Comparative Enzymology of Cholinesterases Underlies the Biochemical Method of Squid Taxonomy

Classification and evaluation is performed of comparative enzymologic parameters of cholinesterase activity (ChEA) in the optic ganglia tissue of individuals of different squid species with different habitats, the parameters used in the biochemical method of Cephalopoda taxonomy. Seven species of oceanic squids belonging to three families, Ommastrephidae (subfamilies Ommastrephinae and Todaradinae), Thysanoteuthidae and Gonatidae, are studied. As cholinesterase effectors, 8 substrates, 8 irreversible organophosphorus inhibitors, and 17 reversible onium inhibitors are tested. Different levels of comparison of the enzymologic parameters of ChEA are proposed. First, the enzymatic homogeneity or heterogeneity of ChEA is a criterion of interspecies difference. Second, the interspecies differences may be evaluated from parameters of the ChEA substrate specificity in different squid species. Third, use of irreversible organophosphorus inhibitors is the test that is essentially simpler, sufficiently sensitive, and reliable for comparison in the enzymologic method. Fourth, the finer nuances of the taxonomy at the level of intraspecies differences can be revealed by using reversible onium inhibitors of different structure. The enzymologic method of the squid taxonomy has allowed revealing isolation of populations of Ommastrephes bartrami individuals from different parts of the broken habitation areal.     

Comparative-enzymologic study of phosphatase activity of Pacific hydrobionts

Activities of acid phosphatase are studied with use as substrates of phenyl phosphate, α- and β-glycerophosphates in various organs and tissues of a large group of marketable hydrobionts of the Pacific basin (12 fish species, 7 invertebrate species, and one mammalian species) and of alkaline phosphatase in various organs of the Commander (Berryteuthis magister) and the New Zealand (Nototodarus sloani sloani) squids. Intertissue and interspecies differences have been revealed in the substrate and inhibitory specificity of the studied enzyme preparations. The method of isolation and a partial purification of preparations of acid phosphatase from tissue of gonads and of alkaline phosphatase from tissues of kidney and liver of individuals of marketable squid species are described.     

Cholinesterase activity in tissues of some crab species from the Japan Sea

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes were compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme of squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.     

Possible imitation of jellyfish by the squid paralarvae of the family Gonatidae (Cephalopoda,oegopsida)

Paralarval behaviour of eight species of the family Gonatidae (Teuthoidea, Cephalopoda) was examined in small 3–1 aquaria on board ship during planktonic surveys, which were carried out above and off the continental slope of the western part of the Bering Sea. Undisturbed paralarvae moved in aquaria with an average frequency of 15–20 mantle contractions per minute. In response to a sudden disturbance (flash of light, impact to the aquarium wall) squids exhibited a defensive body posture, relaxing the mantle and pulling the head with tentacles and arms into the mantle cavity, thereby becoming similar in appearance, size and colour to small jellyfishesAglantha digitalis (Hydromedusae).     

Comparative Study of Substrate–Inhibitor Specificity of Brain Cholinesterase Activity of Pacific Salmon Fish of the Salmonidae Family

Salmon fish of the Salmonidae family, representatives of the Pacific salmon Oncorhynchus genus, the humpback salmon O. gorbuscha Wielb., dog salmon O. keta, coho salmon O. kisutch, blueback salmon O. nerka, and chinook salmon O. tschawytscha, and also of noble salmons genus, Salmo—black salmon S. salar, caught in the northwest water area of the Pacific ocean, are studied in the work. Iodides of acetyl-, propionyl-, and butyrylthiocholine were used as substrates. Carbamate proserine and 5 organophosphorus inhibitors were studied as inhibitors. Testing of homogeneity of cholinesterase activity in the brain tissue has revealed only one enzyme in the coho salmon, while several enzymes, in the dog salmon and humpback salmon, which can be an enzymologic argument for the favor of hypothesis about the presence of interspecies groups among Pacific salmons of the Oncorhynchus genus. Interspecies differences in substrate specificity of the brain tissue in the studied salmonid species are found. The fish enzymes of the fishes have shown a high sensitivity to proserine. Only in the case of diisopropylfluorophosphate, both interspecies and intergenus differences are revealed.     

