Humoral and Cellular Immune Response of European Seabass Dicentrarchus labrax Vaccinated with Heat-Killed Mycobacterium marinum (iipA::kan Mutant).

No vaccine is yet commercially available against Mycobacterium marinum, the etiological agent of fish mycobacteriosis (also known as "fish tuberculosis"). The mycobacterial gene responsible for invasion and intracellular persistence, iipA, is known to moderate M. marinum pathology in Zebrafish Danio rerio. Two doses of heat-killed, wild-type, virulent M. marinum and two doses of a heat-killed, avirulent M. marinum iipA::kan mutant strain were used in parallel to vaccinate European Seabass Dicentrarchus labrax. The fish were then challenged with live, virulent M. marinum, and the pathogenesis of the infection was monitored. High specific immunoglobulin M (IgM) response and an increase in cytokine tumor necrosis factor alpha (TNF-α) messenger RNA expression levels were observed in all vaccinated fish. At 1 month postchallenge, TNF-α expression levels increased in spleen tissues of fish vaccinated with the virulent type and in those of unvaccinated fish, whereas in the head kidney, expression was up-regulated only in unvaccinated fish. The expression then decreased, and at 2 months postchallenge, expression appeared similar in all vaccination types. The highest survival rate (75%) was recorded in the group of fish that were vaccinated with a high dose of avirulent iipA::kan mutant. The iipA::kan mutant induced a strong immune response accompanied by only modest tissue disruption. Coupled with an effective program of booster treatments, the iipA::kan mutant vaccine may be developed into a powerful preventive measure against fish mycobacteriosis.

Cadherin 6 is activated by Epstein-Barr virus LMP1 to mediate EMT and metastasis as an interplay node of multiple pathways in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial malignancy, which is notorious among head-and-neck cancers with its metastatic feature. Epstein-Barr virus (EBV) infection plays a fundamental role in NPC development with the mechanism is not well understood. Here we demonstrate that EBV oncoprotein LMP1 drives EMT and metastasis of NPC by reactivating the adhesion molecule, cadherin 6 (CDH6), which normally occurs in embryogenesis with unknown role in NPC. CDH6 was found to be upregulated in LMP1-positive NPC tissues, and was identified as a target of the epithelium-specific miR-203. LMP1-activated NF-κB transcriptionally repressed the miR-203 expression by binding to the promoter region of miR-203 gene. CDH6 activation in turn induced EMT and promoted metastasis in NPC. CDH6 depletion, NF-κB inhibitor and miR-203 overexpression were able to impair the EMT effects. The miR-203 downregulation in NPC tissues was strongly associated with metastasis clinically. The CDH6 activator, Runt-related transcription factor 2 (RUNX2), was also activated by EBV in the event. For both CDH6 and RUNX2 are components at TGF-ß downstream, CDH6 became a node protein for the interplay of multiple signalings including NF-κB and TGF-ß. Therefore, the switch-on of miR-203 was important for nasopharyngeal epithelial cells to maintain normal phenotype. This study demonstrates that EBV has evolved sophisticated strategies by driving epithelial cells to obtain malignant features, particularly in NPC metastasis, providing novel biomarkers for the therapy and prognosis of EBV-associated NPC.

[Clinicopathologic characteristics and prognosis of early onset prostate cancer].

