Collagen type XI alpha1 may be involved in the structural plasticity of the vertebral column in Atlantic salmon (Salmo salar L.).

Atlantic salmon (Salmo salar L.) vertebral bone displays plasticity in structure, osteoid secretion and mineralization in response to photoperiod. Other properties of the vertebral bone, such as mineral content and mechanical strength, are also associated with common malformations in farmed Atlantic salmon. The biological mechanisms that underlie these changes in bone physiology are unknown, and in order to elucidate which factors might be involved in this process, microarray assays were performed on vertebral bone of Atlantic salmon reared under natural or continuous light. Eight genes were upregulated in response to continuous light treatment, whereas only one of them was upregulated in a duplicate experiment. The transcriptionally regulated gene was predicted to code for collagen type XI alpha1, a protein known to be involved in controlling the diameter of fibrillar collagens in mammals. Furthermore, the gene was highly expressed in the vertebrae, where spatial expression was found in trabecular and compact bone osteoblasts and in the chordoblasts of the notochordal sheath. When we measured the expression level of the gene in the tissue compartments of the vertebrae, the collagen turned out to be 150 and 25 times more highly expressed in the notochord and compact bone respectively, relative to the expression in the trabecular bone. Gene expression was induced in response to continuous light, and reduced in compressed vertebrae. The downregulation in compressed vertebrae was due to reduced expression in the compact bone, while expression in the trabecular bone and the notochord was unaffected. These data support the hypothesis that this gene codes for a presumptive collagen type XI alpha1, which may be involved in the regulatory pathway leading to structural adaptation of the vertebral architecture.

Pathological changes in juvenile Atlantic halibut Hippoglossus hippoglossus persistently infected with nodavirus.

This is the first description of a persistent subclinical nodavirus infection in the Atlantic halibut Hippoglossus hippoglossus. Juvenile fish (1 to 5 g) were sampled at 4, 5 and 8 mo of age at a fish farm in Norway during and after weaning. None showed clinical signs of viral encephalopathy and retinopathy (VER) or other disease. Pathological changes and/or nodavirus were detected by light microscopy, immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and transmission electron microscopy in all fish examined. High numbers of virus particles were found in macrophage-like cells in the central nervous system, including brain and retina (CNS). The virus particles displayed the icosahedral shape and size (approximately 25 nm) characteristic of nodaviruses. The virus-infected cells formed focal cell aggregates and were seen in all regions of the brain and all nuclear cell layers of the retina. The cytoplasm of the infected cells was filled with membrane-enclosed inclusions packed with virus particles. Some virus particles lay along membranes and formed membrane-bound necklace-like arrangements. The virus-infected cells of the retina also contained pigment granula located generally inside virus inclusions and sometimes forming a coating around the virus particles. All frontal parts with the eyes and brain and 50% of the mid-parts, which included the abdominal organs, were found positive for nodavirus with RT-PCR. Pathological changes in these persistently nodavirus-infected fish differ from earlier descriptions in Atlantic halibut during outbreaks of VER. Vertical transmission from infected spawners is believed to be a major route for nodavirus infection. Detection of nodavirus in subclinical infected fish and a better understanding of its pathogenesis are important in order to prevent the spread of nodavirus in the fish-farming industry.

Ontogeny of cholecystokinin-immunoreactive cells in the digestive tract of Atlantic halibut, Hippoglossus hippoglossus, larvae.

