Transcriptomic analysis of immunoglobulin novel antigen receptor (IgNAR) heavy chain constant domains of brownbanded bamboo shark (Chiloscyllium punctatum).

Cartilaginous fish are the evolutionarily oldest group of animals which possess antibodies, T cell receptors and major histocompatibility complex (MHC). The immunoglobulin novel antigen receptor (IgNAR) found in cartilaginous fish is a heavy chain homodimer which lacks light chain. The presence of non-canonical cysteine molecules and lack of CDR2 region make it more significant. To synthesize active binding domains based on variable region of IgNAR (VNAR), knowledge on the constant region dynamics play a significant role. The IgNAR exhibit species variations in its primary sequence features; hence, this study was conducted to determine the IgNAR heavy chain constant domain of the brownbanded bamboo shark (Chiloscyllium punctatum). Peripheral blood leukocytes (PBL) isolated from adult bamboo sharks were used to synthesize a cDNA library. A total of four billion residues of two million sequences (average length 218.41 bp) were obtained. Assembled sequences were aligned with published cartilaginous fish IgNAR constant region sequences. Transcriptome analysis revealed two distinct types of IgNAR in the brownbanded bamboo shark. Also, constant-1 domain sequences displayed 13 unique sequences which may reflect the least number of IgNAR gene clusters. The phylogenetic analysis revealed the closest relationship with the nurse shark (Ginglymostoma cirratum) followed by the wobbegong shark (Orectolobus maculatus) which belong to the same order Orectolobiformes. Analysis of the constant domains of the brownbanded bamboo shark IgNAR revealed an evolutionarily conserved nature and this knowledge can be used to design primers for VNAR cloning. Furthermore, knowledge on the structural features in IgNAR constant domains that increase the stability could be useful in the process of stabilizing human immunoglobulins.

Characterization and antimicrobial susceptibility of motile aeromonads isolated from freshwater ornamental fish showing signs of septicaemia.

A total of 74 phenotypically identified presumptive motile Aeromonas isolates recovered from septicaemic freshwater ornamental fish in Sri Lanka were genetically characterized by sequencing of rpoD and gyrB genes. rpoD/gyrB phylogeny confirmed only 53 isolates as Aeromonas, among which A. veronii was the predominant species (79.2%), followed by A. hydrophila (7.5%), A. caviae (5.7%), A. jandaei (1.9%), A. dhakensis (3.8%) and A. entero pelogenes (1.9%). The aeromonads confirmed by sequencing were further subjected to 16S rDNA PCR-RFLP which substantiated sequencing results for 83% of isolates. Fingerprinting of A. enteropelogenes (n = 42) using ERIC-PCR revealed no dominant clones, and the majority were genetically distinct. All isolates were screened by PCR for 7 virulence determinant genes (aer, act, ast, alt, fla, ser, exu) and 2 integrase encoding genes (intI1, intI2). Each isolate contained ≥3 of the virulence genes tested for, with a heterogeneous distribution. Of the isolates, 77% harboured the intI1 gene, while none had intI2. In vitro antimicrobial susceptibility testing showed highest resistances towards tetracycline (58.5%) and erythromycin (54.7%). Our results indicate the diverse range of aeromonads that could potentially be associated with motile aeromonad septicaemia in ornamental fish. This is the first isolation of A. dhakensis from a septicaemic ornamental fish since its original description from the same host.

Correlation with larval body size of mRNA levels of growth hormone, growth hormone receptor I and insulin-like growth factor I in larval torafugu Takifugu rubripes.

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

Fibre type-specific expression patterns of myosin heavy chain genes in adult torafugu Takifugu rubripes muscles.