Objective: To observe the clinicopathologic features and prognosis of prostate cancer (PCa) in young men. Methods: Twenty-eight early onset (≤55 years) patients with PCa pathologically confirmed in the Peking University Third Hospital and Peking University Shougang Hospital from January 1st 2000 to August 31st 2016 were collected. There were 18 radical prostatectomy (RP) cases and 10 transrectal prostatic biopsy cases. Contemporaneously, 445 elderly (>55 years) patients were collected, of which 385 had detailed pathological information, were chosen as control group. The mean age of young group was 51 years (29-55 years). Follow-up data for 22 cases were available (1-110 months). The correlation of the clinicopathological features and prognosis were analyzed retrospectively. Results: Presurgical prostatic specific antigen (PSA) level was abnormal in young patients, with 18 cases (64.3%) had elevated fPSA level, 26 (92.9%) had increased tPSA level, and 26 (92.9%) had decreased fPSA/tPSA ratio. Gleason score (GS) was 8 in 10.7% (3/28) of cases, and 9 in 42.9% (12/28) of cases. Of the 18 patients with RP, 17 (94.4%) had pT stage ≥pT2c. PSA level (P=0.006) and GS (P=0.001) were positively correlated with pT stage. Family history of PCa in 1st degree relatives was found in 9.1% of the cases. During follow-up, 2 patients died of PCa, 7 patients showed progression within 24 months. There were no significant differences in PSA level and GS between young patients and elderly patients, while the former group was more likely to have incontinence (P=0.023), higher PSA levels (P=0.001), and lower overall survival (P=0.049). Only postsurgical PSA level was found to be negatively associated with overall survival (P=0.030) and cancer specific survival (P=0.021) in young patients. Conclusions: Presurgical PSA level and GS are positively correlated with pT stage of early onset PCa. Compared with elderly patients, young patients are more likely to have incontinence, higher postsurgical PSA level, and lower overall survival. Among all the parameters, only postsurgical PSA level shows an adverse impact on prognosis of early onset PCa. Young patients, especially those with family history, may benefit from studies on the susceptibility loci and phenotype of PCa.

[Patients with prostatic cancer undergoing radical prostatectomy after single positive core biopsy: a clinicopathologic analysis of 62 cases].

OBJECTIVE: To evaluate the clinical and pathological features of patients with prostate cancer who had only one positive biopsy core and underwent radical prostatectomy (RP). METHODS: A total of 62 patients with prostate cancer who had only one positive biopsy core and underwent RP were enrolled. The clinicopathological features of the biopsy and RP were analyzed. Those factors that may cause discordance of Gleason score (GS) and influence pathological T (pT) stage on RP were further evaluated. RESULTS: Out of the 62 patients, 40(64.5%) had pT stage ≥pT2c. The positive surgical margins, extraprostatic extension and seminal vesicle invasion were found in 17.7%(11/62), 9.7%(6/62) and 3.2%(2/62) of patients, respectively. Compared to GS of RP, that of biopsy specimens was concordant in 39 (62.9%), higher in 6 (9.7%), and lower in 17 (27.4%) patients. There was no significant correlation between GS discordance and age, preoperative PSA level, FPSA/TPSA or maximum percentage of cancer per core (P>0.05). The maximum percentage of cancer per core (χ(2)=6.670 7, P=0.009 8) and biopsy GS (χ(2)=4.020 0, P=0.045 0) were significantly related to the pT stage on RP. CONCLUSIONS: Single positive core prostate cancer at biopsy should not be considered as a low-risk disease. Although the preoperative clinicopathological factors cannot predict lower GS on biopsy, the maximum percentage of cancer per core and GS on biopsy are of significance to the pT stage on RP.

Systematic identification and differential expression profiling of MicroRNAs from white and red muscles of siniperca chuatsi.

MicroRNAs (miRNAs) participate in the regulation of myogenesis and muscle physiological function. Most skeletal muscles in vertebrates contain a mixture of fibertypes. So far, the regulatory mechanism of the miRNA in terms of controlling muscle phenotype is poorly understood. In the present study, we use Siniperca chuatsi as a model system and demonstrate that miRNAs are involved in regulating the physiological processes and metabolism of different muscle fibers in vertebrates. The miRNA transcriptomes of the white muscle, red muscle, and five other tissues from Siniperca chuatsi were profiled using Solexa deep sequencing. We characterized 186 conserved miRNAs and 3 novel miRNAs from the two small RNA libraries of white and red muscles. Among the 155 miRNAs overlapped between the two libraries, we identified 60 significantly expressed miRNAs between the two types of muscle fibers. Using integrative miRNA target-prediction and network-analysis approaches, an interaction network of differentially expressed and muscle-related miRNAs and their putative targets were constructed. Sch-miR-181a-5p that could act to control the performance of the different muscle fiber types by targeting the myostatin gene was identified.

