Zymosan-induced immune challenge modifies the stress response of hypoxic air-breathing fish (Anabas testudineus Bloch): Evidence for reversed patterns of cortisol and thyroid hormone interaction, differential ion transporter functions and non-specific immune response.

Fishes have evolved physiological mechanisms to exhibit stress response, where hormonal signals interact with an array of ion transporters and regulate homeostasis. As major ion transport regulators in fish, cortisol and thyroid hormones have been shown to interact and fine-tune the stress response. Likewise, in fishes many interactions have been identified between stress and immune components, but the physiological basis of such interaction has not yet delineated particularly in air-breathing fish. We, therefore, investigated the responses of thyroid hormones and cortisol, ion transporter functions and non-specific immune response of an obligate air-breathing fish Anabas testudineus Bloch to zymosan treatment or hypoxia stress or both, to understand how immune challenge modifies the pattern of stress response in this fish. Induction of experimental peritonitis in these fish by zymosan treatment (200ngg-1) for 24h produced rise in respiratory burst and lysozomal activities in head kidney phagocytes. In contrast, hypoxia stress for 30min in immune-challenged fish reversed these non-specific responses of head kidney phagocytes. The decline in plasma cortisol in zymosan-treated fish and its further suppression by hypoxia stress indicate that immune challenge suppresses the cortisol-driven stress response of this fish. Likewise, the decline in plasma T3 and T4 after zymosan-treatment and the rise in plasma T4 after hypoxia stress in immune-challenged fish indicate a critical role for thyroid hormone in immune-stress response due to its differential sensitivity to both immune and stress challenges. Further, analysis of the activity pattern of ion-dependent ATPases viz. Na+/K+-ATPase, H+/K+-ATPase and Na+/NH4+-ATPase indicates a functional interaction of ion transport system with the immune response as evident in its differential and spatial modifications after hypoxia stress in immune-challenged fish. The immune-challenge that produced differential pattern of mRNA expression of Na+/K+-ATPase α-subunit isoforms; nkaα1a, nkaα1b and nkaα1c and the shift in nkaα1a and nkaα1b isoforms expression after hypoxia stress in immune-challenged fish, presents transcriptomic evidence for a modified Na+/K+ ion transporter system in these fish. Collectively, our data thus provide evidence for an interactive immune-stress response in an air-breathing fish, where the patterns of cortisol-thyroid hormone interaction, the ion transporter functions and the non-specific immune responses are reversed by hypoxia stress in immune-challenged fish.

Melatonin integrates multidimensional regulation of Na+/K+-ATPase in ionocytes and promotes stress and ease response in hypoxia-induced air-breathing fish: lessons from integrative approach.

As circadian regulator, melatonin is involved in many physiological processes including ionosmotic regulation in fishes. Na+/K+-ATPase (NKA), an ubiquitous Na+/K+ transporter in ionocyte epithelia that drives electrochemical Na+ gradients and systemic osmotic integration, is a target of stress in fish. However, it is not certain how melatonin regulates NKA functions in ionocyte epithelia and how it modulates the adaptive response such as stress and ease response in fish particularly in hypoxia condition. We, thus, examined the short-term in vivo action of melatonin on the dynamics of NKA regulation in branchial, renal and intestinal ionocytes of hypoxia-induced air-breathing fish (Anabas testudineus Bloch). Interestingly, we found a rise in plasma melatonin in fish when kept for 30 min of forced submergence in water and that indicates a role for melatonin in hypoxia tolerance. A fall in blood [Na+ , K+] occurred in these hypoxic fish which later showed a recovery after melatonin treatment. Similarly, melatonin favored the fall in NKA activity in branchial and renal epithelia of hypoxic fish, though it remarkably stimulated its activities in non-stressed fish. Likewise, melatonin that produced differential pattern of mRNA expression in nkaα1-subunit isoforms (nkaα1a, nkaα1b and nkaα1c) and melatonin receptor isoforms (mtnr1a, mtnr1bb, mtnr1bb x1x2 ) in the tested ionocyte epithelia, showed reversed expression in hypoxic fish. In addition, the rise in NKAα-protein abundance in branchial and renal epithelia of melatonin-treated hypoxic fish indicated a recovery action of melatonin. A higher NKAα-immunoreactivity was found in the immunohistochemical and immunofluorescent images of branchial ionocytes and renal proximal and distal ionocytes of hypoxic fish treated with melatonin. Furthermore, an activation of PKA and PKG-dependent phosphorylation was found in branchial epithelia of hypoxic fish. The generated integrative parabola model showed that melatonin has a maximum targeted action on NKA function in the renal epithelia, suggesting its lead role in the integration of ionosmotic balance during the recovery or ease response. Over all, the data indicate a multidimensional and preferential action of melatonin on NKA regulation in fish ionocytes that integrate the recovery action against hypoxia, thus pointing to a major role for melatonin in stress and ease response in this fish.

