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PubMed2006

A digital imaging procedure for seven-probe-labeling FISH (Rainbow-FISH) and its application to estuarine microbial communities.

For multi-probe-labeling fluorescence in situ hybridization (FISH), a digital imaging procedure was developed consisting of systematic background noise reduction and target signal equalization using a hue, saturation, value color partitioning technique. By the combined application of seven DNA probes, each labeled with three fluorochromes at maximum, seven kinds of cultured type strains were distinguished in a microscopic field simultaneously. Using this seven-probe-labeling FISH (Rainbow-FISH), several phylogenetic groups of microbes that occur frequently in aquatic environments, such as Alpha-, Beta- and Gammaproteobacteria, Cytophaga-Flavobacterium and Actinobacteria, were identified and quantified. The total counts of cells specified by Rainbow-FISH were in the range of 96-108% of those of general FISH, showing that the method is highly reliable for quantitative population analysis. Analyzing samples obtained at points along a river to a sea, we found a reverse population change in two groups: apparent decreases in Betaproteobacteria but gradual increases in Gammaproteobacteria. This method provides a platform toward the improvement of semiautomatic analysis of aquatic microbes under various metabolic conditions.

Actinobacteria↗
PubMed1989

Presence of the fish pathogen Vibrio salmonicida in fish farm sediments.

The persistence of the fish pathogen Vibrio salmonicida in fish farm sediments was studied by use of fluorescent-antibody techniques. The specificities of the monoclonal antibodies and polyclonal rabbit serum used in the study were tested against a number of Vibrio strains, including 4 isolates from intestinal tracts of healthy fish and 98 isolates from sediments. V. salmonicida was detected in sediment samples from diseased farms several months after an outbreak of the disease. The bacterium was also detected in a sediment sample from a disease-free fish farm. No V. salmonicida could be detected in sediments not influenced by fish farming. The number of positive samples was generally higher with application of rabbit serum as opposed to use of monoclonal antibodies, indicating that the rabbit serum may cross-react with other bacteria.

Animals↗
PubMed1998

An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH).

Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromosomes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.

Chromosome Aberrations↗
PubMed2002

Characterization of supernumerary rings and giant marker chromosomes in well-differentiated lipomatous tumors by a combination of G-banding, CGH, M-FISH, and chromosome- and locus-specific FISH.

Supernumerary ring chromosomes and/or giant marker chromosomes are often seen in soft-tissue tumors of low-grade or borderline malignancy, such as well-differentiated liposarcomas or atypical lipomas. Classic cytogenetic banding techniques have proved insufficient to identify the genomic composition and structure of such rings and markers, but fluorescent in situ hybridization (FISH) studies have shown that they consist mainly of amplified material from chromosome 12, more specifically from bands 12q13-->q15. We have used the new FISH-based screening techniques comparative genomic hybridization (CGH) and multicolor-FISH (M-FISH) in combination with G-banding and analysis by chromosome- and locus-specific fluorescent in situ probes to examine in detail the karyotypic characteristics of 22 lipomatous tumors, most of them classified histologically as well-differentiated liposarcomas, selected because they had been shown to harbor rings and/or marker chromosomes. M-FISH, in contrast to G- banding, was found to be informative with regard to the chromosomal origin of the rings and other markers present, whereas CGH and hybridizations with locus-specific probes helped identify which subchromosomal regions were involved. We found that chromosome bands 12q15-->q21 were always gained, with 12q15-->q21 being amplified (i.e., a green-to-red ratio >2 by CGH) in 14 of 22 tumors. In three tumors, two distinct but close amplicons in 12q could be identified, corresponding to bands 12q13-->q15 and 12q21. The genomic segment 1q21-->q23 was gained in 12 cases, reaching the level of amplification in seven. Bands 6q24 and 7p15, whose pathogenetic involvement in liposarcomas has not been reported previously, were gained in three cases each. In addition, the rings and giant markers often contained interspersed sequences from several other chromosomes that did not give an equally clear impression of being nonrandomly involved.

Biomarkers, Tumor↗
PubMed2006

Multiplex-FISH (M-FISH): technique, developments and applications.

Multiplex FISH (M-FISH) represents one of the most significant developments in molecular cytogenetics of the past decade. Originally designed to generate 24 colour karyotyping, the technique has spawned many variations and an equally diverse range of applications. In tumour and leukaemia cytogenetics, the two groups that have been targeted represent both ends of the cytogenetic spectrum: those with an apparently normal karyotype (suspected of harbouring small rearrangements not detectable by conventional cytogenetics) and those with a complex aberrant karyotype (which are difficult to karyotype accurately due to the sheer number of aberrations). In research, mouse M-FISH provides a powerful tool to characterize mouse models of a disease. In addition, the ability to accurately karyotype single metaphases without selection makes M-FISH the perfect tool in chromosome breakage studies and for characterizing clonal evolution of tumours. Finally, M-FISH has emerged as the perfect partner for the developing genomic microarray (array CGH) technologies, providing a powerful approach to gene discovery.

