Manuel Rey-Méndez

Challenges in the identification of species of canned fish

Abstract The identification of fish species becomes a problem when the usual identifying characteristics are removed on processing and only a portion of flesh is available. When the flesh is raw or cooked under normal conditions, the species is readily established by electrophoresis of the muscle proteins. The procedure cannot be used for heat-sterilised canned fish as the proteins are severely denatured. DNA is also degraded but techniques are now available for targeting and amplifying species-specific fragments. The amplified products can then be analysed by a range of techniques some of which are suitable for food control laboratories.

Fish species identification in canned tuna by PCR-SSCP: validation by a collaborative study and investigation of intra-species variability of the DNA-patterns

Analysis of single strand conformation polymorphism (SSCP) of an amplicon (123 bp) obtained by polymerase chain reaction (PCR) of the mitochondrial cytochrome b gene was used to identify the fish species in canned tuna. Single-stranded DNA (ssDNA) was separated by polyacrylamide gel electrophoresis, and visualised by silver staining. The reliability of the method was tested by a collaborative study in which eight European laboratories participated. Seven unknown samples (five from individual species and two mixtures of two tuna species) of canned tuna had to be identified by comparison with reference material. From a total of 72 cases, 65 (90.3%) were assigned correctly. Intra-species variability of SSCIP patterns was found in the case of Katsuwonus pelamis and Sarda sarda. As specimens from various fishing grounds gave two or three different patterns of ssDNA, the possibility of some variability of the DNA patterns has to be considered in SSCP analysis of these species.

Serotonin upregulates the activity of phagocytosis through 5-HT1A receptors

In this study, we investigated whether serotonin could regulate the in vitro activity of phagocytosis through 5‐hydroxytryptamine or serotonin (5‐HT1A) receptors. Mouse peritoneal macrophages were cultured with serotonin and the activity of phagocytosis was assessed by the uptake of zymosan and latex particles added to the culture media. Specific binding of [3H]8‐OH‐DPAT and immunohistochemistry using an affinity‐purified anti‐5‐HT1A‐receptor antibody were assayed in the macrophages. In addition, we took advantage of the availability of pharmacological inhibitors of nuclear factor‐κB (NF‐κB) to explore its role in the regulation of the 5‐HT1A receptor. Serotonin increased the in vitro activity of phagocytosis in a dose‐dependent manner. The 5‐HT1A receptor agonist (±)‐8‐hydroxy‐2‐(di‐n‐propyl‐amino)‐tetralin (R(+)‐8‐OH‐DPAT) reproduced these effects. Serotonin‐ or R(+)‐8‐OH‐DPAT‐induced increases in phagocytosis were blocked by the 5‐HT1A receptor antagonist WAY100635 and the NF‐κB inhibitor pyrrolidinedithiocarbamate. Moreover, mouse peritoneal macrophages expressed specific binding sites for [3H]8‐OH‐DPAT when cultivated in the presence of zymosan or latex beads. Immunohistochemistry confirmed the expression of the 5‐HT1A receptor protein in the macrophages. These results show that serotonin can upregulate the activity of peritoneal macrophages through 5‐HT1A receptors.

Alterations induced by chronic stress in lymphocyte subsets of blood and primary and secondary immune organs of mice

BackgroundThe immune system is particularly sensitive to stress. Although acute stress generally has positive effects, chronic stress typically provokes immunosuppression. The elucidation of the mechanisms involved in immunosuppression are of interest for the design of therapeutic approaches to avoid the appearance of stress disorders. This study aimed to investigate chronic stress-induced alterations on lymphocyte subset distribution and percentages. The experiments were performed with C57BL/6 mice subjected to chronic immobilization stress.ResultsStress caused a marked increase in apoptosis inside the thymus, and a reduction in the total number of thymocytes. Furthermore, the proportion of immature thymocytes declined significantly suggesting that the increased apoptosis mainly affected cells of immature phenotype. In blood, the total number of lymphocytes diminished but not all lymphocyte populations showed the same tendency: while the relative proportion of B cells declined slightly, the relative proportion of circulating CD3+ cells, and particularly some T cell subsets showing an immature phenotype (CD3+PNA+), increased under stress. The spleen and lymph nodes show a marked reduction in cellularity, but the relative proportion of T cells increased, while no change or only a slight reduction was observed in the relative proportion of B cells. Similarly, the relative proportion of T cells increased in bone marrow.ConclusionsDetailed data on the alterations of lymphoid cell subsets occurring under immobilization stress, both in the bloodstream and in different lymphoid tissues, are obtained. In general, T cells are more affected than B cells and, in particular, a marked increase in the percentage of a subset of circulating PNA+CD3+ T cells is observed.

