Morphological and molecular characterization of Anisakis larvae (Nematoda: Anisakidae) in Beryx splendens from Japanese waters

The third-stage (L3) larvae of Anisakis, which are the etiological agents of human anisakiasis, have been categorized morphologically into Anisakis Type I larvae and Anisakis Type II larvae. Genetic analysis has allowed easy identification of these larvae: Anisakis Type I larvae include the species Anisakis simplex sensu stricto, Anisakis pegreffii, Anisakis simplex C, Anisakis typica, Anisakis ziphidarum, and Anisakis nascettii, whereas Anisakis Type II larvae include the species Anisakis physeteris, Anisakis brevispiculata, and Anisakis paggiae. Since human consumption of raw fish and squid is common in Japan, we investigated Anisakis L3 larvae in 44 specimens of Beryx splendens from Japanese waters. A total of 730 Anisakis L3 larvae collected from B. splendens were divided morphologically into 4 types: Type I, Type II, and 2 other types that were similar to Anisakis Type III and Type IV described by Shiraki (1974). Anisakis Type II, Type III, and Type IV larvae all had a short ventriculus, but their tails were morphologically different. In addition, data from genetic analysis indicated that Anisakis Type II, Type III, and Type IV larvae could be identified as A. physeteris, A. brevispiculata, and A. paggiae, respectively. Therefore, A. physeteris, A. brevispiculata, and A. paggiae can be readily differentiated not only by genetic analysis but also by morphological characteristics of L3 larvae.     

Identification of the polypeptides of the major light-harvesting complex of photosystem II (LHCII) with their genes in tomato.

Using an improved SDS-PAGE system, the polypeptides of the major chlorophyll a/b light-harvesting complex of PSII (LHCII) from tomato leaves were resolved into five polypeptide bands. All the polypeptides were matched with the genes encoding them by comparing amino acid sequences of tryptic peptides with gene sequences. The two major LHCII bands (usually comigrating as a '27 kDa' polypeptide) were encoded by cab1 and cab3 (Type I LHCII) genes. A third strong band of about 25 kDa was encoded by cab4 (Type II) genes. Polypeptides from two minor bands of 23-24 kDa were not N-terminally blocked; their N-terminal sequences showed they were Type III LHCII proteins. One complete cDNA clone and several incomplete clones for Type III polypeptides were sequenced. Combined with the peptide sequences, the results indicate that there are at least four different Type III genes in tomato, encoding four almost identical polypeptides. Thus, all the LHCII CAB polypeptides have been identified, and each type of LHCII polypeptide is encoded by distinct gene or genes in tomato.     

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.     

Influence of host habitat on the occurrence of gill monogeneans Pseudodactylogyrus spp. on wild Japanese eels Anguilla japonica

With technological improvements in otolith microchemical analysis, the flexible use of habitat from coastal marine to fresh waters has been discovered in Japanese eels Anguilla japonica. We examined the occurrence of 3 congeneric gill monogeneans-Pseudodactylogyrus anguillae, P. bini, and P. kamegaii-on wild Japanese eels, in relation to the host's flexibility. From April 2008 to October 2009, 114 eels were collected from a brackish-water cove and 2 rivers flowing into the cove in Ehime Prefecture, western Japan. Based on otolith microchemical analysis, the eels were discriminated according to the following 4 types of habitat use: freshwater residents (Type I), individuals utilizing low-salinity habitats (Type II), downstream habitat-shifters (Type III), and cove residents (Type IV). P. anguillae occurred mainly on Type I and II eels, while P. bini was primarily found on Type I eels. In contrast, P. kamegaii occurred mainly on Type III and IV eels. Thus, we conclude that species composition and infection levels of Pseudodactylogyrus spp. clearly differed with habitat-use patterns of Japanese eels. Also, since P. anguillae was scarcely found on either Type III or IV eels, this study suggests that previous identifications of monogeneans collected from European brackish-water localities as P. anguillae may require verification.     

Complete amino acid sequence of the type III isozyme of rat hexokinase, deduced from the cloned cDNA

Clones containing cDNA coding for the Type III isozyme of rat hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were isolated from a library prepared in lambda gt10 with rat liver mRNA. Three clones were characterized. Their composite sequence includes the entire coding region for Type III hexokinase, 3' untranslated sequence extending into the polyadenylated region, and 80 bp of 5' untranslated sequence. Extensive similarity in sequence of N- and C-terminal halves of the enzyme, previously seen with the Type I isozyme, is consistent with the view that these 100-kDa mammalian hexokinases are the evolutionary result of duplication and fusion of a gene coding for an ancestral hexokinase having a molecular weight of approximately 50 kDa. Extensive similarities are seen between sequences of the Type I and III isozymes, and those reported for mammalian glucokinase (also called Type IV hexokinase) and for the hexokinase and glucokinase of yeast. Residues thought to be involved in catalytic function are highly conserved in all of these enzymes. Based on a quantitative comparison of sequence similarities, it is concluded that the 50-kDa mammalian glucokinase is more closely related to the 100-kDa mammalian enzymes than it is to the 50-kDa enzymes from yeast. One interpretation of this might be that the mammalian glucokinase arose by resplitting of the gene coding for the 100-kDa mammalian hexokinases.     