Comprehensive in silico studies, based on the total fugu genome database, which was the first to appear in fish, revealed that torafugu Takifugu rubripes contains 20 sarcomeric myosin heavy chain (MYH) genes (MYH genes) (Ikeda et al., 2007). The present study was undertaken to identify MYH genes that would be expressed in adult muscles. In total, seven MYH genes were found by screening cDNA clone libraries constructed from fast, slow and cardiac muscles. Three MYH genes, fast-type MYH(M86-1), slow-type MYH(M8248) and slow/cardiac-type MYH(M880), were cloned exclusively from fast, slow and cardiac muscles, respectively. Northern blot hybridization substantiated their specific expression, with the exception of MYH(M880). In contrast, transcripts of fast-type MYH(M2528-1) and MYH(M1034) were found in both fast and slow muscles as revealed by cDNA clone library and northern blot techniques. This result was supported by in situ hybridization analysis using specific RNA probes, where transcripts of fast-type MYH(M2528-1) were expressed in fast fibres with small diameters as well as in fibres of superficial slow muscle with large diameters adjacent to fast muscle. Transcripts of fast-type MYH(M86-1) were expressed in all fast fibres with different diameters, whereas transcripts of slow-type MYH(M8248) were restricted to fibres with small diameters located in a superficial part of slow muscle. Interestingly, histochemical analyses showed that fast fibres with small diameters and slow fibres with large diameters both contained acid-stable myofibrillar ATPase, suggesting that these fibres have similar functions, possibly in the generation of muscle fibres irrespective of their fibre types.

Activation of cytotoxic polymorphonuclear leukocytes by in vivo administration of a streptococcal preparation, OK-432.

Injection ip of a streptococcal preparation, OK-432, into WKA and DONRYU rats induced in vitro cytotoxicity of polymorphonuclear leukocytes (PMN) against tumor cells in terms of their cytostasis and cytolysis. PMN were obtained from peritoneal exudates of WKA and DONRYU rats. Cytotoxicity of activated PMN was immunologically nonspecific, although a certain target selectivity was observed in PMN cytotoxicity. The titer of cytotoxicity of PMN was dependent on the dose of injected OK-432 and on the number of PMN in the cytotoxicity assay. Activated PMN appeared very early (6 hr) after OK-432 injection, and the cytotoxic titer of PMN decreased from 24 hours and disappeared at 96 hours after injection of the reagent. In vitro culture of PMN with OK-432 also evoked cytotoxicity of PMN.

5'-flanking sequences of zebrafish fast myosin heavy chain genes regulate unique expression in the anterior, medial subsection and posterior tail somites of the skeletal muscle.

In zebrafish, fast muscle-specific myosin heavy chain genes have their unique expression patterns in a well-defined and restricted region of the skeletal muscle. However, the transcriptional regulatory mechanisms involved have remained unclear. Here, we examined the regulation of spatio-temporal expression patterns of myhz1 (myhz1.1, myhz1.2 and myhz1.3) and myhz2 during their development by using transient gene and stable transgenic techniques. Embryos microinjected with different length 5'-flanking sequences of myhz1 conjugated with the enhanced green fluorescent protein (EGFP) gene showed EGFP expression in the anterior and medial subsections of somites, but not in the tail somite region. In contrast, embryos microinjected with different length 5'-flanking sequences of myhz2 showed EGFP expression exclusively at the posterior tail somite domain. Promoter deletion analyses demonstrated that reduced EGFP fluorescence typically is correlated with smaller 5'-flanking sequences. The immunohistochemical observation revealed that zebrafish larvae provided with the transient gene and those from stable transgenic lines consistently expressed EGFP in the fast muscle fibers. r-VISTA plot identified one common conserved region of about 140°bp among myhz1.1, myhz1.2 and myhz1.3. Deletion of this conserved region from the 5'-flanking sequence of each myhz1 markedly reduced EGFP expression in its unique spatial somite region. Deletion mutation analysis demonstrated that myhz2 expression in the tail somite region might be mediated by Tbx (family of transcription factors having a common DNA-binding sequence known as T-box) binding elements. In summary, 5'-flanking sequences of myhz1 and myhz2 regulate their unique expression patterns in a well-defined and restricted somite region of the skeletal muscle in zebrafish.

Structure and expression of mRNA for vitellogenin in Bombyx mori.

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81-85) on the biosynthesis of B. mori vitellogenin is also discussed.

