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.

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.

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.

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.

Temperature-dependent enhancement of cell proliferation and mRNA expression for type I collagen and HSP70 in primary cultured goldfish cells.

Goldfish (Carasius auratus) primary culture cells derived from caudal fin were incubated over a temperature range of 20-35 degrees C. The population doubling time of cells cultured at 20, 25, 30 and 35 degrees C were 34, 29, 17 and 14 h, respectively. Interestingly, cDNA-representational difference analysis revealed type I collagen alpha chain (colalpha(I)) as a candidate for a warm temperature-specific gene. mRNA levels of colalpha(I) increased with an increase of incubation temperature and days of culture. Furthermore, the cell growth rate and colalpha(I) mRNA levels were rapidly changed following temperature shifts. To examine the effects of culture temperature shift on the cellular physiological states, mRNA levels of HSP70 were additionally investigated. HSP70 mRNA levels in the cells cultured at 30 and 35 degrees C were again 2-3 times higher than those at 20 and 25 degrees C. When the culture temperature was shifted from 20 to 35 degrees C, HSP70 mRNA levels were rapidly increased within 1 h. Subsequently, mRNA levels of the 35 degrees C-treated cells decreased, but remained doubled compared with those of the 20 degrees C-treated cells, even 4 h following the temperature shift. When the culture temperature was lowered from 35 to 20 degrees C, HSP70 mRNA levels decreased to about 70% of the original levels in 4 h. These results indicate that goldfish cells cultured at different temperatures easily develop temperature-associated steady physiological states within 4 h of temperature shifts.

Molecular and enzymatic properties of a cathepsin L-like proteinase with distinct substrate specificity from northern shrimp (Pandalus borealis).

We purified a cathepsin L-like proteinase to homogeneity from the hepatopancreas of northern shrimp Pandalus borealis by several chromatographic procedures. The purified proteinase showed the highest specificity for leucine residue at P2, a specificity pattern similar to cathepsins S and K whereas proline and arginine residues were not suitable as P2 substrates. However, unlike these proteinases, it accepted valine almost equally to the phenylalanine residue at P2. The shrimp cathepsin was strongly inhibited by E-64, leupeptin and antipain, while benzyloxycarbonyl-Phe-Tyr(t-Bu)-CHN2, a specific inhibitor of cathepsin L, remained largely ineffective. Next, we determined the primary structure of the shrimp enzyme by molecular cloning and investigated the residues constituting the S2 subsite, which is possibly involved in its unusual substrate specificity. The deduced amino acid sequence of the shrimp proteinase shared the highest identity of 65% with a cathepsin L-like proteinase from lobster, but its identity to the well-characterized mammalian cathepsins S, L, and K fell within narrower ranges of 52-55%. However, the shrimp proteinase differed from these cathepsins in some key residues including, for example, the unique occurrence of cysteine and glutamine residues at the structurally important S2 subsite. Interestingly, transcripts of this proteinase were exclusively detected in the shrimp gut coinciding with its broad pH activity and stability profiles, which is also unusual as a cysteine proteinase. These results suggest that the shrimp enzyme is homologous to mammalian cathepsins S, L, and K, but is distinct from each of these proteinases in both enzymatic and structural properties.

In vivo activation of pro-form Bombyx cysteine protease (BCP) in silkmoth eggs: localization of yolk proteins and BCP, and acidification of yolk granules.

The present study was designed to investigate the process of acidification of yolk granules during embryogenesis. In oocytes of mature Bombyx mori silkmoth, yolk proteins and a cysteine protease (pro-form BCP) were found in yolk granules. BCP was localized in small sized yolk granules (SYG, 3-6 microm in diameter) and yolk proteins in large sized granules (LYG, 6-11 microm in diameter), which might result in a spatial separation of protease and its substrates to avoid unnecessary hydrolysis. The granules were isolated on Percoll density gradient centrifugation. Although separation of LYG and SYG was incomplete, the granules sedimented in different fractions when using unfertilized egg extract, in which LYG was recovered from heavier fractions and BCP from lighter fractions. Acid phosphatase, as well as other lysosomal marker enzymes tested, was recovered from LYG-containing fractions. When extracts were prepared from developing eggs (day 3), some BCP-containing granules co-sedimented with LYG. The inactive pro-form BCP was activated in vivo, in parallel with yolk protein degradation, and as demonstrated previously in vitro under acidic conditions (). These results suggest that acidification occurs in yolk granules during embryogenesis. This was also confirmed using acridine orange fluorescent dye. In early development, most yolk granules were neutral, but became acidic during embryonic development. SYG were progressively recovered in heavier density fractions, displaying acidic interior. In this fraction, BCP-containing granules seem to be associated with larger granules (6-11 microm in size). In addition, SYG (BCP containing granules) were likely to be acidified earlier than LYG. Our results suggest that acidification initiates yolk degradation through activation of pro-form BCP.