Burkitt Lymphoma↗
PubMed2006

Relationship between total mercury concentration and fish size in two pelagic fish species: implications for consumer health.

Total mercury concentrations were determined in different size classes of two pelagic fish species of great commercial importance, horse mackerel (Trachurus trachurus) and Mediterranean horse mackerel (Trachurus mediterraneus), to evaluate the relationship between total mercury concentration and fish size and to determine whether any differences might affect the quantitative assessment of mercury exposure for consumers. Mercury concentrations in horse mackerel and in Mediterranean horse mackerel were between 0.16 and 2.41 microg g(-1) of weight wet (mean, 0.68 microg g(-1)) and between 0.09 and 1.62 microg g(-1) (mean, 0.51 microg g(-1)), respectively. The regression curves revealed a significant relationship between mercury concentration and fish size (length and weight) for both species. Concentrations exceeding the proposed limit for human consumption were observed in 33.3% of the samples of both species and were associated with larger specimens. The consumption of the larger specimens could lead to an increase in mercury exposure for consumers. Estimated weekly intakes, calculated on the basis of concentrations relative to each size class, revealed a high exposure associated with the consumption of fish larger than 30 cm (horse mackerel, 11.63 to 20.16 microg/kg of body weight; Mediterranean horse mackerel, 5.86 to 13.55 microg/kg of body weight). An understanding of the factors leading to an increase in mercury exposure can help consumers make informed decisions about eating fish.

Animals↗
PubMed2001

IPM-FISH, a new M-FISH approach using IRS-PCR painting probes: application to the analysis of seven human prostate cell lines.

We have developed an alternative multicolor karyotyping technique based on multiplex fluorescence in situ hybridization (M-FISH) and our own optical device with a specific filter set. The most innovative part of our development is the use of interspersed polymerase chain reaction (IRS-PCR) painting probes that show an R-band pattern simultaneous to the combinatorial labeling. This allows us not only to recognize the origin of chromosomal fragments, but to identify the breakpoints as well. We have used this technique to analyze seven cell lines: four prostate cancer cell lines (CA-HPV-10, LNCaP, DU145, and PC3), and three normal transformed epithelial prostate cell lines (PNT1B, PNT2, and PZ-HPV-7). In order to validate our IRS-PCR multiplex FISH (IPM-FISH) technique and to complement the results, we applied comparative genomic hybridization (CGH) and FISH analysis, showing good correlation with the IPM-FISH results. To date, molecular and cytogenetic studies have identified several chromosomal regions that are altered in human prostate cancer; several candidate genes have been suggested. However, reliable markers for predicting the aggressiveness of early prostate cancer are not yet available. Our results show several common, unbalanced rearrangements in the cell lines. These rearrangements are similar to regions already implicated in prostate cancer, validating these cell lines as a good model system.

Cell Line, Transformed↗
PubMedDate not supplied

A new highly effective ovulating agent for fish reproduction. Practical application of LH-RH analogue for the induction of spawning of farm fishes.

The synthetic analogue of the nonapeptide LH-RH has been found to be highly effective in the induction of spawning of farm fishes (the grass carp, the silver carp, the spotted silver carp, and the black carp.) Its biological activity is many times higher than that of the synthetic decapeptide or natural releasing hormone. Out of a total number of 500 mature fishes treated with the nonapeptide alone and/or combined with a minimum amount of fish pituitary, 396 of them spawned--with an over-all spawning rate of 78%. As far as we know, this is probably the most effective ovulating agent or hormone now available for fishes. This finding is of paramount importance in pisciculture for large-scale production of fry. The recommended dosages for the following farm fishes are: the grass carp--1-10 microgram/kg b.w.; the silver carp--3 microgram or more/kg b.w. in divided doses; the spotted silver carp--1.4 microgram or more/kg b.w.; and the black carp--10 microgram/kg b.w. For the latter species, the efficacy of the peptide could be improved by a concurrent administration of 0.5-2 mg of the pituitary gland. The significance of this work in piscicultural practice and the mechanism of hormonal action are briefly discussed.

Animals↗
PubMed2005

Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study.

Identification of 10 white fish species associated with U.K. food products was achieved using PCR-RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end-point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials.

Animals↗
PubMed2003

Mercury in UK imported fish and shellfish and UK-farmed fish and their products.

