Echinoderms (Echinodermata) from the Gulf of California, Mexico

A systematic list of the echinoderms of the Gulf of California, based on museum specimens of the Colección Nacional de Equinodermos, Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Autónoma de México and the National Museum of Natural History, Smithsonian Institution, Washington, D.C. is presented. A total of 193 echinoderm species is recorded, distributed in 108 genera, 51 families and 19 orders. 12 new records for the Gulf of California are presented: Asteroidea (four), Ophiuroidea (three) and Holothuroidea (five).     

Echinoids (Echinodermata: Echinoidea) from the Gulf of Mexico

The echinoid fauna of the Gulf of Mexico collected during three research cruises (20-1260 m depth) was surveyed from samples were taken at 43 stations. A total of 190 individuals were identified (eight orders, 11 families, 15 genera and 18 species). Six species are new records for the Gulf of Mexico: Stylocidaris lineata, Phormosoma placenta placenta, Plesiodiadema antillarum, Plethotaenia spatangoides, Brissopsis atlantica and Hypselaster limicolus. This adds to the little information available on the echinoid fauna of Tamaulipas, Veracruz, Tabasco, Campeche and Yucatan states in Mexico.     

Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice

As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.     

Muscarinic toxin-like proteins from Taiwan banded krait (Bungarus multicinctus) venom: purification,characterization and gene organization

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37-Ala38 in BM8; Lys37-Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchR-binding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with alpha-neurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the protein-coding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.     

Stomatopods (Crustacea: Hoplocarida) from the Gulf of Tehuantepec, Mexico

A total of 1,173 specimens of Stomatopoda were captured in 27 localities at different depths (20 to 73 m) during three oceanographic cruises in the Mexican Pacific along the continental shelf of the states of Oaxaca and Chiapas. Seven species were identified of the families Eurysquillidae, Lysiosquillidae and Squillidae. Squilla hancocki and Squilla parva were the most abundant species and the most frequently found together. The relationships between total length and carapace length were obtained for these species, which show that males of S. hancocki have a longer carapace length than females having the same total length, whereas for S. parva the opposite occurs. Larger sizes than previously reported were obtained for Lysiosquilla panamica and Squilla mantoidea. Squilla bigelowi was recorded for the first time in the Gulf of Tehuantepec. All the species were found in the intermediate platform (25-60 m); E. veleronis, S. hancocki and S. parva extended their distributions to the external platform (60-120 m), and S. hancocki and S. parva reached the circalittoral zone (10-25 m).     

Deep-water polychaetes (Annelida) from the southeastern Gulf of California, Mexico

Polychaetes inhabiting deep-sea soft bottoms from the southeastern Gulf of California were collected during four oceanographic cruises during 2000 and 2001. Sampling of benthic organisms was performed with a benthic sledge to collect epifauna and a Karling dredge for epifauna and infauna, in a depth range from 732 to 2 250 m. A list of the polychaetes that were collected and their distribution are presented here. A total of 73 species (distributed among 33 families) were identified. Moreover, 11 species were identified only to genus level and 20 species only to family level. With the exception of Ancistrosyllis hartmanae and Melinnampharete eoa, all identified species have been previously reported in soft bottoms of the Gulf of California or in adjacent areas. Additional previously unreported information is provided herein regarding depth ranges, geographical distribution, morphology and tubes inhabited by the organisms. The morphology of the ampharetids Amage sp. and Samytha sp. does not coincide with that of other species in these genera reported for the Gulf of California, which suggests that they are probably undescribed species.     

A novel plasminogen activator from Agkistrodon blomhoffii Ussurensis venom (ABUSV-PA): purification and characterization

A plasminogen activator with arginine ester hydrolysis activity (ABUSV-PA) has been identified and purified to homogeneity from Chinese Agkistrodon blomhoffii Ussurensis snake venom. ABUSV-PA, a monomeric protein with molecular mass of 27815.2 Da, was purified 180-fold with 0.02% recovery for protein and 3.6% recovery for esterase activity. ABUSV-PA reacts optimally with its substrate N(alpha)-tosyl-l-arginine-methyl ester (TAME) at approximately pH 7.5 and at 51 degrees C. Measurement from inductively coupled plasma-atomic emission spectroscopy (ICP-AES) reveals that ABUSV-PA is a Zn(2+)-containing protein with a stoichiometry of 1:1 [Zn(2+)]:[ABUSV-PA]. Analyses of esterase hydrolysis and UV absorption and CD spectra indicate that Zn(2+) plays an important role in maintaining the structural integrity rather than the esterase activity of ABUSV-PA. Divalent metal ions, including Ca(2+), Mg(2+), Cu(2+), Ni(2+), Mn(2+), and Co(2+), increase the TAME hydrolysis activity of ABUSV-PA. A red-shift of the emission wavelengths of the synchronous fluorescence of ABUSV-PA, compared to those of free Tyr and Trp, indicates a conformation where the Tyr and Trp residues are in exposed hydrophilic environments. The presence of zinc increases the hydrophobicity of the conformational environments surrounding the Trp residues of ABUSV-PA and affects the secondary structure of ABUSV-PA, as proved by UV absorption and CD spectroscopy.     