The specificity of the interaction of the cholinesterases in Far Eastern squid and certain vertebrates with reversible inhibitors

Studies have been made of the effect of three groups of ammonia reversible inhibitors on the activity of erythrocyte acetylcholinesterase, serum butyrylcholinesterase, cholinesterase from frog brain, as well as cholinesterases from the optical ganglia of the Pacific and three populations of the commander squids. Determination of kinetic parameters of the reversible inhibition of these enzymes revealed differences resulting from the specific structure of their catalytic centers. Tetramethylammonium assay confirmed different properties of cholinesterases in individuals of the commander squid from various habitats in the Bering Sea; this finding may be taken as an indication of intraspecific differentiation of these cephalopods. Certain similarity was noted in the inhibitory specificity of cholinesterases from the Pacific and "southern" commander squids with the overlapping habitats.     

Structure-function relationship of substrate length specificity of dextran glucosidase from <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an α-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms of substrate chain length specificity. SMDG efficiently hydrolyzes both short-and long-chain substrates, while O16G acts on only short-chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short β → α loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced by a smaller amino acid, alanine, asparagine or proline; and (ii) short β → α loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates, as implied by structural analysis.     

Statolith microstructure and age of early life stages of planktonic squids Galiteuthis phyllura and Belonella borealis (Oegopsida, Cranchiidae) from the northern North Pacific

Statolith morphology and microstructure were studied in twocommon species of panktonic cranchiid squids, Belonella borealis[four juveniles with mantle length (ML) 375–450 mm] andGaliteuthis phyllura (13 paralarvae and juveniles, ML 9–235mm),caught near the bottom and in pelagic layers over the continentalslope of Siberia in the northwest Bering Sea. The total numberof growth increments within the statoliths ranged from 277 to294 in B.borealis and from 10 to 209 in G.phyllura. Assumingthat these increments were produced daily, both species growrapidly in length (daily growth rate = 1.13mm day^–1 duringthe first 8–10 months of their juvenile phase in the mesopelagiclayers, prior to migration into deeper waters for maturation.     

Effect of the substrate structure on the mechanism of reversible inhibition of cholinesterases of different origin

The mechanism of reversible inhibition of human erythrocyte acetylcholinesterase, horse blood serum butyrylcholinesterase, cholinesterase from optical ganglia of the squids, PacificTodarodes pacificus and CommodoreBerryteuthis magister, from different zones of habitation area is studied in the presence of substrates of various structures (acetylcholine, butyrylcholine, acetylthiocholine, butyrylthiocholine, phenylacetate, indophenylacetate, 2,6-dichlorophenylindophenylacetate). Tested as reversible inhibitors were tetramethylammonium iodide, tetraethylammonium iodide, choline iodide, and two derivatives of α,ω-bis(trimethylammoniommethyl)oligodimethylsiloxane dichloride. It has been revealed that the mechanism of the reversible anticholinesterase action depends essentially both on the enzyme nature and on the structures of substrate and inhibitor. The transfer from cation-containing to hydrophobic substrates increased essentially the contribution of uncompetitive component of the inhibitory constant. In the presence of butyric acid esters (butyrylcholine, butyrylthiocholine), the potency of inhibitors was lower than at hydrolysis of the corresponding acetates. The effect of the substrate structure on the mechanism of reversible inhibition was revealed to a greater extent in reactions with participation of squid cholinesterases.     

Evolutionary History of B1 Retroposons in the Genus Mus

Short interspersed DNA elements (SINEs) amplify by retroposition either by (i) successive waves of amplification from one or a few evolving master genes or by (ii) the generation of new master genes that coexist with their progenitors. Individual, highly conserved, elements of the B1 SINE family were identified from the GenBank nucleotide database using various B1 subfamily consensus query sequences to determine their integration times into the mouse genome. A comparison of orthologous loci in various species of the genus Mus demonstrated that four subfamilies of B1 elements have been amplifying within the last 1–3 million years. Therefore, B1 sequences are generated by coexisting source genes. Additionally, three B1 subfamilies have been concurrently propagated during subspecies divergence and strain formation in Mus, indicating very recent activity of this retroposon family. The patterns of intra- and interspecies variations of orthologous loci demonstrate the usefulness of B1 integrations as a phylogenetic tool. A single inconsistency in the phylogenetic trends was depicted by the presence of a B1 insert in an orthologous locus exclusively in M. musculus and M. pahari. However, DNA sequence analysis revealed that these were independent integrations at the same genomic site. One highly conserved B1 element that integrated at least 4–6 million years ago suggests the possibility of occasional function for B1 integrations. Received: 25 February 2000 / Accepted: 5 June 2000     