Total mercury concentrations were measured in fish and shellfish and their products imported into the UK and also in UK-produced farmed salmon and trout. Three hundred and thirty-six samples were collected using a two-stage sampling plan. The sample plan was weighted to reflect consumption, but with some bias towards fish that might accumulate higher levels of mercury, such as large predatory fish at the top of the food chain. The highest levels of total mercury were found in billfish (swordfish and marlin) and shark. Mercury concentrations in the five samples of fresh/frozen shark ranged from 1.006 to 2.200 mg kg(-1), all above the European Commission limit for the species, and concentrations in 20 samples of fresh/frozen billfish ranged from 0.153 to 2.706 mg kg(-1) with 13 samples above the 1.0 mg kg(-1) limit for the species. One sample of Antarctic ice fish was collected and had a mercury concentration of 0.664 mg kg(-1). The limit for this species was 0.5 mg kg(-1). One sample of fresh/frozen tuna out of the 20 collected had a mercury concentration above the limit of 1.0 mg kg(-1) (1.5 mg kg(-1)), but all other fresh tuna samples were well within the regulatory limit (average 0.4 mg kg(-1)). Mercury concentrations in canned tuna were lower with concentrations on average half that measured in fresh/frozen tuna. Mercury concentrations in UK-farmed salmon and trout were relatively low. The maximum concentration found in 46 samples of fresh/frozen or smoked trout and salmon was 0.103 mg kg(-1).

Animals↗
PubMed1985

Effect of dietary intake of fish oil and fish protein on the development of L-azaserine-induced preneoplastic lesions in the rat pancreas.

The effect of dietary intake of fish (menhaden) oil and fish (cod) protein on the development of pancreatic preneoplastic lesions was examined in male Wistar rats. Fourteen-day-old animals were given a single ip injection of 30 mg L-azaserine/kg body weight [CAS: 115-02-6; diazoacetate serine (ester)]. At 21 days of age they were weaned and maintained on dietary treatment for 4 months. Fish protein did not appear to produce a significantly different preneoplastic response when compared to casein as a protein source. However, a 20% menhaden oil diet, rich in omega 3 fatty acids, produced a significant decrease in the development of both the size and number of preneoplastic lesions when compared to a 20% corn oil diet rich in omega 6 fatty acids. This study provides evidence that fish oils, rich in omega 3 fatty acids, may have potential as inhibitory agents in cancer development.

Animals↗
PubMed1988

The effect of diets containing fish and fish oils on disease risk factors in humans.

Fish and fish oils are rich in omega 3 polyunsaturated fatty acids which are capable of acting as both a source of active eicosanoids and as inhibitors of synthesis of eicosanoids from arachidonic acid by a variety of human cells. There is an extensive literature on the role of omega 3 polyunsaturates in ameliorating the risk factors associated with coronary heart disease, including both atherosclerotic and thrombotic factors. In addition, there is a limited number of studies which report the results of trials testing the effects of fish oils on other clinical conditions including diabetes, arthritis, migraine and psoriasis. There appears to be sufficient evidence to suggest that patients at risk from heart disease could benefit from low dose (1-6 g/day) of fish oil in conjunction with a prudent diet.

Animals↗
PubMed1997

Survey for Gnathostoma nipponicum larvae in gobiid freshwater fish and infectivity of the larvae to a gobiid fish (Chaenogobius urotaenia).

From June 1993 to November 1996, a total of 977 gobiid fish consisting of three species (Tridentiger brevispinis, Chaenogobius urotaenia, and Rhinogobiu brunneus) collected from eastern Aomori Prefecture, were examined for Gnathostoma nipponicum larvae infection. Only one species, C. urotaenia was infected with advanced third-stage larvae (AdL3), and a total of 22 larvae were recovered from 17 (3.4%) of 500 fish. The infected fish were larger than 12 cm in body length and collected in the May-June and September-November seasons. Experimental studies confirmed that C. urotaenia was susceptible to both the early third-stage larvae (EaL3) obtained from Eucyclops serrulatus and AdL3 from Misgurnus anguillicaudatus. Eight of 10 C. urotaenia inoculated orally with 10 EaL3 were positive, and 36 AdL3 were recovered from them (recovery rate: 36.0%) at 30 days postinoculation (PI). All 10 C. urotaenia inoculated with 10 AdL3 were also positive, and a total of 63 AdL3 were recovered (recovery rate: 63.0%) at 10 days PI. The main location of the larvae was the body muscles of the fish. No morphological alterations or death of the larvae were observed in this study. From these results, it seems that the C. urotaenia has characteristics suitable to be the host to the larvae and they may serve as the second intermediate and paratenic host in the natural life cycle of this nematode.

Animals↗
PubMed1992

Occurrence of infectious fish diseases in fish farms in northern Finland.

A total of 47 fish located in 10 lake and river systems in northern Finland were examined for furunculosis, enteric redmouth diseases (ERM), viral fish diseases and the parasite Gyrodactylus salaris. Furunculosis was found in 2 fish farms in different watercourses, ERM in 8 fish farms in 3 watercourses and viral diseases were not found at all. G. salaris was looked for only in salmon and rainbow trout and was found in both species in 3 farms belonging to 2 watercourses.