The novel sequences of major plasma apolipoproteins in the eel Anguilla japonica.

cDNAs encoding major plasma apolipoproteins (apo) were cloned from the eel Anguilla japonica liver and their nucleotide sequences determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that eel lipoproteins contain apolipoproteins of 28 kDa and 14 kDa as major components. Each of the two apolipoproteins showed two isoforms having different isoelectric points as demonstrated by two-dimensional electrophoresis. The two 28 kDa components had different N-terminal amino acid sequences, whereas the two 14 kDa components had an identical one. Then cDNA clones encoding these apolipoproteins were isolated from a cDNA library constructed from the eel liver. An acidic 28 kDa component (28 kDa-1) consisted of 259 amino acids including a putative signal peptide of 27 residues, whereas a basic 28 kDa component (28 kDa-2) was composed of 260 amino acids containing a putative signal peptide of 23 residues. The tandem repeating units, which are characteristic of apolipoproteins, for 28 kDa-1 showed 27.8% identity to that of porcine apoA-IV, although mammalian apoA-IV is about 40 kDa and much larger than 28 kDa-1. However, the repeating units of 28 kDa-2 showed 52.5% identity to that of Atlantic salmon apoA-I. The 14 kDa apolipoprotein consisted of 142 amino acids containing a putative signal peptide of 20 residues. It has a novel sequence differing from apolipoproteins of other vertebrates. The transcriptional expressions of 28 kDa-1, 28 kDa-2, and 14 kDa components were all restricted to the liver, except for the transcripts of 28 kDa-2 which were also slightly expressed in the intestine.

Feasibility of measuring 5-fluorouracil catabolic potential by oral loading.

The efficacy of 5-fluorouracil (5-FU) treatment and the incidence of adverse events differ among patients and depend to some extent on individual variations in drug catabolism. This feasibility study aimed to determine the optimum conditions for a 5-FU oral load test, which would allow the simple evaluation of individual differences in 5-FU catabolism. Patients with colon cancer were given oral 5-FU (200 mg/day) for 3 days (n = 36) or a single 100 mg dose (n = 14). Serum concentrations of uracil, dihydrouracil, 5-FU and 5-fluoro-5,6-dihydrouracil were measured before and after 5-FU administration. The results suggested that a decline in 5-FU metabolism was associated with continuous administration and increasing age. We conclude that a continuous load of 5-FU is necessary in order to predict the efficacy and side-effects of the drug. The 3-day regimen, with its ease of administration, merits further study to assess its possible clinical application.

Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases.

Propeptides of papain-like cysteine proteinases such as papain, cathepsins B, L and S are potent inhibitors of their cognate cysteine proteinases with Ki values in the nanomolar range, and they exhibit highest inhibition selectivity for enzymes from which they originate. Recent studies have identified novel inhibitor proteins that are homologous to the proregions of papain-like cysteine proteinases. Mouse activated T-lymphocytes express cytotoxic T-lymphocyte antigen (CTLA-2), which is homologous to the proregion of mouse cathepsin L. CTLA-2 exhibits inhibitory activities to several cysteine proteinases. We have also identified a similar propeptide-like cysteine proteinase inhibitor, Bombyx cysteine proteinase inhibitor (BCPI), in the silkmoth Bombyx mori. BCPI is a slow and tight binding inhibitor of cathepsin L-like cysteine proteinases with Ki values in picomolar range, and the inhibition is highly selective towards these proteinases just like the propeptides. Recent genome analyses have shown the expression of similar propeptide-like proteins in Drosophila and rat, suggesting the presence of a novel class of cysteine proteinase inhibitors in a variety of organisms. Studies of the gene structures and phylogenetic analysis have shown that genes of the propeptide-like cysteine proteinase inhibitors have emerged from ancestor genes of their parental enzymes.

Characterization of the carp myosin heavy chain multigene family.