The appearance and distribution of cholecystokinin (CCK)-producing cells were investigated in the digestive tract of developing larvae of cultured Atlantic halibut, Hippoglossus hippoglossus. The CCK-producing cells were detected immunohistochemically, by use of a primary antiserum against CCK cloned for the Japanese flounder, Paralichthys olivaceus. No CCK-immunoreactive (IR) cells were detected in first-feeding larvae (33 days after hatching, DAH). Forty-five DAH or 12 days after first feeding, there were a few scattered CCK-IR cells in the epithelium of the anterior midgut in about 30% of the examined larvae. All larvae older than 52 DAH had CCK-IR cells in the anterior midgut, particularly frequent in the most anterior region adjacent to the pyloric caeca. No CCK-IR cells were detected in the foregut, the hindgut, or the midgut posterior to the first curvature. The CCK-IR cells spanned the intestinal epithelium from the basal lamina to the lumen and were triangular in shape, with the nucleus in the basal part and a thin apex toward the lumen. The mechanisms controlling release of bile, pancreatic enzymes, and peristalsis during the 12 days between first feeding and the first detection of CCK-IR cells remain to be clarified.

Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids.

Morphological and biochemical effects were induced at the subcellular level in the skeletal muscle, heart and liver of male rats as a result of feeding with EPA, DHA, and 3-thia fatty acids. The 3-thia fatty acid, tetradecylthioacetic acid (TTA) and EPA induced mitochondrial growth in type I muscle fibers in both the diaphragm and soleus muscle, and the size distribution of mitochondrial areas followed a similar pattern. Only the 3-thia fatty acid induced mitochondrial growth in type II muscle fibers. The mean area occupied by the mitochondria and the size distribution of mitochondrial areas in both fiber types were highly similar in DHA-treated and control animals. Only the 3-thia fatty acid increased the gene-expression of carnitine palmitoyltransferase (CPT)-II in the diaphragm. In the heart, however, the gene expression decreased. In hepatocytes an increase in the mean size of mitochondria was observed after EPA treatment, concomitant with an increase in mitochondrial CPT-II gene expression. Administration of 2-methyl-substituted EPA (methyl-EPA) induced a higher rate of growth of mitochondria than EPA. At the peroxisomal level in the hepatocytes a 3-thia fatty acid, EPA, and DHA increased the areal fraction concomitant with the induction of gene expression of peroxisomal fatty acyl-CoA oxidase (FAO). In the diaphragm, mRNA levels of FAO were not affected by EPA or DHA treatment, whereas gene expression was significantly increased after 3-thia fatty acid treatment. In the heart, both 3-thia fatty acid, EPA and DHA tended to decrease the levels of FAO mRNA. The areal fraction of fat droplets in all three tissue types was significantly lower in the groups treated with 3-thia fatty acid. In the group treated with EPA a lower areal fraction of fat droplets was observed, while the DHA group was similar to the control. This indicates that EPA and DHA have different effects on mitochondrial biogenesis.

Characterization of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay.

A 1349 nucleotide fragment of the RNA2 from a nodavirus affecting Atlantic halibut Hippoglossus hippoglossus was characterised and the nuclotide sequence (accession no. AJ245641) was employed to develop an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay. The sequenced part of the RNA2 of Atlantic halibut nodavirus (strain AH95NorA) was highly similar in organisation to that of the RNA2 of striped jack nervous necrosis virus (SJNNV), and comprised features common to all nodaviruses. These characteristics confirmed that the virus that causes viral encephalopathy and retinopathy (VER) in Atlantic halibut is a nodavirus. The nucleotide sequence of the 1349 nucleotide fragment of Atlantic halibut nodavirus RNA2 was 80% identical to the RNA2 of SJNNV. The T2 region (830 nucleotides) of the RNA2 of Atlantic halibut nodavirus shared 98% of the nucleotide sequence when compared with the homologous region of barfin flounder nervous necrosis virus (BFNNV), while the nucleotide sequence identity to SJNNV in this region was 76%. Phylogenetic analysis based on the nucleotide sequences of the T4 region (421 nucleotides) of Atlantic halibut nodavirus and of other fish nodaviruses revealed a close relationship to the nodaviruses of the barfin flounder clad that have been found in other cold-water species (Pacific cod Gadus macrocephalus and barfin founder Verasper moseri). The nucleotide sequence of the RNA2 of Atlantic halibut nodavirus included some features that differ from that of SJNNV. The ORF of the RNA2 of Atlantic halibut nodavirus lacked 6 nucleotides through a single deletion and a 5-nucleotide deletion, separated by 4 nucleotides. The 3'-non-encoding region contained a 21 nucleotide insert and a 3 nucleotide deletion when compared with SJNNV. In comparison with the RNA2 of SJNNV, the 3'-non-encoding region showed a nucleotide sequence identity of 84.5%. A primer set based on the Atlantic halibut nodavirus nucleotide sequence was employed in order to design an optimal RT-PCR. The detection limit of the PCR was 10 to 100 copies of plasmid, while the detection limit of the RT-PCR assay was 100 to 1000 copies of in vitro transcribed viral RNA.