Expression of a cystine-rich fish antifreeze in transgenicDrosophila melanogaster

We have usedDrosophila melanogaster as a model system for the transgenic expression of cystine-rich Type II antifreeze protein (AFP) from sea raven. This protein was synthesized and secreted into fly haemolymph where it migrated as a larger species (16 kDa) than the mature form of the protein (14 kDa) as judged by immunoblotting.Drosophila-produced Type II AFP demonstrated antifreeze activity both in terms of thermal hysteresis (0.13 °C) and inhibition of ice recrystallization. Recombinant AFP was purified and N-terminal sequencing revealed a 17 aa extension that began at the predicted signal peptide cleavage point. The expression of all three AFP types in transgenicDrosophila has now been achieved. We conclude that the globular Type II and Type III AFPs are better choices for antifreeze transfer to other organisms than is the more widely used linear Type I AFP.     

The external morphology and distribution of cuticular hair organs on the claws of the American lobster, Homarus Americanus (Milne-Edwards)

Four classes of microscopic cuticular hair organs were found on the chelae of Homarus americanus (Milne-Edwards). Type I and Type II organs possess long single sensillae, 30–60 μm and 70–130 μm, respectively. Type III organs are toroid bumps 20–30 μm in diameter, with a small tuft of fibers projecting from the center. Type IV organs are small conical hairs ≈ 1 μm in length.Type IV organs were uniformly distributed over the claws. The distributions of Types I, II, and III organs were analysed through multivariate analysis of variance (MANOVA) with respect to claw, side, and area. Type I organs showed significance of the three-way interaction only. Type II organs showed significance of side, area, and the claw by area interaction. Type III organs showed significance of area, and all interactions. Changes in number and density of Types I, II and III organs, and in number of Type IV organs, were found with growth.     

Neural organization of the lamina neuropil of the larva of the tiger beetle (Cicindela chinensis)

Six neural elements, viz., retinular axons, a giant monopolar axon, straight descending processes (type I), lamina monopolar axons (type II), processes containing clusters of dense-core vesicles (type III), and processes coursing in various directions with varicosities (type IV), have been identified at the ultrastructural level in the lamina neuropil of the larval tiger beetle Cicindela chinensis. Retinular axons make presynaptic contact with all other types of processes. Type I and II processes possess many pre-and postsynaptic loci. Type II processes presumably constitute retinotopic afferent pathways. It remains uncertain whether type I processes are lamina monopolar axons or long retinular axons extending to the medullar neuropil. Type III processes may be efferent neurons or branches of afferent neurons contributing to local circuits. A giant monopolar axon extends many branches throughout the lamina neuropil; these branches are postsynaptic to retinular axons, and may be nonretinotopic and afferent. Type IV processes course obliquely in the neuropil, being postsynaptic to retinular axons, and presynaptic to type I processes.     

Correlation of apoproteins with the genes of the major chlorophyll a/b binding protein of photosystem II in Arabidopsis thaliana. Confirmation for the presence of a third member of the LHC IIb gene family

The major light-harvesting complex in higher plants is LHC IIb. The LHC IIb of Arabidopsis thaliana contains 2 pigment-binding apoproteins of 28 and 25 kDa. To determine the relationship between them and the LHC IIb gene family members, each protein was purified to homogeneity, subjected to direct protein sequencing, and the sequences compared with those deduced from LHC IIb genes in this organism. The 28 kDa protein is the product of Type I LHC IIb genes. The 25 kDa LHC IIb component is distinctly different from the 28 kDa LHC IIb protein, and is more closely related to the type III LHC IIb gene product of barley. Type III gene products lack the first 9-11 residues found in proteins of the Type I and II genes, a region that contains a phosphorylatable threonine residue. The lack of the N-terminal residues explains why this LHC IIb apoprotein has never been seen to be phosphorylated, and partly or wholly why it is smaller. The implication of the missing N-terminus on uptake of LHC II precursor proteins into the plastid and of the relative organization of the LHC IIb subunits in the PS II antenna is discussed.     

Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings

Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.     