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.

Bombyx cysteine proteinase inhibitor (BCPI) homologous to propeptide regions of cysteine proteinases is a strong, selective inhibitor of cathepsin L-like cysteine proteinases.

Bombyx cysteine proteinase inhibitor (BCPI) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregions of certain cysteine proteinases. Here we report the mechanism of its inhibition of several cysteine proteinases. BCPI strongly inhibited Bombyx cysteine proteinase (BCP) activity with a K(i) = 5.9 pM, and human cathepsin L with a K(i) = 36 pM. The inhibition obeyed slow-binding kinetics. The inhibition of cathepsin H was much weaker (K(i) = 82 nM), while inhibition of papain (K(i) > 1 microM) and cathepsin B (K(i) > 4 microM) was negligible. Following incubation with BCP, BCPI was first truncated at the C-terminal end, and then gradually degraded over time. The truncation mainly involved two C-terminal amino acid residues. Recombinant BCPI lacking the two C-terminal amino acid residues still retained substantial inhibitory activity. Our results indicate that BCPI is a stable and highly selective inhibitor of cathepsin L-like cysteine proteinases.

A novel heat stress-responsive gene in the marine diatom Chaetoceros compressum encoding two types of transcripts, a trypsin-like protease and its related protein, by alternative RNA splicing.

We investigated the change of mRNA expression patterns in the laboratory-grown diatom Chaetoceros compressum under heat-stress conditions by mRNA arbitrarily primed (RAP) RT-PCR. Cells grown at 20 degrees C were subjected to heat treatment at 30 degrees C for 15 min and subsequently maintained at 20 degrees C for 8 h. Four genes including HI-5 were detected as heat stress-responsive genes by fingerprint analysis of RAP RT-PCR. Cloning for full-length cDNA sequences of HI-5 transcripts and related genomic DNA analysis revealed that two types of mRNA, HI-5a and HI-5b, were transcribed from the single HI-5 gene. While the HI-5a protein contained a catalytic domain characteristic to trypsin-like proteases, the HI-5b protein lacked this domain due to an insertion in the associated mRNA of 112 nucleotides; this insertion sequence contained a stop codon near the central region. Quantitative RT-PCR was performed to investigate the changes in expression levels of the two types of mRNA following heat treatment. The HI-5b transcripts were constitutively expressed in both unstressed and heat-stressed cells. In contrast, the number of HI-5a transcripts markedly increased in cells immediately after heat stress, reaching levels 19-fold higher at 8 h after heat stress than that in unstressed cells. These results suggest that RNA splicing plays a key role in heat stress-dependent expression of the HI-5a and HI-5b transcripts from the single HI-5 gene in the diatom.

Topical application of HIV DNA vaccine with cytokine-expression plasmids induces strong antigen-specific immune responses.

The topical application of DNA vaccine to the skin is a useful method of immunization because of its simplicity, painlessness and economy. But the immune responses that it elicits are relatively low. In this study, we administered human immunodeficiency virus type-1 (HIV-1) DNA vaccine with cytokine-expressing plasmids to the skin of mice by a new topical application technique involving prior elimination of keratinocytes using fast-acting adhesive. Our results revealed that the topical application of HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. Co-administration of the DNA vaccine with cytokine expression plasmids of IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by this new method raised the levels of both the HIV-specific cytotoxic T lymphocyte (CTL) response and delayed-type hypersensitivity (DTH) and facilitated the induction of substantial immune responses by DNA vaccine. Skin biopsy sections, thus, immunized showed significant increases of S-100 protein-positive dendritic cells (DCs). These results suggest that the topical application method described here is an efficient route of DNA vaccine administration and that the immune response may be induced by DNA plasmids taken in by DCs, Langerhans cells (LCs), or others such as antigen-presenting cells. This new topical application is likely to be of benefit in clinical use.

Protection against influenza virus challenge by topical application of influenza DNA vaccine.