中国算盘子属(叶下珠科)一些种的分类学处理

算盘子属(Glochidion J.R.G.Forst.)是叶下珠科(Phyllanthaceae)叶下珠族(Phyllantheae)中一个分类极为困难的类群。基于广泛野外考察与馆藏标本查阅,对中国该属部分物种进行分类学处理。其中,长柱算盘子[G.khasicum(Müll.Arg.)Hook.f.]与倒卵叶算盘子(G.obovatum SieboldZucc.)在中国的分布予以排除,菲岛算盘子[G.philippicum(Cav.)C.B.Rob.]在中国被发现仅分布于台湾地区;G.bodinieri H.Lév.,G.pseudo-obscurum var.glabrum Pamp.与G.pseudo-obscurum var.lanceolatum Pamp.这三个名称被处理为湖北算盘子(G.wilsonii Hutch.)的新异名;G.vaniotii H.Lév.被排除在算盘子属外,并接受为芸香科臭常春(Orixa japonica Thunb.)的异名。另外,对G.khasicum(Müll.Arg.)Hook.f.,G.obovatum SieboldZucc.,G.philippicum(Cav.)C.B.Rob.,G.pseudo-obscurum var.lanceolatum Pamp.及G.wilsonii Hutch.这五个名称进行了后选模式的指定。     

Catalytical properties of liver monoamine oxidases of the Commander squid <Emphasis Type="Italic">Berryteuthis magister</Emphasis> from various habitation zones

There has been performed kinetic analysis of enzymatic reactions of deamination of tyramine, tryptamine, serotonin, benzylamine, β-phenylethylamine, and histamine under action of liver monoamine oxidase (MAO) of the Commander squid Berryteuthis magister from various habitation zones in the Bering and Japan Seas. A substrate inhibition by high concentrations of all studied substrates has been revealed, which seems to indicate mutual effect of various MAO forms present in liver of the studied squids. Analysis of kinetic parameters of enzymatic reactions of deamination of six studied substrates and the substrate-inhibitory analysis with use of two derivatives of acridine and deprenyl indicate the enzyme heterogeneity, the presence of at least two MAO-A forms, and the absence of intraspecies differences in MAO of the Commander squids from various habitation zones. The most active was the MAO form responsible for serotonin deamination. There were obtained quantitative difference in substrate specificity and reaction ability with respect to inhibitor of proflavin for the liver MAO of the Commander and Pacific squids.     

Distribution of cephalopods in the upper epipelagic northwestern Bering Sea in autumn

In the northwestern Bering Sea in autumn, the epipelagic cephalopod community was represented by the boreal fauna, and was found to be composed of three families and nine species of the order Teuthida: Gonatidae (Berryteuthis magister, Boreoteuthis borealis, Gonatopsis japonicus, Gonatus madokai, Gonatus kamtschaticus, Gonatus onyx, and Gonatus pyros), Chiroteuthidae (Chiroteuthis calyx) and Onychoteuthidae (Onychoteuthis borealijaponica). Two pelagic gonatid species (B. borealis and G. kamtschaticus) dominated the cephalopod community in the upper 50 m. The distribution patterns of B. borealis and G. onyx were associated with diel vertical migrations of these squid. The distribution of two distinct size groups of G. kamtschaticus suggested ontogenetic migration of larger squid to deeper layers, and adds to previous data suggesting that this species may be a heterogeneous assemblage. Demersal B. magister rarely occurred in the surface waters. The occurrence of maturing O. borealijaponica in the southern marine area indicated that these were occasional seasonal migrants from the ocean. The occurrence of juvenile C. calyx suggested that these squid may conduct vertical forage migrations from deep waters to the surface layers.     