Time-course of the murine lymphoid tissue involution during and following stressor exposure

Groups of 35-day-old male C57BL/6 mice were stressed 1 hour per day by immobilization for 1, 2, 3, 5, 8, 11 or 14 consecutive days. Control groups were left undisturbed. The animals were then killed and body weight and the weights of the thymus, spleen and axillary lymph nodes determined. Chronic immobilization stress caused involution of the thymus, spleen and lymph nodes to an extent depending on the number of days of stress. The thymus showed the fastest response: thymus weight was significantly lower in stressed animals than in controls by the third day of stress while significant effects on spleen and lymph node weight were not observed until day 5. Fast recovery of lymphoid organ weight was observed after the stress period. The thymus recovered most quickly: control values were re-attained approximately 8 days after cessation of stress, and indeed by day 20 thymus weight was about 12% higher than in normal animals. The spleen and lymph nodes recuperated weight more slowly, re-attaining control values after about 20 days.

Effects of amphetamine on cell mediated immune response in mice

Mice injected with amphetamine showed a dose-related suppression of the natural killer cell activity. The capacity of T-cells to generate cytotoxic T-lymphocytes (CTL) in mixed lymphocyte cultures and in vivo was also assayed and amphetamine was found to inhibit CTL responses.

Effects of fluoxetine on the immunosuppressive response to stress in mice

Mice exposed to a chronic auditory stressor and treated with fluoxetine (5 mg/kg) showed a reduction in stress-induced suppression of thymus and spleen cellularity, and in peripheral T lymphocyte population. The blastogenic response of spleen lymphoid cells and the delayed type hypersensitivity response (DTH) to sheep red blood cells (SRBC) were also assessed and fluoxetine was found to partially reverse the inhibitory effect of stress on both parameters.

Identification of European Hake Species (Merluccius merluccius) Using Real-Time PCR

A rapid and precise method for identifying European hake (Merluccius merluccius) based on TaqMan technology is presented. The method can be applied to fresh, frozen, and processed fish products to detect the fraudulent or unintentional mislabeling of this species. Specific primers and a minor groove binding (MGB) TaqMan probe were designed for this purpose based on partial sequences of the mitochondrial DNA control region. Combinations of primers and probe concentrations that gave the lowest Ct value and the highest final fluorescence value were selected to carry out efficiency, specificity, and cross-reactivity assays. The method was successfully tested on 31 commercial hake samples. A Ct value of about 16 was obtained when Merluccius merluccius was present; however, the fluorescence signal was not detected most of the time (Ct value 40) or presented significantly higher Ct values (38.2 +/- 0.96) for the nonhake species.

Effects of fluoxetine on the development of lung metastases induced by operative stress in rats

Experiments were performed in order to evaluate the effects of fluoxetine, a selective inhibitor of neural serotonin transporter antidepressant, on the development lung metastases in rats subjected to laparotomy and injected (i.v.) with 10(4) Walker 256 (W-256) carcinosarcoma cells. The number of metastatic nodules on the surface of the lungs, as well as the percentage-area of metastases in the frontal section through pulmonary hilus were increased in rats subjected to sham-surgery or laparotomy. Treatment with fluoxetine (5 mg/kg) partially reversed those adverse effects of surgery, but the difference was clearer when it was administered before surgery was performed. Survival periods were also assessed and fluoxetine was found to decrease the lethality of rats exposed to surgery.

Effects of nefazodone on voluntary ethanol consumption induced by isolation stress in young and aged rats

Late-onset ethanol (EtOH) consumption is related to life and social stressors of aging. The stress system (hypothalamic-pituitary-adrenal, HPA, axis) coordinates the adaptive response of the organism to stressors, but age-related deficits in HPA function seem to be associated with disorders such as late-onset EtOH consumption, anxiety and depression. In the present study, we examined whether HPA dysfunction is associated with stress-related EtOH consumption in aged rats and whether the treatment with nefazodone hydrochloride, a phenylpiperazine antidepressant, partially reverses the adverse effects of isolation (ISOL) stress. The animals were offered two-bottle choice consumption of 0.2% saccharin and 10% EtOH/0.2% saccharin, and then exposed to 4 days of ISOL stress on an irregular, unpredictable schedule. ISOL stress-induced increases in corticosterone secretion and EtOH consumption both during and following the stress (recovery period) in aged rats. Nevertheless, this effect at the recovery period was not evident in young stressed rats. Nefazodone caused a significant decrease in plasma corticosterone levels and EtOH consumption. The attenuation of stress-induced corticosterone by nefazodone was correlated with reduced EtOH consumption. These findings link the effect of ISOL stress to the induction of voluntary EtOH consumption following the end of the stressor and the limitation of aged HPA to down-regulated corticosterone.