Duplication of Dio3 genes in teleost fish and their divergent expression in skin during flatfish metamorphosis.

Deiodinase 3 (Dio3) plays an essential role during early development in vertebrates by controlling tissue thyroid hormone (TH) availability. The Atlantic halibut (Hippoglossus hippoglossus) possesses duplicate dio3 genes (dio3a and dio3b). Expression analysis indicates that dio3b levels change in abocular skin during metamorphosis and this suggests that this enzyme is associated with the divergent development of larval skin to the juvenile phenotype. In larvae exposed to MMI, a chemical that inhibits TH production, expression of dio3b in ocular skin is significantly up-regulated suggesting that THs normally modulate this genes expression during this developmental event. The molecular basis for divergent dio3a and dio3b expression and responsiveness to MMI treatment is explained by the multiple conserved TREs in the proximal promoter region of teleost dio3b and their absence from the promoter of dio3a. We propose that the divergent expression of dio3 in ocular and abocular skin during halibut metamorphosis contributes to the asymmetric pigment development in response to THs.

Timecourse of oocyte development in saithe Pollachius virens.

Wild caught North Sea saithe Pollachius virens were monitored for growth, sex steroid profiles and oocyte development pre-spawning and measured for egg size and group fecundity during the spawning season in the laboratory. Vitellogenesis commenced in late October-early November, at a leading cohort size (CL ) of c. 250 µm, after which oocytes grew rapidly in size until spawning started in February. Notably, a distinct cortical alveoli stage was virtually absent with yolk granules observed in developing oocytes at the very beginning of vitellogenesis. Little atresia was observed pre-spawning, but atretic re-absorption of remnant oocytes containing yolk granules was found in all females immediately post-spawning. As expected, concentrations of sex steroids, oestradiol-17ß (females), testosterone (both sexes) and 11-ketotestosterone (both sexes), increased pre-spawning before dropping post-spawning. The present experiment provides the first validation of sex steroid levels in P. virens. Post-ovulatory follicles were visible in histological sections from female gonads 9-11 months post-spawning, but then disappeared. Spawning commenced around a CL of c. 750 µm (700-800 µm). Hydrated oocytes (eggs) measured between 1·04 and 1·31 mm (mean = 1·18 mm) with decreasing sizes towards the end of spawning. The average estimated realized fecundity was c. 0·84 million eggs (median female total length, LT = 60 cm). Spawning lasted from 13 February to 29 March.

Angiogenic factors in relation to clinical effect in a phase II trial of weekly paclitaxel.

BACKGROUND: Several anticancer agents including paclitaxel have an inhibitory effect on angiogenesis. AIMS: To compare the overall response rate and time to progression with changes in circulating angiogenic factors during palliative treatment with weekly paclitaxel. MATERIAL AND METHODS: Patients with metastatic BC, ECOG 0-2, received weekly paclitaxel, concomitant with trastuzumab if HER2+ BC (n = 7). Circulating vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were determined at base-line and before start of new course. RESULTS: Fifty-five of 63 included patients were evaluable. The overall response rate including stable disease ≥24 weeks (CR + PD + SD) was obtained in 25 of the evaluable patients (45%). The median time to progression (TTP) was 5.3 months and overall survival (OS) 16.7 months. Patients with triple negative breast cancer (TNBC) showed a trend towards higher base-line VEGF compared with hormone receptor positive or HER2+ tumours and had shorter TTP. Significant differences in VEGF and bFGF levels at 12 weeks were found between patients with longer versus shorter TTP (VEGF: p = 0.046, bFGF: p = 0.005) and between patients gaining versus lacking clinical benefit (VEGF: p = 0.05, bFGF: p = 0.02). CONCLUSIONS: The clinical utility of circulating VEGF may be a useful tool for monitoring treatment efficacy.

Natural sex change in the temperate protogynous Ballan wrasse Labrus bergylta.

Wild Ballan wrasse Labrus bergylta were sampled monthly over 2 years in western Norway to identify the natural process of sex change in this species. Light microscopy of standard histological-stained and immunohistochemistry-treated gonad tissue showed that spermatogonial germ cells tended to proliferate around the periphery of the lamellae before filling into the slowly receding, apoptotic central areas of the lamellae. Sex change occurred following the breeding season. From July to September, fish were most often in an early state of gonadal transition (ET), characterized by degenerating previtellogenic oocytes and pockets of proliferating spermatogonia in the germinative epithelia. The majority of fish with late transitional gonads, that were typically dominated by spermatogenic cells, developing efferent ducts and the beginning of lobule formation, were found between October and November. Sex steroid profiles of fish representing the different sexual phases showed that breeding females had the highest concentrations of 17ß oestradiol (E2 ) and the lowest concentration of 11 ketotestosterone (11KT). Concentrations of E2 decreased greatly in ET fish at the beginning of sex change and remained low in all subsequent phases. The opposite trend was demonstrated in 11KT profiles. Initial-phase female fish had minimal concentrations of 11KT, but these increased during subsequent transitions. Sex change occurred most often in fish 34-41 cm total length (L(T)) and the median of fish in the size-frequency overlap of female and male fish was 36 cm L(T).