Isolation and properties of extracellular β-xylosidases from fungi <Emphasis Type="Italic">Aspergillus japonicus</Emphasis> and <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Homogeneous β-xylosidases with molecular mass values 120 and 80 kDa (as shown by SDS-PAGE), belonging to the third family of glycosyl hydrolases, were isolated by anion-exchange, hydrophobic, and gel-penetrating chromatography from enzyme preparations based on the fungi Aspergillus japonicus and Trichoderma reesei, respectively. The enzymes exhibit maximal activity in acidic media (pH 3.5–4.0), and temperature activity optimum was 70°C for the β-xylosidase of A. japonicus and 60°C for the β-xylosidase of T. reesei. Kinetic parameters of p-nitrophenyl β-xylopyranoside and xylooligosaccharide hydrolysis by the purified enzymes were determined, which showed that β-xylosidase of A. japonicus was more specific towards low molecular weight substrates, while β-xylosidase of T. reesei preferred high molecular weight substrates. The competitive type of inhibition by reaction product (xylose) was found for both enzymes. The interaction of the enzymes of different specificity upon hydrolysis of glucurono- and arabinoxylans was found. The β-xylosidases exhibit synergism with endoxylanase upon hydrolysis of glucuronoxylan as well as with α-L-arabinofuranosidase and endoxylanase upon hydrolysis of arabinoxylan. Addition of β-xylosidases increased efficiency of hydrolysis of plant raw materials with high hemicellulose content (maize cobs) by the enzymic preparation Celloviridine G20x depleted of its own β-xylosidase.     

Development of four phylogenetically-arrayed BAC libraries and sequence of the APA locus in Phaseolus vulgaris

The APA family of seed proteins consists of three subfamilies, in evolutionary order of hypothesized appearance: phytohaemagglutinins (PHA), α-amylase inhibitors (αAI), and arcelins (ARL). The APA family plays a defensive role against mammalian and insect seed predation in common bean (Phaseolus vulgaris L.). The main locus (APA) for this gene family is situated on linkage group B4. In order to elucidate the pattern of duplication and diversification at this locus, we developed a BAC library in each of four different Phaseolus genotypes that represent presumptive steps in the evolutionary diversification of the APA family. Specifically, BAC libraries were established in one P. lunatus (cv. ‘Henderson: PHA⁺ αAI⁻ ARL⁻) and three P. vulgaris accessions (presumed ancestral wild G21245 from northern Peru: PHA⁺ αAI ⁺ ARL⁻; Mesoamerican wild G02771: PHA⁺ αAI ⁺ ARL⁺; and Mesoamerican breeding line BAT93: PHA⁺ αAI ⁺ ARL⁻). The libraries were constructed after HindIII digestion of high molecular weight DNA, obtained with a novel nuclei isolation procedure. The frequency of empty or cpDNA-sequence-containing clones in all libraries is low (generally <1%). The Henderson, G21245, and G02771 libraries have a 10× genome coverage, whereas the BAT93 library has a 20× coverage to allow further, more detailed genomic analysis of the bean genome. The complete sequence of a 155 kbp-insert clone of the G02771 library revealed six sequences belonging to the APA gene family, including members of the three subfamilies, as hypothesized. The different subfamilies were interspersed with retrotransposon sequences. In addition, other sequences were identified with similarity to chloroplast DNA, a dehydrin gene, and the Arabidopsis flowering D locus. Linkage between the dehydrin gene and the D1711 RFLP marker identifies a potential syntenic region between parts of common bean linkage group B4 and cowpea linkage group 2     

The screening of the enzyme and isoenzyme patterns in seeds ofAllium cepa L.

The screening of enzyme patterns in seeds ofAllium cepa cv. Všetatská revealed the presence of the following enzymes: alcohol dehydrogenase, lactate dehyd ogenase, NAD+- and NADP+-glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase NAD+- and NADP+-malate dehydrogenase, NADH₂- and NADPH₂-tetrazolium reductase catalase, Superoxide dismutase, acid and alkaline phosphatase, L-leucine aminopeptidase, glutamate dehydrogenase, non-specific esterase, and cholinesterase. Altogether 17 enzymes were detected in onion seeds, nine of which had more than three isoenzymes, NAD+-malate dehydrogenase had 8, and non-specific esterase 9 isoenzymes. The demonstration of cholinesterase and Superoxide dismutase activities is remarkable.     