Effect of deletions at structural domains of group II intron bI1 on self-splicing in vitro

Some group II introns can undergo a protein-independent splicing reaction with the basic reaction pathway similar to nuclear pre-mRNA splicing and the catalytic functions of some of the structural components have been determined. To identify further functional domains, we have generated an ensemble of partial and complete deletions of domains I, II, III and IV of the self-splicing group II intron bI1 from yeast mitochondria and studied their effects on the splicing reaction in vitro. Our results indicate that domains II and IV, which vary considerably in length and structure among group II introns, do not play a direct role in catalysis but mainly help to ensure the proper interaction between upstream and downstream catalytically active structural elements. Deletions of sub-domains of domain I and domain III indicate that these elements are involved in 5' cleavage by hydrolysis and in a reaction in trans (exon reopening), and that this function can be inhibited without affecting the normal 5' cleavage by transesterification. Yet, we infer that the helical structures affected by the mutational alterations might not contribute to this reaction mode per se but that changes within local secondary structures perturb the internal conformation of the ribozyme. Furthermore, we have designed an abbreviated version of intron bI1, with a length of 542 nucleotides, which is still catalytically active.     

Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u

Four kinds of thermostable chitinase were isolated from the cell-free culture broth of Bacillus licheniformis X-7u by successive column chromatographies on Butyl-Toyopearl, Q-Sepharose, and Sephacryl S-200. We named the enzymes chitinases I(89 kDa), II(76 kDa), III(66 kDa) and IV(59 kDa). Chitinases II, III and IV possessed extremely high optimum temperatures (70-80 degrees C), showing remarkable heat stability. Chitinases II, III and IV produced (GlcNAc)2 and GlcNAc from colloidal chitin and chitinase I predominantly produced (GlcNAc)2. The action pattern of chitinase I on PN-(GlcNAc)4 also showed a stronger propensity to cleave off the (GlcNAc)2 unit from the non-reducing end than the other three chitinases. Chitinases II, III and IV catalyzed a transglycosylation reaction that converted (GlcNAc)4 into (GlcNAc)6.     

A whole range of fine structural criteria for immunohistochemically identified LH cells in rats

Summary Pituitaries from normal, young and adult male rats were fixed either in sublimate-formalin or in glutaraldehyde-osmium. In adjacent Paraplast sections, almost all the gonadotrophs were immunostained with both LH and FSH antisera. The rat LH and FSH antisera used were shown to be highly specific by the absorption test and by double antibody radioimmunoassay. Thin and thick adjacent Epon sections were prepared for EM and immunohistochemical examination. Cells stained with the rat LH antiserum were identified by LM, and then observed in detail by EM. On the basis of these observations we suggest that the LH cells are arranged in a sequence of basophils, i.e., Types II/III, III, III/IV and IV: Type II/III basophils are elongate with a cytoplasmic process and less vesiculated. They have morphological features of Type II (classical thyrotrophs) and also of Type III basophils. Type III basophils are oval in shape and moderately vesiculated. Both Types II/III and III basophils can be divided into two classes of cell characterized mainly by the existence of only small secretory granules (150–220 nm in diameter) (Type A) or by the coexistence of small and large (350–500 nm) (Type B). Type III/IV basophils are cells intermediate between types III and IV basophils, and moderately vesiculated with an abundance of secretory granules (150–300 nm in diameter). Type IV basophils are large, spherical or oval cells whose RER cisternae are conspicuously dilated; they contain less numerous secretory granules (150–300 nm in diameter). It is concluded that LH cells are not a single cell type, but include a wide range of subtypes.     

Isolation and partial characterization of the entire human pro alpha 1(II) collagen gene.

Using a cDNA probe specific for the bovine Type II procollagen, a series of overlapping genomic clones containing 45 kb of contiguous human DNA have been isolated. Sequencing of a 54 bp exon, number 29, provided direct evidence that the recombinant clones bear human Type II collagen sequences. Localization of the 5' and 3' ends of the gene indicated that the human Type II collagen gene is 30 kb in size. This value is significantly higher than that of the homologous avian gene. The segregation of a polymorphic restriction site in informative families conclusively demonstrated that the Type II gene is found in a single copy in the human haploid genome. Finally, sequencing of a triple helical domain exon has confirmed that a rearrangement leading to the fusion of two exons occurred in the pro alpha 1(I) gene, following the divergence of the fibrillar collagens.     

Enzymopenic hereditary methemoglobinemia: a clinical/biochemical classification

Recessively inherited NADH-cytochrome B5 reductase deficiency, when present in the homozygous or doubly heterozygous form, is manifested by two different clinical presentations, depending on the nature and cellular distribution of the mutant enzyme. The observations supporting a clinical and biochemical classification of enzymopenic hereditary methemoglobinemia are summarized. Type I, with deficiency demonstrable only in the erythrocytes, presents as uncomplicated, benign methemoglobinemia. Type II, generalized cytochrome B5 deficiency demonstrable in all of the tissues that have been examined, is accompanied by severe, lethal, progressive neurological disability, in addition to methemoglobinemia. Type III deficiency is limited to hematopoietic cells and resembles Type I clinically. Type IV, also clinically like Type I, is associated with deficiency of the cofactor, cytochrome B5. Except for Type IV, the different types appear to be the result of mutations in paired alleles of a gene on chromosome 22 that affect the catalytic activity or stability of the cytochrome B5 reductase.     

Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.