Surface disinfection of Atlantic halibut Hippoglossus hippoglossus eggs with ozonated sea-water inactivates nodavirus and increases survival of the larvae.

Disinfection by ozonation of sea-water may reduce the risk of transmission of nodavirus, a major fish pathogen, via Atlantic halibut Hippoglossus hippoglossus eggs. In the present study, eggs at 4 d prior to hatching were exposed to nodavirus and then to ozonated sea-water using different concentrations (0.3 to 10 mg l-1) and exposure times (0.5 to 10 min). None of the larvae from virus-exposed eggs washed with ozonated sea-water developed viral encephalopathy and retinopathy (VER), which was detected in all dead larvae from eggs exposed to nodavirus but not washed with ozonated sea-water. In the non-treated control group about 20% of the dead larvae developed the disease. This suggests that the halibut eggs taken from a large-scale production facility were already contaminated with nodavirus. The egg groups which had been treated with 4 mg O3 l-1 for 0.5 min or with lower total ozone exposures had a higher survival and no adverse effects on the development of the larvae after hatching were observed. Although a slight delay in hatching was found, after 2 d the cumulative hatching had normalised. In the egg groups with high total exposure (4 mg O3 l-1 for 1 min or higher total ozone exposures) a pronounced negative effect on hatching was observed. Our results indicate that the egg surface may be important in the transfer of nodavirus and that nodavirus associated with the surface of the egg may be inactivated by ozonated sea-water.

Transmission of viral encephalopathy and retinopathy (VER) to yolk-sac larvae of the Atlantic halibut Hippoglossus hippoglossus: occurrence of nodavirus in various organs and a possible route of infection.

The susceptibility of the Atlantic halibut Hippoglossus hippoglossus yolk-sac larvae to viral encephalopathy and retinopathy (VER) was investigated by waterborne challenge experiments with nodavirus. Transfer of VER was indicated by several lines of evidence. A significantly higher cumulative mortality was observed after challenge with virus compared to mock challenge, and increasing doses of virus resulted in shorter incubation periods. When the challenge was performed on the day after hatching, the time from inoculation to the time when 50% of the larvae were dead (LT50) ranged from 26 to 32 d. Postponement of challenge for 13 d reduced the LT50 to 14 d, indicating that the susceptibility of the larvae to the present nodavirus strain was low during the first 2 wk after hatching. The progression of the infection was monitored by sequential immunohistochemistry and electron microscopy. On Day 18 after hatching the initial signs of infection were observed as a prominent focus of immunolabelling in the caudal part of the brain stem. In the same larvae immunolabelled single cell lesions were observed in the stratified epithelium of the cranial part of the intestine. The portal of entry into the larvae may thus have been the intestinal epithelium, while the route of infection to the CNS may have been axonal transport to the brain stem through cranial nerves such as the vagus nerves. Later in the infection, lesions became more severe and widespread and were also found throughout the brain and spinal cord and in the retina, cranial ganglia, intestine, liver, olfactory epithelium, yolk-sac epithelium, gills and pectoral fins. The mortality in all virus-challenged groups was 100%. This study thus demonstrates that the present nodavirus strain is able to replicate and cause VER in Atlantic halibut yolk-sac larvae at temperatures as low as 6 degrees C.