Aqua-{6,6'-dimeth-oxy-2,2'-[ethane-1,2-diylbis(nitrilo-methyl-idyne)]diphenolato}(4-hydroxy-benzoato)manganese(III).

The title compound, [Mn(C(18)H(18)N(2)O(4))(C(7)H(5)O(3))(H(2)O)], was synthesized by a template reaction of ethane-1,2-diamine and 3-methoxy-salicylaldehyde in presence of manganese(II) 4-hydroxy-benzoate. The Jahn-Teller-distorted manganese(III) centre has an octa-hedral geometry. Extensive O-H⋯O hydrogen-bonding inter-actions generate a two-dimensional sheet structure parallel to (103).

Hypohydrotic ectodermal dysplasia.

Hypohydrotic ectodermal Dysplasia (Christ-Siemens Touraine syndrome) is a rare genetic disorder that affect several ectodermal structures. The condition is usually inherited as X-linked recessive trait, in which gene is carried by females and manifested in males. The manifestations may vary in individuals and usually involves skin, hair, nail, sweat and sebaceous glands. Hypohydrotic Ectodermal Dysplasia with classical features in two siblings is reported here.

Dietary interventions for multiple sclerosis.

BACKGROUND: Clinical and experimental data suggest that certain dietary regimens, particularly those including polyunsaturated fatty acids (PUFAs) and vitamins might improve outcomes in people with multiple sclerosis (MS). Diets and dietary supplements are much used by people with MS in the belief that they might improve disease outcomes. OBJECTIVES: We performed a Cochrane review of all randomised trials of dietary regimens for MS with the aim of answering MS consumers' questions regarding the efficacy and safety of these interventions. SEARCH STRATEGY: We searched the Cochrane MS Group trial register (February 2006), Cochrane Central Register of Controlled Trials (CENTRAL), Cochrane Library, Issue 1, 2006, MEDLINE (PubMed) (1966 to March 2006), EMBASE (1974 to March 2006) and the bibliographies of papers found. SELECTION CRITERIA: All randomised controlled trials comparing a specific dietary intervention, diet plan or dietary supplementation, with no dietary modification or placebo, were eligible. DATA COLLECTION AND ANALYSIS: Two reviewers independently selected articles, assessed trial quality and extracted data. Trial quality was poor, particularly as regards descriptions of randomisation, blinding and adverse event reporting. Some studies had large numbers of drop-outs; dropouts were never included in the analyses. MAIN RESULTS: PUFAs did not have a significant effect on disease progression, measured as worsening of Disability Status Scale. Omega-6 fatty acids (11-23 g/day linoleic acid) had no benefit in 75 relapsing remitting (RR) MS patients (progression at two years: relative risk (RR)=0.78, 95% CI [0.45 to 1.36]) or in 69 chronic progressive (CP) MS patients (RR=1.67, 95% CI [0.75 to 3.72]. Linoleic acid (2.9-3.4 g/day) had no benefit in CPMS (progression at two years: RR=0.78, 95% CI [0.43 to 1.42]). Slight decreases in relapse rate and relapse severity were associated with omega-6 fatty acids in some small studies, however these findings are limited by the limited validity of the endpoints.Omega-3 fatty acids had no benefit on progression at 12 months in 14 RRMS patients or at 24 months in 292 RRMS patients (RR=0.15, 95% CI [0.01 to 3.11], p= 0.22 at 12 months, and 0.82 95% CI [0.65 to 1.03], p=0.08, at 24 months). The low frequency of reported adverse events suggests no major toxicity associated with PUFA administration. No studies on vitamin supplementation and allergen-free diets were analysed as none met the eligibility criteria. AUTHORS' CONCLUSIONS: PUFAs seem to have no major effect on the main clinical outcome in MS (disease progression), and does not substantially affect the risk of clinical relapses over 2 years. However, the data available are insufficient to assess any potential benefit or harm from PUFA supplementation. Evidence bearing on the possible benefits and risks of vitamin supplementation and antioxidant supplements in MS is lacking. More research is required to assess the effectiveness of diets interventions in MS.