The effect of dietary lipid content and stress on egg quality in farmed Atlantic cod Gadus morhua.

The present study assessed differences in fecundity and egg quality from Atlantic cod Gadus morhua fed isoproteic diets containing 13% fat (low fat, LF) or 20% fat (high fat, HF) and either stressed or left unstressed as a control over the spawning season. Each diet was fed to triplicate groups of G. morhua from June 2009, through to first maturation and spawning. In January 2010 sub-groups of G. morhua were moved to land-based spawning tanks where the experimental trial was carried out. At the start of the experiment, G. morhua fed the high-fat diet were significantly larger than G. morhua fed low-fat diet. These differences were maintained through the spawning season, although with a loss of mass in both dietary groups. Relative fecundity through the season was significantly lower in stressed G. morhua fed LF compared to unstressed G. morhua fed the same diet. Stressed G. morhua had a higher variability in weekly amount of eggs spawned, spawning occurred more irregularly, and the spawning period lasted longer than in unstressed G. morhua. Several egg quality variables were also affected: eggs from G. morhua fed LF and exposed to stress had lower fertilization and hatching rates compared to the unstressed G. morhua fed the same diet as well as all G. morhua fed HF. Gadus morhua fed a low-fat diet appeared less tolerant to stress than fish fed a high-fat diet.

Sex-biased miRNA expression in Atlantic halibut (Hippoglossus hippoglossus) brain and gonads.

The role of miRNA in fish sexual development is not elucidated yet. We profiled miRNAs in gonads and brains of Atlantic halibut using SOLiD sequencing technology. We found tissue- and sexually dimorphic expression of several miRNAs, including miR-29a, miR-34, miR-143, miR-145, miR-202-3p, miR-451, and miR-2188. miR-9 and miR-202 were abundant in brain and gonads, respectively. In the next step, we selected some miRNAs showing differential expression patterns between sexes and performed RT-qPCR on 3 age groups: juveniles, 3-year-, and 5-year-olds. In brains, miR-451 was significantly down-regulated in juveniles compared to adults. let-7a, miR-143, and miR-202-3p were up-regulated in gonads of mature males compared to immature females at the same age. We investigated the effect of suppressing aromatase cytochrome P450 enzyme on miRNA expression at the onset of sex differentiation through masculinization with Fadrozole or 17-α-methyltestosterone. We found significant differences in miRNA expression between masculinized individuals and untreated controls. miR-202-3p was significantly down-regulated in female juveniles compared to male juveniles. The expression levels of let-7a and miR-451 were restored after termination of the masculinization treatment. Our data give a first insight into miRNA involvement in sexual development in teleosts.

Pegylated liposomal doxorubicin as first-line monotherapy in elderly women with locally advanced or metastatic breast cancer: novel treatment predictive factors identified.

We investigated the efficacy and safety of single-agent pegylated liposomal doxorubicin (PLD) as first-line treatment for elderly women with advanced breast cancer and evaluated predictive markers for response and toxicity. Twenty-five women ≥ 65 years received 40 mg/m(2) PLD every 28 days. Time to treatment failure (TTF), response rate, time to progression (TTP) and overall survival (OS) was calculated. The ABCB1 single nucleotide polymorphisms (SNP), tumor MRN complex, and TOPOIIα were analyzed. A mean of 7.4 cycles PLD were administered and TTF was 5.5 months and OS 20.6 months. ABCB1 SNPs were found to correlate to both efficacy and toxicity, while tumor expression of the MRN complex and TOPOIIα correlated to TTP. PLD is a safe and effective treatment for elderly breast cancer patients. Also potential predictive markers were identified.

The reproductive cycle of female Ballan wrasse Labrus bergylta in high latitude, temperate waters.