Oncaea frosti,a new species (Copepoda: Poecilostomatoida) from the Liberian coast and the Gulf of Mexico

A new species, Oncaea frosti, is described and compared to O. venusta Philippi and O. venella Farran. The three species are closely related and difficult to distinguish, except for the differences in size of females and males and dorsal dilation of pediger 2 of females of O. frosti.     

The purification and characterization of a necrotoxin from tarantula, Dugesiella hentzi (Girard), venom

The major toxin, a necrotoxin, of the venom of Dugesiella hentzi (Girard) has been purified by gel filtration. The purified toxin was homogeneous by gel filtration, polyacrylamide gel electrophoresis, and an isoelectric focusing procedure. The molecular weight estimation was 6700 and the isoelectric pH was 10.0. The amino acid composition shows 16 lysine, 8 cysteine, and one tryptophan residues, with no tyrosine, methionine, alanine, arginine, or histidine residues. The purified protein is toxic to certain insects and mice with the primary site of action being muscle tissue in the mouse. Modification of the single tryptophan residue resulted in a loss of toxicity.A significant increase of serum creatine phosphokinase activity was observed in mice injected with the necrotoxin. Histological examination showed the primary lesions were acute focal areas of myocardial necrosis, and no histological differences in myocardial lesions were seen between mice injected with the purified necrotoxin or with the whole venom.     

Isolation, purification and characterization of collagenase from hepatopancreas of the land snail Achatina fulica

Collagenase (matrix metalloproteinase-1, EC:3.4.24.7) was isolated from the hepatopancreas of Achatina fulica and characterized for its enzymatic activity and immunological properties. Procollagenase was isolated using ammonium sulphate precipitation and gel filtration, followed by purification by reverse-phase high performance liquid chromatography in the presence of trifluoroacetic acid and by dialysis in neutral buffer. In the presence of SDS and beta-mercaptoethanol, the procollagenase resolved into two subunits with molecular masses of 63 and 28 kDa, respectively. The 63 kDa fragment retained its ability to bind and degrade gelatin, but the 28 kDa was inactive. Analysis by 2D gel electrophoresis revealed that the 63 kDa fragment was basic (pIs 7.6, 7.8 and 8.15), while the 28 kDa fragment was acidic (pI 4.7 and 5.1). Western blot analysis confirmed the identity of collagenase, as only matrix metalloproteinase-1 rabbit antibodies against human matrix metalloproteinase-1 (N-terminal region) recognized both the isolated procollagenase and the 63 kDa fragment.     

Thermostable dipeptidase from Bacillus stearothermophilus: its purification, characterization, and comparison with aminoacylase

Dipeptidase (dipeptide hydrolase [EC 3.4.13.11]) has been purified to homogeneity and crystallized from the cell extract of Bacillus stearothermophilus IFO 12983. The enzyme has a molecular weight of about 86,000, and is composed of two subunits identical in molecular weight (43,000). The enzyme contains 2 g atoms of zinc per mol of protein. A variety of dipeptides consisting of glycine or only L-amino acids serve as substrates of the enzyme; Km and Vmax values for L-valyl-L-alanine are 0.5 mM and 68.0 units/mg protein, respectively. The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturants such as urea and guanidine hydrochloride. The enzyme also catalyzes hydrolysis of several N-acylamino acids with Vmax values 3-30% of those for the hydrolysis of dipeptides. The thermostable dipeptidase shares various properties with bacterial aminoacylase [EC 3.5.1.14]: their subunit molecular weight, metal content and requirement, amino acid composition, and amino acid sequence in the N-terminal region are very similar.     

Glutamine synthetase from a cyanobacterium, Phormidium lapideum: purification, characterization, and comparison with other cyanobacterial enzymes

Glutamine synthetase has been purified to homogeneity from cell extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The subunit molecular weight of the enzyme was determined as about 59,000 by sodium dodecyl sulfate gel electrophoresis. Electron micrographs of the Phormidium enzyme revealed a two-layered structure of regular hexagons (12 subunits per molecule), which markedly resembles the three-dimensional polypeptide backbone structure of the Salmonella typhimurium glutamine synthetase established by X-ray crystallography (Almassy, Janson, Hamlin, Xuong, & Eisenberg (1986) Nature 323, 304-309). The N-terminal amino acid sequence of the Phormidium enzyme shows very high similarity with that of the enzyme from an N2-fixing cyanobacterium, Anabaena 7120; 18 residues are common in 23 residues compared. Strong immunocross-reactions between the antibody against the purified Phormidium glutamine synthetase and other cyanobacterial enzymes except the Anacystis enzyme were observed. The apparent Michaelis constants for NH3, L-glutamate, and ATP were determined to be 0.29, 7.4, and 1.7 mM, respectively. Divalent metal ions such as Mg2+ and Mn2+ activated the enzyme in the biosynthetic reaction, whereas various amino acids and glutamate analogs strongly inhibited the enzyme.     