Early development of forebrain gonadotrophin-releasing hormone (GnRH) neurones and the role of GnRH as an autocrine migration factor.

Normal migration of the gonadotrophin-releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio-temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post-fertilisation and by 4-5 days post-fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino-modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo, that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.

Characterization and functional analysis of the 5' flanking region of myosin light chain-2 gene expressed in white muscle of the gilthead sea bream (Sparus aurata).

The promoter region ( approximately 1400 bp) of myosin light chain 2 gene of fast skeletal muscle from the marine fish Sparus aurata was cloned, sequenced and characterized. It contains a consensus sequence for TATA box, six perfect E-boxes known as binding sites to myogenic basic helix-loop-helix transcription factors and four putative MEF2-binding sites. Three genomic fragments (truncated at their upstream region) of 244, 650 and 1400 bp showed promoter activity evidenced by muscle-specific reporter gene activity using transient expression of green fluorescent protein in microinjected zebrafish embryos and in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA. The three genomic fragments also directed luciferase activity in skeletal muscle of S. aurata fry following intramuscularly injection of plasmid DNA showing a 60 to 150-fold higher luciferase activity compared to that obtained with pGL3-Basic. These experiments show that the three genomic fragments are functional muscle-specific promoters which will be useful for directing myostatin and follistatin expression in fish muscle in order to study their effect on fish muscle growth.

Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos.

Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.

Gli2 mediation of hedgehog signals in slow muscle induction in zebrafish.

Zebrafish skeletal muscles are composed of two major types of muscle fibers, broadly classified as fast or slow fibers. Recent studies have demonstrated that members of the Hedgehog (Hh) family induce the formation of slow muscle fibers. Hedgehog signals are secreted proteins that function through the transcription factor Glis. We report here the characterization of a zebrafish Gli2 expression in slow and fast muscle cells and the study of the roles of Hedgehogs and Gli2 in zebrafish muscle development using two mutant strains; sonic-you (syu) and you-too (yot), respective for sonic hedgehog (shh) and Gli2 mutation. We have demonstrated that Shh and Gli2 mutation causes similar defects in slow muscle formation. There is, however, a difference in the degree of defect between these two mutants. In yot mutant embryos, development of slow muscles was completely blocked, whereas in syu mutant embryos, a small number of slow muscle cells could still form, suggesting that other Hhs were also involved in slow muscle induction. Induction of slow muscles by other Hhs appeared to require Gli2, because ectopic expression of Echidna hedgehog (Ehh) and Tiggy-winkle hedgehog (Twhh) failed to induce slow muscles in yot mutant embryos. Together, these data suggest that further Hhs, other than Shh, are also involved in the induction and differentiation of slow muscle cells and that Gli2 is required by Shh, Twhh, and Ehh, thus playing a key role in the induction and differentiation of slow muscle cells.

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.

Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-beta gene families.

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-beta signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-beta superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-beta signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-beta signals exert competing positive and negative influences on the development of muscle pioneer cells.

Postnatal morphologic changes and glial fibrillary acidic protein immunoreactivity in the anteroventral cochlear nucleus of the acoustically-deprived gerbil.