This 2 year study examined the reproductive cycle of wild female Ballan wrasse Labrus bergylta in western Norway as a precursor to captive breeding trials. Light microscopy of ovarian histology was used to stage gonad maturity and enzyme-linked immuno-absorbent assay (ELISA) to measure plasma concentrations of the sex steroids testosterone (T) and 17beta-oestradiol (E(2)). Ovarian recrudescence began in late autumn to early winter with the growth of previtellogenic oocytes and the formation of cortical alveoli. Vitellogenic oocytes developed from January to June and ovaries containing postovulatory follicles (POF) were present between May and June. These POF occurred simultaneously among other late maturity stage oocytes. Plasma steroid concentration and organo-somatic indices increased over winter and spring. Maximal (mean +/-s.e.) values of plasma T (0.95 +/- 0.26 ng ml(-1)), E(2) (1.75 +/- 0.43 ng ml(-1)) and gonado-somatic index (I(G); 10.71 +/- 0.81) occurred in April and May and decreased greatly in July when only postspawned fish with atretic ovaries occurred. Evidence indicates that L. bergylta are group-synchronous multiple spawners with spawning occurring in spring and peaking in May. A short resting period may occur between late summer and autumn when previtellogenic oocytes predominate and steroid levels are minimal.

Molecular characterization and quantification of the gonadotropin receptors FSH-R and LH-R from Atlantic cod (Gadus morhua).

In order to elucidate regulatory mechanisms during puberty final oocyte maturation and spawning, full-length sequences coding for the receptors for follicle-stimulating hormone (FSH-R) and luteinizing hormone (LH-R) were isolated from female Atlantic cod (Gadus morhua) by a RACE-PCR based strategy. The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species but contained some distinct differences. Conserved features important for functionality, such as a long N-terminal extracellular domain (ECD), seven transmembrane domains and a short C-terminal intracellular domain, were identified in both predicted proteins. Partial genomic sequences for these genes were also determined, allowing the design of mRNA-specific quantitative PCR assays. Due to suspected alternative splicing during expression of these genes, additional real-time PCR assays detecting variants containing the membrane-anchoring domain were established. Besides the expected expression of FSH-R and LH-R mRNA in the gonads similarly strong signals for LH-R were also obtained in male gill, and in female and male brain. When relative expression was analysed at different stages of sexual maturation, levels for FSH-R increased moderately during gonadal growth whereas those of LH-R showed a high peak at spawning.

20 beta-hydroxysteroid dehydrogenase and CYP19A1 are differentially expressed during maturation in Atlantic cod (Gadus morhua).

In order to better quantify the molecular mechanisms regulating final oocyte maturation and spawning, complete coding sequences with partially or fully untranslated regions for the steroidogenic enzymes, cytochrome P450 aromatase and 20 beta-hydroxysteroid dehydrogenase, were cloned from ovaries of Atlantic cod (Gadus morhua). The nucleotide and amino acid sequences showed high homologies with the corresponding sequences of other fish species, and conserved features important for functionality were identified in both predicted proteins. The sequences of the corresponding genomic loci were also determined, allowing the design of mRNA-specific quantitative PCR assays. As a reference gene for the real-time RT-PCR assays, eukaryotic elongation factor 1 alpha was chosen, and the mRNA as well as the genomic sequence was determined. In addition, a real-time quantitative PCR assay for the 18S rRNA was adapted to be used in cod. Analysis of immature and maturing female cod from July to January respectively showed that the enzyme genes showed the expected quantitative changes associated with physiological regulation. However, mRNA for eukaryotic elongation factor 1 alpha, and to a lesser extent even 18S rRNA, showed variable expression in these samples as well. To find accurate standards for real-time PCR in such a dynamic organ as the cod ovary is not an easy task, and several possible solutions are discussed.

Lysine intake affects gene expression of anabolic hormones in Atlantic salmon, Salmo salar.

Nutritional factors influence regulation of the growth hormone (GH) and the insulin-like growth factor (IGF) system in fish, but so far there are no published studies describing how single indispensable amino acids influence these systems. Therefore, the present study aimed to test whether lysine (Lys) intake at low (LL=2.85 g/16 gN), medium (ML=4.91 g/16 gN) and high levels (HL=9.19 g/16 gN) affected the expression of genes related to the GH-IGF system (i.e. GH receptor, GH-R, IGF-I, IGF-II, IGF binding protein 1, IGFBP-1, IGF-I receptor IGF-IR) in Atlantic salmon during seawater growth phase. Salmon fed the HL diet significantly up-regulated hepatic IGF-I mRNA level by a factor of 2.2 as compared to those with medium Lys intake. In addition a significant up-regulation of 2.7-fold in muscle IGF-II mRNA was present. Low Lys intake decreased the nitrogen deposition and muscle protein accretion in fish and significantly down-regulated hepatic IGFBP-1 as well as muscle GH-R and IGF-II, as compared to those fed the ML diet. mRNA of IGF-IR on the other hand was not affected by Lys intake. High Lys intake resulted in a 7-fold up-regulation of muscle IGF-II mRNA level as compared to low Lys intake, and thus might be an important local anabolic regulator in fast muscle tissue. The single indispensable amino acid Lys indeed affected signalling through the genes of IGF-I, IGFBP-1 in hepatic tissue and GH-R, IGF-II in fast muscle in Atlantic salmon. Concomitantly the higher Lys intake increased nitrogen deposition to a certain level.

