiplatelet phospholipase with a conserved Glu 6 residue was found in each of the venoms. Basic phospholipases with Trp 6 (W6) but without detectable enzyme activities were also isolated from the venom of C. rhodostoma, H. hypnale, and T. wagleri. These W6 enzymes showed strong heparin-binding affinity and capable of inducing edema in rat paws. The fact that the venoms of Callosellasma and Hypnale contain similar types of phospholipases is in accord with recent reports that these two taxa formed a clade. Deinagkistrodon venom does not contain phospholipase variants other than the Glu-6 subtype as Trimeresurus, Agkistrodon, and Protobothrops venoms do. Interestingly, the Glu-6 enzyme from T. wagleri venom has a molecular weight of 15,319 Daltons, higher than those of most other venom phospholipases. Our results show that new types of the enzyme are more likely to be found in the venom of monotypic species; the amino acid sequence data or the subtypes of venom-phospholipases are potentially useful as markers or a character system for studying higher-order systematics of venomous snakes.

Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

Crystallization and preliminary X-ray diffraction studies of the neurotoxic, heterodimeric phospholipase A2 from the Taiwan viper (Vipera russelli formosensis).

The 28 kDa heterodimeric complex from Taiwan viper (F4/F7 complex) is composed of a neurotoxic phospholipase A2 (F4) and a non-toxic PLA2-like component (F7). Despite a high sequence identity (65%), the biological and pharmacological activities of F4 and F7 are contrasting. The complex is a structural analogue of Vipoxin found in the venom of the Bulgarian viper Vipera ammodites meridionalis. It is unclear how and why such varied bioactivities are expressed in these similar components. The F4/F7 complex has been crystallized using hanging-drop vapour diffusion and macroseeding techniques. The space group is monoclinic P21 with unit-cell dimensions a = 74.92, b = 85.13, c = 78.16 A and beta = 95.12 degrees. X-ray intensity data to 2.0 A resolution have been collected at 120 K and the structure has been solved using the molecular-replacement method. There are four F4/F7 complex molecules in the asymmetric unit, which do not exhibit any local point-group symmetry.

Effect of site directed mutagenesis on the activity of recombinant trimucrotoxin, a neurotoxic phospholipase from Trimeresurus mucrosquamatus venom.

Trimucrotoxin, the basic phospholipase A2 from Trimeresurus mucrosquamatus venom, is neurotoxic and myotoxic, and structurally similar to crotoxin B subunit. To investigate the amino acid residues responsible for its neurotoxicity, we have mutated its interface-recognition residues including a conserved Asn6 in all the Crotalinae neurotoxic phospholipases. The wild-type and the mutants were expressed in E. coli as fusion-proteins and activated in vitro by factor Xa cleavage after folding. The completion of folding and activation were checked with electrospray ionization mass spectrometry and circular dichroism measurement. Enzymatic activities and neurotoxicities toward the chick tissue of four trimucrotoxin mutants (N6A, N6E, N6R and 6E7T8L) were compared with those of the wild type which was as active as that was isolated from the venom. Mutants N6A and N6E retained more than half of the original enzymatic activity but their neurotoxicities reduced to 33% and 10% that of the wild type, respectively. Mutants N6R and 6E7T8L retained 20-25% of the enzyme activity toward the anionic micellar substrate but were inactive toward the zwitterionic micellar substrate, and their neurotoxicities were less than 3% of that of the wild type. These results demonstrate the importance of residues 6-8 in trimucrotoxin for its neuronal specificity and the specificity toward potential substrates.

The hemolymph clottable proteins of tiger shrimp, Penaeus monodon, and related species.

A clottable protein was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion-exchange chromatography. The protein formed stable clots in the presence of Ca2+ and the transglutaminase in hemocyte lysate. It is thermostable at temperatures up to 66 degrees C. The molecular mass of the clottable protein was determined to be 380 kDa by SDS-PAGE and MALDI-TOF mass spectrometry, and the protein exists as disulfide-linked homodimers and oligomers. The size and amino acid composition of the clottable protein are similar to those of several other shrimps, prawns, lobster and crayfish, and their N-terminal amino acid sequences are 60-80% identical. Monosaccharide analysis of the clottable protein revealed the presence of mannose, glucosamine or N-acetylglucosamine and possibly glucose in this glycoprotein of about 5% sugar content. Lipid in the protein upon electrophoresis was hardly detectable with the Oil Red O staining method. In immunodiffusion and immunoblotting analyses, the anti-clottable protein antibodies reacted with the clottable proteins from the penaeid shrimps but not with those from other crustaceans.