Echinoderms (Echinodermata) from the Mexican waters of the Gulf of Mexico

We present a systematic list of the echinoderms from Gulf of Mexico's Mexican waters based on specimens of the Colecci6n Nacional de Equinodermos, Instituto de Ciencias del Mar y Limnologia, Universidad Nacional Aut6noma de México and the National Museum of Natural History, Smithsonian Institution, Washington, D.C. This list reveals an important echinoderm biodiversity present in the Gulf of Mexico, where five of the six echinoderm classes are represented. A total of 209 echinoderm species is recorded, distributed in 129 genera, 63 families and 25 orders. 31 new records for the Gulf of Mexico are presented: Asteroidea (16), Ophiuroidea (nine), Echinoidea (one) and Holothuroidea (five).     

A new genus of Cletopsyllidae (Copepoda,Harpacticoida) from Gulf of Mexico

A new genus and new species of the family Cletopsyllidae Huys & Willems, 1989 from deep-sea sediment in the Gulf of Mexico, are reported and fully described and illustrated. The new genus Pentacletopsyllus (type species: P. montagni sp. n.) can be distinguished from the four known genera of the family by antennule segmentation, length ratio of first and second endopodal segments of P1, and armature pattern on P5 exopod. It also differs from its sister genera by the rostrum being bifid at the tip, third segment of the female antennule having a smooth posterior margin, the baseoendopod of P5 with biarticulate outer setophore bearing basal seta, and female caudal rami without lobate expansion. A revised key to species of the family Cletopsyllidae Huys & Willems, 1989 is provided.     

Expression,purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta

BmTXKbeta, a scorpion toxin isolated from the Chinese scorpion Buthus martensii Karsch (BmK), was expressed as a GST fusion protein in BL21 (DE3) strain. The recombinant GST-BmTXKbeta protein was purified by affinity chromatography. When treated with enterokinase, the GST-BmTXKbeta fusion protein released an approximate 6.5kDa protein which was the expected size for correctly processed. About 2mg purified recombinant BmTXKbeta protein (rBmTXKbeta) was produced from 1l bacterial culture, using this expression and purification system. The function of rBmTXKbeta was studied on the rabbit atrial myocyte by whole-cell patch clamp technique. The results showed that rBmTXKbeta inhibited the transient outward current (I(to)) of rabbit atrial myocyte with recovery after washout and the inhibition was concentration-dependent. The rBmTXKbeta prolonged the action potential duration of rabbit atrial myocyte in a concentration-dependent manner, whereas it did not affect the action potential amplitude.     

Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae)

Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8–Cys23 and Cys11–Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.     

A new species of baleen whale (Balaenoptera) from the Gulf of Mexico,with a review of its geographic distribution

Bryde's-like whales are a complex of medium-sized baleen whales that occur in tropical waters of all three major ocean basins. Currently, a single species of Bryde's whale, Balaenoptera edeni Anderson, 1879, is recognized, with two subspecies, Eden's whale, B. edeni edeni and Bryde's whale, B. edeni brydei (Olsen, 1913), although some authors have recognized these as separate species. Recently, a new, evolutionarily divergent lineage of Bryde's-like whale was identified based on genetic data and was found to be restricted primarily to the northern Gulf of Mexico (GOMx). Here, we provide the first morphological examination of a complete skull from these whales and identify diagnostic characters that distinguish it from the other medium-sized baleen whale taxa. In addition, we have increased the number of genetic samples of these Bryde's-like whales in the GOMx from 23 to 36 individuals, all of which matched the GOMx lineage. A review of Bryde's-like whale records in the Caribbean and greater Atlantic supports an isolated distribution for this unique lineage, augmenting the genetic and morphological body of evidence supporting the existence of an undescribed species of Balaenoptera from the Gulf of Mexico.     

A novel blood coagulation factor IX/factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake): isolation and characterization

Using affinity chromatography on a column of factor X-Cellulofine, we have isolated a novel blood coagulation factor X-binding protein with anticoagulant activity from the venom of Trimeresurus flavoviridis (Habu snake). This anticoagulant protein was also purified by chromatography on Sephadex G-75 and S-Sepharose Fast Flow. The yield of the purified protein was approximately 16 mg from 400 mg of crude venom. The purified protein gave a single band on both analytical alkaline disc-gel electrophoresis and SDS-PAGE. This protein had a relative molecular weight (Mr) after SDS-PAGE of 27,000 before reduction of disulfide bonds and 14,000 after reduction of disulfide bonds. The protein prolonged the clotting time induced by kaolin or factor Xa. In the presence of Ca2+, it formed a complex with factor X, the molar ratio being 1 to 1. Similar complex formation was observed with factor Xa and factor IX/factor IXa, but not with other vitamin K-dependent coagulation factors, i.e., prothrombin, factor VII, protein C, protein S, and protein Z. The interaction of this anticoagulant protein with factor IX/factor X was dependent on gamma-carboxyglutamic acid (Gla) domains, since Gla-domainless derivatives of factor X and factor IXa beta' did not interact with this anticoagulant protein.