Primary Structure, sequence analysis, and expression of the thermostable d - hydantoinase from Bacillus stearothermophilus SD1

The gene coding for the thermostable d-hydantoinase from the thermophilic bacterium Bacillus stearothermophilus SD1 was cloned and its nucleotide sequence was completely determined. The d-hydantoinase protein showed considerable amino acid sequence homology (20–28%) with other hydantoinases and functionally related allantoinases and dihydroorotases. Strikingly the sequence of the enzyme from B. stearothermophilus SD1 exhibited greater than 89% identity with hydantoinases from thermophilic bacteria. Despite the extremely high amino acid homology among the hydantoinases from thermophiles, the C-terminal regions of the enzymes were completely different in both sequence and predicted secondary structure, implying that the C-terminal region plays an important role in determining the biochemical properties of the enzymes. Alignment of the sequence of the d-hydantoinase from B. stearothermophilus SD1 with those of other functionally related enzymes revealed four conserved regions, and five histidines and an acidic residue were found to be conserved, suggesting a close evolutionary relationship between all these enzymes. Received: 20 December 1996 / Accepted: 12 March 1997     

Identification, distribution and relative abundance of paralarval gonatid squids (Cephalopoda: Oegopsida: Gonatidae) from the Gulf of Alaska, 2001 2003

Paralarvae of the family Gonatidae were sampled in the Gulfof Alaska during spring 2001–2003. Taxonomic characterswere determined to allow identification of the specimens tospecies. The dorsal head chromatophore pattern (DHCP) was themost robust character and allowed identification to speciesfor the first time without requiring the removal and examinationof the radula. Six different DHCPs were found among the sixspecies in the study area. The 1140 specimens collected consistedof the following six species: Berryteuthis anonychus (759),Berryteuthis magister (71), Gonatopsis borealis (155), Gonatuskamtschaticus (1), Gonatus madokai (4) and Gonatus onyx (143).The specimens had a size range of 3.0–20.63 mm dorsalmantle length with the majority of specimens smaller than 10 mm.All species showed an increasing trend in abundance from theshelf (0–200 m) to the slope (200–1000 m)to the basin (>1000 m) except G. onyx in 2001 and 2002.Wide variation in distribution and abundance was found for thefour most abundant species; however, in general, B. anonychuswas most abundant and widely distributed, followed by Gonatopisborealis, Gonatus onyx and B. magister. (Received 28 April 2006; accepted 1 February 2007)     

Comparative enzymological study of catalytic properties of liver monoamine oxidases in frogs

Comparative enzymological study of catalytical properties of monoamine oxidase (MAO) of liver of the marsh frog Rana ridibunda and common frog Rana temporaria has revealed certain features of similarity and differences between these enzymes. The MAOs from both studied biological sources show catalytic properties resembling those of the classical MAO of terrestrial vertebrates: they deaminate tyramine, tryptamine, serotonin, and benzylamine and do not deaminate histamine, have sensitivity to clorgyline, the specific inhibitor of the MAO A form, and deprenyl, the specific inhibitor of the MAO B form, and are not inhibited by 10⁻² M semicarbazide. Based on data of substrate-inhibitor analysis, a suggestion is put forward about the existence of two molecular forms of the enzyme in liver of the studied frog species. Quantitative interspecies differences have been revealed between liver MAO of Rana ridibunda and Rana temporaria in values of kinetic parameters of reactions of deamination of several substrates and in sensitivity to the inhibitors, deprenyl and clorgyline. In the species Rana temporaria the MAO activity in reaction of deamination of serotonin and benzylamine were virtually identical, whereas in the species Rana ridibunda these parameters for serotonin were almost one order higher than for benzylamine. In the species Rana ridibunda, selectivity of action of deprenyl was expressed many times weaker, while selectivity of the clorgyline—one order of magnitude stronger than in the species Rana temporaria. The catalytic activities towards all studied substrates of liver MAO of both studied amphibian species were several times lower as compared with the enzyme of rat liver.