Molecular cloning and deduced primary structures of acidic and basic phospholipases A2 from the venom of Deinagkistrodon acutus.

This paper presents the nucleotide sequences of the cDNAs encoding three acidic phospholipases A2 and one basic phospholipase A2 from Deinagkistrodon acutus venom. The deduced primary structure of the basic enzyme is closest to that of the basic neurotoxic enzyme from Trimeresurus mucrosquamatus venom, while the acidic phospholipases from D. acutus have highest sequence similarity to that from Agkistrodon halys pallas. The phylogeny of this monotypic species is discussed.

LYS-49 phospholipase A2 homologs from venoms of Deinagkistrodon acutus and Trimeresurus mucrosquamatus have identical protein sequence.

The complete amino acid sequences of the Lys-49 PLA2s from the venom of Deinagkistrodon acutus (from Taiwan and China) and Trimeresurus mucrosquamatus (Taiwan habu) were solved by a facile cDNA cloning and sequencing method. The deduced amino acid sequences of the Lys-49 PLA2s of both venoms are identical, suggesting close phylogenic relationship between this two snake species of different genera. In addition, by cloning and cDNA sequencing, the mRNA coding for a Arg-49 PLA2 homolog of low expression level was also found in the venom gland of T. mucrosquamatus.

Functional and sequence characterization of coagulation factor IX/factor X-binding protein from the venom of Echis carinatus leucogaster.

A new coagulation factor IX/factor X-binding protein (IX/X-bp) from Echis carinatus leucogaster venom has been purified and designated ECLV IX/X-bp. ECLV IX/X-bp binds factor IX and X in a Ca(2+)-dependent manner and is devoid of thrombin-inhibitory and platelet-aggregating activities. The apparent dissociation constants (Kd) for binding of ECLV IX/X-bp to factor IX and factor X are 6.6 and 125 nM, respectively. Upon the addition of Mg2+, the required Ca2+ concentration for optimal binding of ECLV IX/X-bp to factor IX and factor X was prominently reduced. Mg2+ also increases the affinity of factor X for the venom protein. Direct binding of IX/X-bp to factor IX and X could also be detected by far-Western blotting, and results of the experiment ruled out the lectin-like mechanism of ECLV IX/X-bp. The complete amino acid sequence and the disulfide pattern of ECLV IX/X-bp was deduced by enzymatic hydrolysis and automated sequencing of the S-pyridylethylated protein. The venom protein is a heterodimer with one subunit of 131 amino acid residues and another of 125 residues. Both subunits are homologous to each other and to other snake venom proteins of the C-type lectin superfamily.

Two types of Russell's viper revealed by variation in phospholipases A2 from venom of the subspecies.

This study compared the phospholipases A2 (PLA2S) present in four commercially available venoms of Russell's viper subspecies by HPLC fractionation and partial sequence analysis. A potent heterodimeric PLA2 neurotoxin (designated a Russtoxin) was found in the venoms of all Russell's vipers except Daboia russelli (Sri Lanka and South India). The venom PLA2S of D. r. russelli (southern India) used in a previous study appear to be the same as those of D. r. pulchella (Sri Lanka), while the venom PLA2S of D. r. russelli (Pakistan) and D. r. siamensis (Burma and Thailand) resemble those of D. r. formosensis (Taiwan). This study provides evidence for the presence of two types of Russell's viper. Daboia russelli formosensis (Taiwan). D. r. siamensis (Thailand and Burma) and D. r. russelli (Pakistan) represent one type whose venom contains PLA2S having an Asn residue at the N-terminus, while D. r. pulchella (South India and Sri Lanka) represents the other type, whose venom contains PLA2S with a N-terminal residue Ser.

Molecular cloning and characterization of a neurotoxic phospholipase A2 from the venom of Taiwan habu (Trimeresurus mucrosquamatus).