We isolated partial coding sequences for 29 carp myosin heavy chain genes (MyoHCs) and determined the nucleotide sequences around the region encoding the loop 2 of the myosin molecule. The predicted amino acid sequences from the isolated genes all showed very high similarity to those of skeletal and cardiac muscles from higher vertebrates, but not to those of smooth and non-muscle counterparts. Among all clones isolated, carp MyoHC10, MyoHCI-1-3 and MyoHC30 showed exon-nucleotide sequences identical to those of cDNAs encoding the loop 2 region of the 10 degrees C-, intermediate- and 30 degrees C-type fast skeletal isoforms [Hirayama and Watabe, Euro. J. Biochem. 246 (1997) 380-387]. The loop 2 of 28 types of carp MyoHCs was encoded by two exons separated by an intron corresponding to that of the 16th in higher vertebrate MyoHCs, whilst this intron was not found in carp MyoHC30. Although carp MyoHC30 had a gene organization different from those of higher vertebrates and other carp MyoHCs, its predicted amino acid sequence for loop 2 showed the highest homology to those of higher vertebrates among carp MyoHCs. In the 28 carp MyoHCs containing the intron, a combination of different nucleotide sequences for the two resulted in 14 distinct series for the combined coding sequence. These different nucleotide sequences encoded nine distinct amino acid sequences. Phylogenetic analysis for the present loop 2 and light meromyosin previously reported for carp MyoHCs [Imai et al., J. Exp. Biol. 200 (1997) 27-34] revealed that carp MyoHCs have recently diverged and are more closely related to each other than to MyoHCs from other species.

An ice nucleation active gene of Erwinia ananas. Sequence similarity to those of Pseudomonas species and regions required for ice nucleation activity.

The ice nucleation active gene, inaA, of Erwinia ananas IN-10 has been sequenced. This gene encodes a protein composed of 1322 amino acid residues. The inaA protein contains a 1120-residue segment consisting of 70 repeats of closely related 16 amino acid motifs (R-domain), which is flanked by N- and C-terminal sequences (N- and C-domains, respectively). Its primary structure is similar to, but not identical with, those of Pseudomonas inaW and inaZ gene products. By truncating the inaA gene to various extents, it was found that deletion of the C-domain resulted in complete loss of the ice nucleation activity, whereas removal of the N-domain led to a moderate decrease in the activity. Complete loss of the activity was also observed when the N-domain plus a large part of the P-domain were deleted. It is suggested that the C-domain is required for the assembly of inaA protein to form a functional ice nucleus.

A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases.

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.

ATP-dependent protease in human placenta.

We have found that human placental mitochondria contain ATP-dependent protease in a soluble form. The molecular weight of the protease have been up to 108,000, which is the same as ATP-dependent protease in bovine adrenal cortex. Since this protease has been distributed among steroid hormone-producing tissues such as testis and adrenal cortex and ATP-dependent protease can degrade cytochrome P-450scc, a key enzyme in steroid hormone biosynthesis, we suggest that the protease may have an important role in the regulation of steroid hormone metabolism.

Postoperative bronchopleural fistula: clinical and experimental study.

It is a well-known fact that in pulmonary tuberculosis patients treated by resection, the quality of the suture material used for closing the bronchial stump plays an important role in the pathogenesis of postoperative bronchopleural fistula. Of 426 cases treated surgically and in whom silk suture thread was used, 23 developed bronchopleural fistula, whereas none of the 220 cases sutured with nylon monofilament developed abnormality. Statistical analysis of 100 surgical cases with silk thread suture and of 100 cases with nylon monofilament suture showed that the two groups had no marked differences as to background factors. Howevers, as compared with the silk-thread suture group, the nylon-monofilament suture group revealed more consistently favorable postoperative bronchoscopic findings. Experimental studies with dogs showed a similar lack of complications when the monofilament suture material was used, as contrasted were conducted in hospital by the same surgical personnel using the same procedures, it can be said that, to insure prevention of complications, the suture material for bronchial stump closure should be of non-irritating nature and preferably of monofilament strength and quality, such as nylon monofilament.

The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic ACh receptors.

Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimer's disease.

A new enzyme, NADPH-dihydropteridine reductase in bovine liver.