This study investigated the morphological changes and glial fibrillary acidic protein immunoreactivity (GFAP-IR) in the anteroventral cochlear nucleus (AVCN) of acoustically-deprived gerbils during postnatal development. The mongolian gerbil, Meriones unguiculatus, had been acoustically deprived on the right side or left side by a surgical ligation of the external auditory canal at postnatal day 12-14. No discernible microcysts were located in the ipsilateral AVCN at one, three, six and nine months after monaural ligation. Also, no discernible microcysts were located in the contralateral AVCN at one and three months after monaural ligation. Numerous microcysts were located in the contralateral AVCN at six months after monaural ligation and were slightly reduced in number at nine months after monaural ligation. Some of the microcysts closely apposed to and connected with the blood vessels through a leakage route or channel. A foamy region was found in the superficial granule cell cap of the AVCN. The foamy region became evident in the ipsilateral AVCN at three months after monaural ligation. However, the foamy region became evident in the contralateral AVCN at three and nine months after monaural ligation. Vacuoles were mainly found in the neuronal cells at the junction of the superficial and deep layers in the AVCN. These vacuoles were found in the contralateral AVCN at one, three, six, and nine months after monaural ligation. However, vacuoles were found in the ipsilateral AVCN only at three months after monaural ligation. Morphological changes of the myelin sheath were found to be more severe in the contralateral AVCN than in the ipsilateral. GFAP-IR was located in the superficial layer of the contralateral AVCN at three and nine months after monaural ligation. However, GFAP-IR was found in the superficial and deep layers of the ipsilateral AVCN at three and nine months after monaural ligation. GFAP-IR was also found in the superficial layers of the ipsilateral AVCN at six months after monoaural ligation. Microcysts are presumably derived from the detachment of the myelin sheath from the retracted axons, protrusion of the myelin sheath, and disruption of the myelin sheath. The major conclusions were that (1) microcysts were greatly reduced following acoustical ligation during postnatal development, and (2) blood vessels and GFAP-immunoreactive astrocytes may be involved in the depletion of microcysts for maintaining the homeostasis of the microenvironment in the cochlear nuclei.

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene. The interactions between CREs and the transcription factor Pit-1 in mediating GH gene expression were also examined. In HeLa cells, a human cervical cancer cell line deficient in Pit-1, both basal and cAMP-induced expression of pGH.CAT were apparent only with the cotransfection of a Pit-1 expression vector. These results taken together indicate that the two CREs in the chinook salmon GH gene are functionally associated with the cAMP-dependent pathway and that their promoter activity is dependent on the presence of Pit-1

Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos.

Wnts are secreted signaling factors which influence cell fate and cell behavior in developing embryos. Overexpression in Xenopus laevis embryos of a Xenopus Wnt, Xwnt-8, leads to a duplication of the embryonic axis. In embryos ventralized by UV irradiation, Xwnt-8 restores expression of the putative transcription factor goosecoid, and rescues normal axis formation. In contrast, overexpression of Xwnt-5A in normal embryos generates defects in dorsoanterior structures, without inducing goosecoid or a secondary axis. To determine whether Xwnt-4 and Xwnt-11 fall into one of these two previously described classes of activity, synthetic mRNAs were introduced into animal caps, normal embryos, and UV-treated embryos. The results indicate that Xwnt-4, Xwnt-5A, and Xwnt-11 are members of a single functional class with activities that are indistinguishable in these assays. To investigate whether distinct regions of Xwnt-8 and Xwnt-5A were sufficient for eliciting the observed effects of overexpression, we generated a series of chimeric Xwnts. RNAs encoding the chimeras were injected into normal and UV-irradiated Xenopus embryos. Analysis of the embryonic phenotypes and goosecoid levels reveals that chimeras composed of carboxy-terminal regions of Xwnt-8 and amino-terminal regions of Xwnt-5A are indistinguishable from the activities of native Xwnt-8 and that are the reciprocal chimeras elicit effects indistinguishable from overexpression of native Xwnt-5A. We conclude that the carboxy-terminal halves of these Xwnts are candidate domains for specifying responses to Xwnt signals.

Genomic structure of growth hormone genes in chinook salmon (Oncorhynchus tshawytscha): presence of two functional genes, GH-I and GH-II, and a male-specific pseudogene, GH-psi.