Using gel-filtration chromatography and reverse-phase (RP) HPLC we have purified a presynaptic neurotoxin (designated as trimucrotoxin) from the crude venom of Taiwan habu (Trimeresurus mucrosquamatus). Its complete primary structure was solved by an automated N-terminal sequencing and cDNA sequencing method. The enzyme inhibited the twitch of the chick biventer cervicis muscle at 0.1-1 micrograms/ml and showed lethality in mice (LD50 = 1.2 micrograms/g, when given intravenously). Trimucrotoxin exists mainly as a homodimer of 14 kDa subunits as shown by a gel-filtration experiment, and dissociates into monomers during SDS/PAGE in the absence of Ca2+. However, most of trimucrotoxin migrated as slowly as a trimer during nondenaturing SDS/PAGE in the presence of Ca2+ or Sr2+. Its amino acid sequence identity to crotoxin B and agkistrodotoxin is about 75%, and its cDNA sequence is 82% identical to that of crotoxin B. Rabbit antiserum against trimucrotoxin also cross-reacted with the other crotalid neurotoxic phospholipases A2. Furthermore, the purified acidic subunit of crotoxin potentiated the neurotoxicity of trimucrotoxin. A comparison of the sequences of these crotalid neurotoxins revealed some common features of the possible neurotoxic sites, including residues 6, 11, 76-81 and 119-125.

Functional and sequence characterization of agkicetin, a new glycoprotein Ib antagonist isolated from Agkistrodon acutus venom. offf2p4.

A new glycoprotein Ib (GPIb) antagonist, agkicetin, was purified from the venom of Agkistrodon acutus and characterized. It is a disulfide-linked heterodimer consisting subunits of 15 and 14 kDa. The subunits are homologous to each other and to other snake venom proteins of the C-type (Ca(2+)-dependent) lectin superfamily. Agkicetin behaved as a potent antagonist of von Willebrand Factor (vWF)-induced platelet agglutination (IC50 = 12.5 nM) and bound specifically to GPIb of fixed platelets with high affinity (Kd = 38 nM). It did not bind coagulation factor IX and thrombin. Monoclonal antibody against epitope on the N-terminal domain of GPIb competed the binding of agkicetin with platelets. Reduced and alkylated agkicetin lost most of its inhibitory efficacy toward vWF-induced platelet agglutination.

Determination of the structure of two novel echistatin variants and comparison of the ability of echistatin variants to inhibit aggregation of platelets from different species.

Two new variants of short disintegrins were purified from the venom of Echis carinatus leakeyi and named echistatin beta and gamma. These proteins were found to be about 85% similar in amino acid sequence to echistatin alpha which has been well studied. The disulphide pattern of echistatin gamma appeared to be identical with that of echistatin alpha. They all contain the adhesive recognition sequence Arg-Gly-Asp (RGD) but inhibit the aggregation of platelets from human and other mammals with different potencies. Echistatin beta and alpha are far more effective on platelets from humans and guinea pigs than those from rabbits and rats whereas echistatin gamma is less discriminating of the platelets of the species tested. This species-dependent platelet sensitivity to echistatin beta and gamma could be attributed to the variations in residues 15, 21, 22 and 27, which are close to or within the RGD loop, rather than to the C-terminal variations after residue 46. Taking advantage of the presence of methionine residues flanking both sides of the ARGDDM motif in echistatin gamma, we deleted this hexapeptide by CNBr cleavage to produce des-(23-28)-echistatin gamma. The modified protein showed c.d. and fluorescent spectra grossly similar to the intact echistatin but its antiplatelet potency decreased more than 200-fold. We thus propose that a favourable conformation of the RGD region is responsible mainly for the high-affinity binding of echistatin to the platelet glycoprotein IIb-IIIa as shown previously for the binding of medium-size disintegrin.

Characterization of a cDNA encoding the precursor of platelet aggregation inhibition and metalloproteinase from Trimeresurus mucrosquamatus venom.

This paper presents the nucleotide sequence of a cDNA encoding a full-length precursor of a novel platelet aggregation inhibitor named trimucrin and a hemorrhagic metalloproteinase from Trimeresurus mucrosquamatus snake venom. The deduced structure of the precursor protein is compared with those of other members of the metalloproteinase/disintegrin family.