An enzyme designated as NADPH-dihydropteridine reductase was found in the extract of bovine liver and partially purified. In contrast to NADH-dpendent dihydropteridine reductase [EC 1.6.99.7], the enzyme catalyzes the reduction of quinonid-dihydropterin to tetrahydropterin in the presence of NADPH. The two enzymes were separated by column chromatography on DEAE-sephadex. Tyrosine formation in the phenylalanine hydroxylation system was also stimulated by NADPH-dihydropteridine reductase. The existence of these two dihydropteridine reductases suggests that the tetrahydro from ofpteridine cofactor may be regenerated in two different ways in vivo.

The pH-dependency of myosin ATPases from yellowtail ordinary and dark muscles.

Myosins were isolated from the ordinary (white) and dark (red) muscles of yellowtail, Seriola quinqueradiata, and the pH-dependency of ATPase activity, along with some physicochemical properties, was examined. The ordinary muscle myosin contained three kinds of light chain (A1, DTNB and A2 light chains), the molecular weights of which were 28,000, 20,000, and 16,000, respectively. The dark muscle myosin possessed only two kinds of light chain (D1 and D2), the molecular weights of which were 26,000 and 20,000 respectively. These ordinary and dark muscle myosins resemble the fast and slow muscle myosins of the higher vertebrate, respectively, in light chain pattern. The pH optima of the ordinary muscle myosin Ca2+-ATPase activity appeared at 6-6.5 and 9-10, irrespective of whether the enzyme reaction was started by the addition of ATP to the preincubated reaction mixture containing myosin (method I), or vice versa (method II). In the case of the dark muscle myosin, a small peak appeared at around pH 8.5 on the alkaline side when the activity was assayed by method I, whereas a prominent peak appeared at around 9.5 when it was assayed by method II, suggesting instability of this myosin under alkaline conditions. In connection with this, the reaction mixture at pH 9.5 showed a very small and slow increase in turbidity, suggesting a change in the physical state of myosin. The ordinary muscle myosin exhibited approximately three times higher actin-activated Mg2+-ATPase activity than the dark muscle myosin. Superprecipitation activity was also higher in the former than the latter actomyosin. However, both actomyosins showed similar pH-superprecipitation activity profiles.

Myosin subfragment-1 isoforms having different heavy chain structures from fast skeletal muscle of thermally acclimated carp.

Three heavy chain isoforms of chymotryptic myosin subfragment-1 (S1) with different molecular sizes of 96 kDa (H1), 94 kDa (H2), and 92 kDa (H3), were detected in the fast skeletal muscle from thermally acclimated carp. In total, six S1 isoforms were present, including two S1 isoforms for each heavy chain due to associated A1 and A2 light chains. H1 heavy chain was dominant in the 10 degrees C-acclimated carp and responsible for high acto-S1 Mg(2+)-ATPase activity and low thermostability. In contrast, H3 heavy chain predominating in the 30 degrees C-acclimated carp showed low acto-S1 Mg(2+)-ATPase activity and high thermostability. H2 heavy chain was found in the 10- and 20 degrees C-acclimated fish. H3 heavy chain featured three tryptic fragments with normal molecular masses of 25, 50, and 20 kDa in order from the N-terminus. However, H1 heavy chain contained an unusual, longer "20 kDa" peptide whose molecular size was estimated to be about 23 kDa.

Characterization of porcine adrenocortical lysosomes.

Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase were found exclusively in the lysosomes, while alpha-mannosidase and beta-glucuronidase were in both the lysosomal and microsomal fractions. The activity of cathepsin D was remarkably high, amounting to more than 6 times that in porcine liver and to more than 10 times that in liver of Sprague-Dawley rats in terms of units per g wet tissue. Porcine adrenocortical lysosomes showed a modal isopycnic density value of 1.155, but mitochondria a value of 1.145. The validity of these values was studied by investigating the possibilities of agglutination of organelles, damage to lysosomal membranes, disruption of mitochondria due to the hydrostatic pressure and by applying the same procedures of isopycnic centrifugation to hog and rat livers. After these validity tests, porcine adrenocortical lysosomes were concluded to be unique in their strikingly high content of cathepsin D as well as in their low modal isopycnic density which is very close to that of porcine adrenocortical mitochondria.