We studied the use of a DNA vaccine expressing the matrix (M) gene of the influenza virus A/PR/8/34. Mice were immunized by painting the DNA vaccine three times on the skin after removal of its keratinocytic layers. Immunization by this method produced M-specific antibodies and cytotoxic T lymphocyte (CTL) response, and acquired resistance against influenza virus challenge. This protection was abrogated by the in vivo injection of anti-CD8 or anti-CD4 monoclonal antibody. We further found that simultaneous topical application (t.a.) of GM-CSF expression plasmid (pGM-CSF) or liposomes plus mannan produced stronger immune response competence and enhanced the protective effect against influenza virus challenge. The present study revealed that administering DNA vaccine by topical application can elicit both humoral and cell-mediated immunity (CMI).

Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene.

DNA vaccination is characterized by its preferential induction of the cytotoxic T cell lymphocyte (CTL) response and is expected to be a useful means of protection against viral infection. We examined the protective effect of an expression plasmid (pME18S-M) containing M1 and M2 genes of influenza A/PR/8/34. We detected the CTL activity by introducing these plasmids into BALB/c mice by either the intramuscular or the intranasal route. The influenza-specific antibody response was also induced, although its neutralizing effect against influenza virus was not observed. From 70 to 80% protection was observed in the mice immunized with the pME18S-M plasmid followed by lethal infection with influenza viruses of the A/WSN/33 and A/PR/8/34 strains, whereas all mice without the plasmid vaccination failed to survive. This protective activity was significantly weakened when the CD8(+) cells of these immunized mice were eliminated by several injections of anti-CD8 antibody. The protective activity was also weakened when anti-CD4 antibody was injected in the early phase of DNA vaccination. These data suggest that the pME18S-M plasmid is useful as a DNA vaccine for overcoming highly mutational influenza viruses.

Kinetic and structural properties of two isoforms of trypsin isolated from the viscera of Japanese anchovy, Engraulis japonicus.

Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25 degrees C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.

Phosphorylation of molluscan twitchin by the cAMP-dependent protein kinase.

Catch in certain molluscan muscles is released by an increase in cAMP, and it was suggested that the target of cAMP-dependent protein kinase (PKA) is the high molecular weight protein twitchin [Siegman, M. J., Funabara, J., Kinoshita, S., Watabe, S., Hartshorne, D. J., and Butler, T. M. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 5384-5388]. This study was carried out to investigate the phosphorylation of twitchin by PKA. Twitchin was isolated from Mytilus catch muscles and was phosphorylated by PKA to a stoichiometry of about 3 mol of P/mol of twitchin. There was no evidence of twitchin autophosphorylation. Two phosphorylated peptides were isolated and sequenced, termed D1 and D2. Additional cDNA sequence for twitchin was obtained, and the D2 site was located at the C-terminal side of the putative kinase domain in a linker region between two immunoglobulin C2 repeats. Excess PKA substrates, e.g., D1 and D2, blocked the reduction in force on addition of cAMP, confirming the role for PKA in regulating catch. Papain proteolysis of (32)P-labeled twitchin from permeabilized muscles showed that the D1 site represented about 50% of the (32)P labeling. Proteolysis of in-situ twitchin with thermolysin suggested that the D1 and D2 sites were at the N- and C-terminal ends of the molecule, respectively. Thermolysin proteolysis also indicated that D1 and D2 were major sites of phosphorylation by PKA. The direct phosphorylation of twitchin by PKA is consistent with a regulatory role for twitchin in the catch mechanism and probably involves phosphorylation at the D1 and D2 sites.

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.

Molecular cloning and characterization of two isoforms of trypsinogen from anchovy pyloric ceca.

Complementary DNA clones encoding two isoforms of trypsinogen were isolated from the pyloric ceca of anchovy by rapid amplification of cDNA ends (RACE). Nucleotide sequences of isolated clones encoded, in addition to characteristic signal and activation peptides, two isoforms of trypsin containing 220 and 221 amino acid residues. Both enzymes contained the catalytic triad of a serine protease, together with the residues determining substrate specificity. The anchovy trypsins showed a high amino acid identity of about 80% to those of other fish species. Southern blot analysis with a probe cross-reactive to both isoforms showed a complex genomic pattern. Northern blot analysis with the same probe revealed the highest expression of messenger RNA in the pyloric ceca. Structural parameters possibly involved in higher catalytic properties of fish trypsin were examined by three-dimensional modeling, which included deletion in the autolysis loop, lack of Tyr-151 at the entrance of the S1 pocket, and distribution of charged residues.

Immunologic characterization of HIV-specific DNA vaccine.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.