Medical intelligence in Sweden. Vitamin B12: oral compared with parenteral?

BACKGROUND: Sweden is the only country in which oral high dose vitamin B12 has gained widespread use in the treatment of deficiency states. OBJECTIVE: The aim of the study was to describe prescribing patterns and sales statistics of vitamin B12 tablets and injections in Sweden 1990-2000.Design, setting, and sources: Official statistics of cobalamin prescriptions and sales were used. RESULTS: The use of vitamin B12 increased in Sweden 1990-2000, mainly because of an increase in the use of oral high dose vitamin B12 therapy. The experience, in statistical terms a "total investigation", comprised 1,000,000 patient years for tablets and 750,000 patient years for injections. During 2000, 13% of residents aged 70 and over were treated with vitamin B12, two of three with the tablet preparation. Most patients in Sweden requiring vitamin B12 therapy have transferred from parenteral to oral high dose vitamin B12 since 1964, when the oral preparation was introduced. CONCLUSION: The findings suggest that many patients in other post-industrial societies may also be suitable for oral vitamin B12 treatment.

Survival after treatment for breast cancer in a geographically defined population.

BACKGROUND: South East Sweden with 976 000 inhabitants is served by nine hospitals with specialized breast surgeons. Population-based mammographic screening was introduced in 1986 for women aged 40-74 years. Patients with primary breast cancer were treated according to a joint management programme. METHODS: All patients were reported to a regional cancer registry from which breast cancer incidence, treatment and survival in this defined population were reported. RESULTS: A total of 7892 women had their first invasive breast cancer diagnosed between 1986 and 1999. The median tumour size was 17 mm and 29.9 per cent had axillary metastases. Some 49.8 per cent of these women had a modified radical mastectomy and 31.9 per cent had a segmental resection with axillary clearance. Postoperative radiotherapy was given to 40.3 per cent of the women after mastectomy and to 87.1 per cent after breast-conserving surgery. Tamoxifen and chemotherapy were used as adjuvant treatment except in low-risk patients. Breast cancer-specific survival rate for all stages was 83.5 per cent at 5 years and 74.0 per cent at 10 years. Respective values were 95.8 and 90.9 per cent for patients with stage T1 N0 M0 tumours, and 77.7 and 62.4 per cent for those with T1-2 N1 M0 tumours. CONCLUSION: Breast specialists treating women with breast cancer according to a joint management programme have achieved very good survival rates.

Kinetics and fates of ammonia, urea, and uric acid during oocyte maturation and ontogeny of the Atlantic halibut (Hippoglossus hippoglossus L.).

Considering that amino acids constitute an important energy fuel during early life of the Atlantic halibut (Hippoglossus hippoglossus L.), it is of interest to understand how the nitrogenous end products are handled. In this study we focused on the kinetics and fates of ammonia, urea and uric acid. The results showed that ammonia (T(Amm): NH(3)+NH(4)(+)), and urea-N contents increased during final oocyte maturation. Urea-N excretion dominated the total nitrogenous end product formation in early embryos. Later, yolk T(Amm) levels increased in embryos and ammonia excretion was low. In the last part of the embryonic stage T(Amm) accumulation dominated, and was apparently due to yolk storage. Around hatching, the larval body tissues (larva with yolk-sac removed) accounted for 68% of whole animal urea-N accumulation, while T(Amm) levels increased predominately by yolk accumulation. Afterwards, ammonia excretion dominated and uric acid accumulation accounted for less than 1%. Urea, synthesised either through the ornithine-urea cycle, argininolysis or uricolysis, accounted for approximately 8% of total nitrogenous end product formation in yolk-sac larvae. The results suggested that a sequence occurred regarding which nitrogenous end products dominated and how they were handled. Urea excretion dominated in early embryos (<7 dPF), followed by yolk ammonia accumulation (7-12 dPF), and finally, ammonia excretion dominated in later embryonic and yolk-sac larval stages (>12 dPF).