Pathogenicity of nodavirus strains from striped jack Pseudocaranx dentex and Atlantic halibut Hippoglossus hippoglossus, studied by waterborne challenge of yolk-sac larvae of both teleost species.

The present study shows that differences in pathogenicity exist among fish nodavirus strains. In challenge trials, a Japanese strain (SJ93Nag) was highly virulent to larvae of the striped jack Pseudocaranx dentex but replication was not detected in larvae of Atlantic halibut Hippoglossus hippoglossus at 6 degrees C. Conversely, a Norwegian nodavirus strain (AH95NorA) that was highly virulent to the Atlantic halibut larvae did not replicate in striped jack larvae at 20 degrees C. Occurrence of the disease viral encephalopathy and retinopathy (VER) and cumulative mortality were significantly different in the 2 species when challenged with the 2 nodavirus strains. The presence of nodavirus in nervous tissue was monitored by immunohistochemical methods. Our results support the view that the genetic diversity among nodavirus strains reflects the existence of different viral phenotypes which may be adapted to infect different host species and/or for replicating at different temperatures. Fish nodaviruses represent surveyable pathogens well suited for studying the relation between viral genotypic and phenotypic properties such as host specificity, temperature optima, neuroinvasiveness and neurovirulence.

Mitochondrion is the principal target for nutritional and pharmacological control of triglyceride metabolism.

Fish oil polyunsaturated fatty acids and fibrate hypolipidemic drugs are potent hypotriglyceridemic agents that act by increasing fatty acid catabolism and decreasing triglyceride synthesis and secretion by the liver. A major unresolved issue is whether this hypotriglyceridemic effect can occur independent of induction of peroxisomal beta-oxidation, a predisposing factor for hepatocarcinogenesis. The present study was undertaken to determine which component of fish oil, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), is responsible for its triglyceride-lowering effect. We demonstrate that EPA and not DHA is the hypotriglyceridemic component of fish oil and that mitochondria and not peroxisomes are the principal target. Results obtained by fenofibrate feeding support the hypothesis that the mitochondrion is the primary site for the hypotriglyceridemic effect. In contrast to fibrates, EPA did not affect hepatic apolipoprotein C-III gene expression. Therefore, increased mitochondrial beta-oxidation with a concomitant decrease in triglyceride synthesis and secretion seems to be the primary mechanism underlying the hypotriglyceridemic effect of EPA and fibrates in rats, rabbits and possibly also in humans. In addition, these data show that lowering of plasma triglycerides can occur independently of any deleterious peroxisome proliferation.

A hypolipidemic peroxisome proliferating fatty acid induces polydispersity of rat liver mitochondria.

Primary rat hepatocyte cultures exposed to tetradecylthioacetic acid for periods up to 96 h significantly increased fatty acid oxidation and decreased triacylglycerol synthesis and secretion. During the same period the mean areal fraction (%) and polydispersity of both mitochondria and peroxisomes increased, indicating growth and proliferation. In rats fed tetradecylthioacetic acid for 12 weeks, the fatty acid oxidation increased with a concomitant hypolipidemic effect. In addition, the areal fraction of both mitochondria and peroxisomes increased significantly and the number of lipid droplets decreased. The results suggest that tetradecylthioacetic acid affects mitochondria and peroxisomes both in vitro and in vivo. It is concluded that tetradecylthioacetic acid reduces secretion of triacylglycerol from rat hepatocytes both in vitro and in vivo mainly by stimulating fatty acid oxidation.

Tetradecylthioacetic acid incorporated into very low density lipoprotein: changes in the fatty acid composition and reduced plasma lipids in cholesterol-fed hamsters.