Two chinook salmon (Oncorhynchus tshawytscha) growth hormone genes (a functional GH-I gene and a pseudogene, GH-psi) were isolated and characterized. The GH-I gene sequence consists of 1.9 kb of 5'-flanking sequence, 4.1 kb of transcribed region, and 64 bp of 3'-flanking sequence, and contains 6 exons and 5 introns. The pseudogene, GH-psi, spanning 4.1 kb, has a similar structure as the GH-I gene. However, it has one wrong splicing sequence at the intron 1/exon 2 junction, one premature termination codon in exon 5, and a deletion in the last half of exon 5 and the first part of intron 5. In addition to GH-I gene and GH-psi, a third GH gene, GH-II, was identified by the polymerase chain reaction (PCR) and subsequently shown to be the second functional GH-II gene. To study the linkage arrangement of these three GH genes, 50 unrelated chinook salmon (25 males and 25 females) and one chinook salmon family were analyzed by PCR. The results showed that GH-psi exists only in males and that it segregates from father to sons. These results suggest that GH-psi is sex specific and probably resides on the Y chromosome. Together these results indicate that there are three GH genes in the genome of male chinook salmon, and only two GH genes in the females. The extra GH gene in the male is, however, a pseudogene.

Development of an all-fish gene cassette for gene transfer in aquaculture.

To develop an all-fish gene cassette suitable for gene transfer in aquaculture, the antifreeze protein (AFP) gene promoter from the ocean pout (Macrozoarces americanus) was analyzed for its ability to direct exogenous gene expression both in vitro and in vivo. The ocean pout AFP (opAFP) gene promoter fused to the bacterial chloramphenicol acetyltransferase (CAT) was functionally analyzed in two fish cell lines and in Japanese medaka embryos. The opAFP gene promoter was active in these systems, as demonstrated by the transient expression of CAT activity. These results suggest that the opAFP gene promoter is useful for many other gene transfer experiments. To facilitate use of the opAFP gene promoter as a common and versatile vehicle for fish gene transfers, an expression vector, opAFP-V, was constructed by linking the 2.1-kb opAFP gene promoter, the 63-bp opAFP gene 5' untranslated sequence, and the 1.2-kb opAFP gene 3' sequence by two unique restriction sites, Bg/II and HpaI, respectively. Thus, genes of interest can be inserted into either the Bg/II site or the HpaI site depending on the length of their 5' untranslated sequence. The complete DNA sequence of opAFP-V was determined to facilitate future detailed analysis of integration and expression of the transgene.

Growth enhancement in transgenic Atlantic salmon by the use of an "all fish" chimeric growth hormone gene construct.

We have developed an "all fish" growth hormone (GH) chimeric gene construct by using an antifreeze protein gene (AFP) promoter from ocean pout linked to a chinook salmon GH cDNA clone. After microinjection into fertilized, nonactivated Atlantic salmon eggs via the micropyle, transgenic Atlantic salmon were generated. The presence of the transgene was detected by polymerase chain reaction (PCR) using specific oligonucleotide primers. A number of these transgenic fish showed dramatic increases in their growth rate. At one year old, the average increase of the transgenic fish was 2 to 6 fold and the largest transgenic fish was 13 times that of the average non-transgenic control.

[Combined methods of dialysis, cooked meat medium enrichment and laboratory animal toxicity for screening Clostridium botulinum spores in honey and infant food].

One hundred and fifty two samples of honey were purchased from apiaries and markets in Taiwan, meanwhile fifty samples of infant food including powders of infant milk, wheat, rice and commercial mixed cereals, as well as juice had been bought from supermarkets in Taipei city from July 1988 through April 1989. The samples were used for detecting the presence of Clostridium botulinum spores. Honey samples were prepared by dialysis to obtain the bacterial spores; however, infant food had been innoculated directly into the cooked meat medium. The suspicious isolated colonies were identified by Vitek automatic microbial identification system. The supernatants of cooked meat medium enrichment were performed for typing botulinum toxins via the laboratory animal toxicity test. The results showed that two samples of honey contained the spores of C. botulinum type B, but none in samples of infant food. The specific response of clinical signs in mice, after being administered the supernatant of suspected cultural broth, were also observed and described in this article.