Animals↗
PubMed2004

[Omega-3 fatty acids, fish, fish oil and cardiovascular disease--a review with implications to Israeli nutritional guidelines].

Evidence from epidemiological and randomized controlled trials shows beneficial effects of omega-3 (n-3) fatty acids from fish and plant sources on cardiovascular disease (CVD), especially in patients with preexisting CVD. The optimal dose of n-3 is not yet determined, but prospective secondary prevention studies suggest that the addition of 0.5-1.8 grams/day of marine-derived eicosapentaenoic acid and docosahexaenoic acid, or plant derived alpha-linolenic acid at a dose of 1.5-3 grams/day significantly reduce subsequent cardiac events and mortality. These data have led the American Heart Association Dietary Guidelines committee to recommend to the general population the consumption of at least two servings of fatty fish per week, in addition to vegetable oils high in alpha-linolenic acid. The risk of adverse effects and toxicity from contaminants at this dose is low. The amount of daily n-3 fatty acids recommended for patients with coronary heart disease is 1 gram/day. In patients who cannot consume this dose of n-3 fatty acids through diet alone, addition of n-3 supplements should be considered. Higher doses of contaminant-free n-3 supplements, 2-4 grams/day, can be used in the treatment of hypertriglyceridemia. Data on the content of n-3 fatty acids and contaminants in Israeli bred fish is limited. Thus, caution should be exercised when applying these recommendations to the Israeli fish market.

Animals↗
PubMed1990

Occurrence of monogeneans on freshwater fishes in Iran: Dactylogyrus spp. on cultured Iranian fishes.

Thirteen Dactylogyrus spp. were found on cultured fishes in five Iranian fish farms. Besides typical Dactylogyrus vastator, specimens with small anchors were often found in goldfish, which were designated as D. vastator forma minor. Rutilus frisii kutum, a fish cultured only in Iran, was infected by two Dactylogyrus spp. during its short period of prerearing in Iranian fish farms. All Dactylogyrus found are new for the Iranian fauna.

Animals↗
PubMed1990

Pericyte of a teleost fish: ultrastructure, position, and role in neoplasia as revealed by a fish model.

The morphology and position of the pericyte, a periendothelial cell, is described for a teleost fish, Cyprinodon variegatus. This cell was found attached to the abluminal surfaces of capillaries, venules, and arterioles of the submucosa of the midgut of the fish. The cell was encompassed by a thin basal lamina, possessed numerous plasmalemmal vesicles, a "sole region" which contained thinner actin-like filaments and possibly thicker myosin-like filaments, and ranged in form from ovoid to stellate, with long cytoplasmic extensions that partially covered the endothelium of the associated microvessel. The pericyte of C. variegatus has been shown to give rise to hemangiopericytomas (experimentally induced with diethylnitrosamine) and possibly to pericytomas. The range of phenotypic expression of these pericyte-derived neoplasms is broad, and dependent upon the degree of differentiation of their constituent cells which range from clear cell pericytes to myofilamentous laden cells that resemble smooth muscle cells. In this regard and in regard to its normal ultrastructural morphology, and anatomical position, in relationship to microvasculature in this fish, the cell is very similar to other vertebrate pericytes. Limited evidence suggests that small fish species may be excellent study models for further elucidation of pericyte form, function, and role in disease.

Animals↗
PubMed2006

Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils.

Neutrophil extracellular traps (NETs), which are extracellular DNA structures released from neutrophils, are described and characterized for the first time in fish using fluorescent confocal microscopy. Confocal images of fish neutrophil suspensions stained with 6'-diamino-2-phenylindole, dihydrochloride DNA fluorescent stain (DAPI) revealed the presence of NETs which appeared as fibrous structures connecting several cells. Co-localization of NETs with neutrophil granular proteins and actin was investigated using specific antibodies and probes. Double staining of neutrophils with SYTOX green and DAPI revealed that SYTOX stain applied to living cells stained extracellular DNA, but not nuclei. NETs are actively released from stimulated living cells, associated with granular proteins, but not with cytoskeleton, and are not a product of nuclear degradation seen in late apoptotic stages. Additionally, a fluorometric microtiter plate assay to quantify the release of NETs was adopted for use with fish neutrophils, and the effect of stress on NETs release was studied. This assay detected the inhibition of DNA release during stress conditions. In summary, NETs were released from living fish kidney neutrophils upon stimulation, characterized using fluorescence DNA-binding dyes, specific antibodies and probes, and quantified using a microtiter plate fluorometric assay that can rapidly measure a large number of samples. Detection of NETs can be used as an additional assay to an existing battery of functional tests, and as a new research model to study the effects of stress, immunomodulators, and diseases.

Animals↗