The mechanism behind the hypolipidemic effect of tetradecylthioacetic acid (CMTTD, a non-beta-oxidizable 3-thia fatty acid) was studied in hamsters fed a high cholesterol diet (2%), which resulted in hyperlipidemia. Treating hyperlipidemic hamsters with CMTTD resulted in a progressive hypocholesterolemic and hypotriacylglycerolemic effect. Decreased plasma cholesterol was followed by a 39% and 30% reduction in VLDL-cholesterol and LDL-cholesterol, respectively. In contrast, the HDL-cholesterol content was not affected, thus decreasing the VLDL-cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol ratios. 3-Hydroxy-3-methylglutaryl- (HMG) CoA reductase activity and its mRNA level were unchanged after CMTTD administration. Also, the LDL receptor and LDL receptor-related protein (LRP-4) mRNAs were unchanged. The decrease in plasma triacylglycerol was accompanied by a 45% and 56% reduction in VLDL-triacylglycerol and LDL-triacylglycerol, respectively. The hypolipidemic effect of CMTTD was followed by a 1.4-fold increase in mitochondrial fatty acid oxidation and a 2.3-fold increase in peroxisomal fatty acid oxidation. CMTTD treatment led to an accumulation of dihomo-gamma-linolenic acid (20:3n-6) in liver, plasma, very low density lipoprotein, and heart. Noteworthy, CMTTD accumulated more in the heart, plasma, and VLDL particles compared to the liver, and in the VLDL particle alpha-linolenic acid (18:3n-3) decreased whereas eicosatetraenoic acid (20:4n-3) increased. In addition, linoleic acid (18:2n-6) and the total amount of polyunsaturated fatty acids decreased, the latter mainly due to a decrease in n-6 fatty acids. The present data show that CMTTD was detected in plasma and incorporated into VLDL, liver, and heart. The relative incorporation (mol%) of CMTTD was heart > VLDL > liver. In conclusion, CMTTD causes both a hypocholesterolemic and hypotriacylglycerolemic effect in hyperlipidemic hamsters.

The complete nucleotide sequence of the Atlantic salmon growth hormone I gene.

Two closely related genes encoding growth hormone were isolated from Atlantic salmon by genomic cloning. From one of these genes a total of 6500 nucleotides were determined including 3900 nucleotides in exons and introns and about 600 and 2000 nucleotides in 5' and 3' flanking regions. The gene is organized in six exons and encodes a polypeptide of 210 amino acids including a 22 amino acids signal sequence. The promoter region contains a typical TATA box 21 nucleotides upstream from the transcription start site. At the 3' end, three putative poly(A) signal sequences are present. The last two are within a 121 nt inverted repeat.

Distribution patterns of muscle fibre types in major muscles of the bull (Bos taurus).

The study describes the variations in distribution and cross-sectional area (fibre size) of three muscle fibre types (I, IIA, IIB) in 34 of the largest muscles of the bull (Bos taurus). The animals had been kept strictly unexercised for one year before slaughter. Representative sampling was done at 15 positions within each muscle, and from 2700 to 4500 fibres were analysed in each muscle. Different intermuscular patterns are described. The overall volume fraction (%) of type I fibres was about 10% higher in the forepart muscles than in the hindpart muscles (41% and 31%, respectively), while the mean content of type IIB fibres was similar. Type I fibres were particularly abundant in antigravity muscles. Of these, the hindlimb muscles contained 50% more type I fibres (by weight) than those of the forelimb. Typical antigravity antagonists contained very few type I fibres. In the thigh cross-section the proportion of type I fibres was highest in the anterior and medial parts, while the IIB fibres tended to be concentrated in the superficial and posterior parts. Intramuscular patterns were revealed, with type I fibres becoming gradually more abundant from superficial to deep regions, while IIB fibres had an opposite distribution. This was particularly evident in the thigh proper and in the scapular region. Within each fasciculus of all the muscles, the muscle fibre types formed a general spatial pattern. Type I fibres in the muscles of the forepart were on average about 15% larger than those of the muscles in the hindpart. The IIB fibres were on average about 10% larger in the hindpart than in the forepart muscles. A covariation between the proportion of type I and IIB fibres and their cross-sectional area was indicated.