Pyrazolidine-3,5-dione angiotensin-II receptor antagonists.

The crystal structures of three angiotensin-II receptor antagonists involving different spacer groups (CO, CONH and NHCO) between the aryl rings are presented, namely 2-[4-[(3-butyl-1,4-dioxo-2,3-diazaspiro[4.4]non-2-yl)methyl]benzoyl]benzoic acid, C(26)H(28)N(2)O(5), (I), 2-[4-[(3-butyl-1,4-dioxo-2,3-diazaspiro[4.4]non-2-yl)methyl]benzamido]benzoic acid, C(26)H(29)N(3)O(5), (II), and 2-[4-[(3-butyl-1,4-dioxo-2,3-diazaspiro[4.4]non-2-yl)methyl]anilinocarbonyl]benzoic acid monohydrate, C(26)H(29)N(3)O(5) x H(2)O, (III). The aryl rings of (II) are almost coplanar, in contrast with compounds (I) and (III). The conformation of (II) is induced by an intramolecular N-H.O hydrogen bond between the amide and carboxylic acid groups.

Vitamin B12 in primary health care and geriatrics--attitudes, knowledge and competence.

OBJECTIVES: The objective of the study was to test attitudes, knowledge and competence of Swedish general practitioners and geriatricians concerning B12-associated problems in 1998. METHODS: Postal questionnaires were sent to a random sample of 485 GPs and a total sample of 613 geriatricians. The response rates were 70% in the GP group and 69% in the geriatrician group. The questionnaire contained 24 statements to be evaluated by a visuo-analogue scale. RESULTS: There were small numerical differences between the two physician groups. The geriatricians were more aware of risk groups for B12 deficiency. GPs were less categorical concerning low hit rate in the laboratory testing of clinical conclusions. There were statistical differences in both directions for statements on pitfalls in laboratory diagnostics. GPs were somewhat less prone to give risk groups prophylactic B12 therapy. CONCLUSIONS: GPs and geriatricians appeared to be familiar with the current debate on B12-associated problems, suggesting that health care quality will be unaffected by patient transfer from hospital care to primary health care.

Shifts in B12 opinions in primary health care of Sweden.

AIMS: The diagnosis and management of vitamin B12 deficiency varies between countries and within countries. The aim of the study was to map current attitudes and values behind clinical decision-making in Swedish primary health care, which has a unique B12 tradition: two patients out of three are treated with oral high-dose cyanocobalamin. Most patients with B12-associated problems are managed in primary health care by general practitioners (GPs). METHODS: The study was designed to elucidate possible opinion shifts among GPs during the period 1996-1998. GPs (n=499), stratified and randomized, received a questionnaire with 24 statements on B12-associated clinical and laboratory problems, to be evaluated by a visuo-analogue scale. RESULTS: The majority of GPs in primary health care in Sweden accepted homocysteine and methylmalonic acid (MMA) as markers for functional deficiency of vitamin B12. The evaluation of classical markers of B12 deficiency was wary and balanced. There was a consensus of the need for B12 therapy to risk groups such as patients with atrophic gastritis or previous gastric surgery. The answers also appeared to reflect an improvement of professional knowledge and competence concerning B12-associated problems among Swedish GPs between 1996 and 1998. CONCLUSIONS: The overriding conclusion was that B12-associated opinions of Swedish GPs were stable within the period studied, with marginal improvements of knowledge and competence.

Effects of photoperiod on sexual maturation and somatic growth in male Atlantic halibut (Hippoglossus hippoglossus L.).

A major obstacle in modern, intensive aquaculture is the precocious maturation of male fish, leading to decreased somatic growth and reduced filet quality. Effects of photoperiod on sexual maturation and growth in male Atlantic halibut were therefore examined. In June 1996, 1300 1+ fish of both sexes were distributed in two indoor tanks supplied with continuous light (LL) or a simulated natural photoperiod (SNP). In December 1996 and June 1997, 200 individuals were exchanged between the tanks creating six experimental groups that were followed until June 1998. LL stimulated growth and accelerated timing of first maturation by approximately 3 months. LL also appeared to interrupt circannual rhythmicity in sexual maturation. Sexual maturation led to reduced growth from 3 months pre-spawning and throughout the spawning season. Males that did not mature during the experiment attained the highest final body weight. All males reared on LL from June 1997 reached sexual maturity the following season. In contrast, only 26% of the males matured in the group transferred from LL to SNP in June 1997, and this group also had the highest final body weight. The results indicate a possible route for reducing the problem of precocious maturation in male halibut.