A general, computer-based method for study of the spatial distribution of muscle fiber types in skeletal muscle.

The present method provides detailed quantitative information on the spatial distribution of the muscle fiber types in skeletal muscle. This is accomplished by comparing the measured spatial distribution of the fiber types with a computer-simulated random pattern. The method is based on a registration of the absolute frequency for six principal categories of fiber contacts (I-I, I-IIA, I-IIB, IIA-IIA, IIA-IIB, IIB-IIB). A computer program was designed to simulate a random pattern of fibers. The simulations were performed with high accuracy with regard to fiber type proportion and the number of neighbouring fibers. The computer then calculated the frequency for each of the different categories of fiber contacts in the simulated random pattern. The measured distribution of fiber contacts could thus be compared to the simulated random pattern. In three bovine muscles studied, the spatial distribution of the muscle fiber types showed a similar pattern. The muscle fibers had a distinct tendency to be surrounded by fibers of a different type. In all three muscles the difference between the measured and the simulated random pattern was statistically significant (p less than 10(-3).

Composition of muscle fibre types and connective tissue in bovine M. semitendinosus and its relation to tenderness.

The distribution of muscle fibre types and connective tissue in bovine M. semitendinosus is described. A parallel increase in the volume fraction of type I muscle fibres (from 10% to 30%) and a decrease in the IIB volume fraction (from 58% to 34%) was recorded from superficial to deep layers. A positive correlation was observed between the frequency and the cross-sectional area of both type I and IIB fibres. The elastic fibres formed irregularly shaped bundles that made up about 50% of the volume of the perimysium. Thin elastic fibres extended into the endomysium. The relative proportion of elastic fibres in the perimysial connective tissue increased towards the deeper layers of the muscle. A taste panel evaluation of the sensory properties was performed and the data were correlated to the histological observations. A gradual decrease in scores of four tenderness-related traits was recorded from the superficial to the deep layer of the muscle. The superficial layer was rated as most tender, whereas the consecutive layers were rated less tender. The possible relationship between the composition of muscle and the meat quality is discussed.

Substructure of solitary cilia in mouse kidney.

Recent scanning electron microscopic studies confirm the presence of solitary cilia on most epithelial cells along the mammalian nephron and collecting ducts. By transmission electron microscopy we have found that the axonemata of such cilia consist of a maximal number of 9 doublet and no singlet filaments. 10% of the cross-sectioned cilia contain 9 doublets arranged in peripheral ring (9 + 0 pattern). 30% of the cross-sections contain 8 or 7 doublets in peripheral ring and 1 or 2 doublets in the central region (8 + 1 and 7 + 2 patterns). Serial sections and goniometer tilt reveal the central doublets to originate as dislodged peripheral doublets. 60% of the sectioned cilia contain filament numbers between 8 and 4. In patterns of 5 and 4 filaments single microtubules predominate. The functional significance of these atypical cilia is discussed.

Three muscle fibre types in the axial muscle of axolotl (Ambystoma mexicanum Shaw): a quantitative light- and electron microscopic study.

Morphometric analysis by light microscopy of p-phenylene-diamine stained semithin sections of axolotl tail muscle revealed differences in the cross-sectional area of the fibres and in the number of mitochondria and of lipid inclusions per fibre, and indicated the presence of three distinct types of fibres. The tripartition was found to be statistically highly significant. Representative fibres from each group established by light microscopic morphometry were subjected to an ultrastructural morphometric analysis. The volume content of mitochondria amounted to 9.8% of the fibre volume for red, 4.0% for intermediate and 0.8% for white fibres. The myofibrils composed 60%, 70% and 83% in the same fibres. The volume of the sarcotubular system (t-tubuli and sarcoplasmic reticulum) was 2.5% in red, 4.5% in intermediate and 11.7% in white fibres. The three fibre types also demonstrated differences in myofibrillar cross-striation pattern and number of triads. The reliability of the light microscopic morphometry was tested by correlation with EM montages of the representative fibres.