Spontaneous chromosome loss and colcemid resistance in lymphocytes from patients with myotonic dystrophy type 1.

Myotonic Dystrophy type 1 (DM1) is one of the many inherited human diseases whose molecular defect is the expansion of a trinucleotide DNA sequence. DM1 shares with fragile X syndrome (FMR1), another "unstable triplet syndrome", several molecular features not present in the remaining triplet diseases. As FMR1 is also characterised by chromosome instability at the site of the expanded triplet, lymphocytes from DM1 patients and healthy donors were cultured for micronucleus (MN) analysis, in order to verify if DM1 is also prone to chromosome instability. A FISH analysis was also carried out to detect the presence of centromeric sequences in the observed MN. The data indicate that DM1 patients present a percentage of centromere-positive MN significantly higher than controls, suggesting that chromosome loss is the main mechanism underlying the origin of the increased spontaneous instability. To further assess the proneness to instability of cells of DM1 patients, cultures from patients and controls were treated in vitro with growing concentrations of two different mutagens: colcemid, a "pure" aneugen compound whose target is tubulin, and mytomicin C, a strong clastogen. The results show that the patient group is significantly less sensitive to colcemid. These data, together with FISH analysis, suggest the presence, in DM1 patients, of an already damaged tubulin, which becomes no more sensitive to the effect of colcemid and which could be the main defect underlying the aneugenic effects in DM1.

No statistical association between fragile sites and constitutional chromosome breakpoints.

Ten thousand four hundred ninety-two constitutional breakpoints available from the cytogenetic literature were analyzed for their coincidence with known fragile sites (FS) at 303-band resolution. In this analysis we have taken into account the stochastic connections of some features of chromosome bands with both the presence of FS and constitutional breakage. Our results suggest that there is no particular association between FS and constitutional chromosome rearrangements.

Folate-sensitive fragile sites in Chinese hamster cell lines.

The expression of fragile sites in three different Chinese hamster cell lines was studied. Results showed that folate-sensitive fragile sites were expressed in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 7 and in band 1q22. A comparison of the breakpoints involved in formation of chromosome rearrangements in some established Chinese hamster cell lines was also made. Results showed that while the specific type of rearrangement was random, the breakpoints were not. Three of the chromosomal sites most frequently involved in breaks were regions in which fragile sites were expressed.

Specific chromosomal aberrations correlated to transformation in Chinese hamster cells.

Cytogenetic changes were investigated during the spontaneous progression of CHEF18 Chinese hamster cells towards tumorigenicity. We further report the chromosomal characterization of a series of spontaneous anchorage-independent clones, as well as of a series of tumor-derived cell lines resulting from injection of late passage cells in nude mice. The high karyotypic homogeneity (presence of four marker chromosomes strictly associated in all the metaphases analyzed) in all clones and tumor-derived cell lines prompted us to alter the specific pattern of chromosomal aberrations in order to identify which if any of the aberrations were more strictly related to transformation. For this purpose we treated a tumor-derived cell line with Colcemid and analyzed the reversion of anchorage-independent phenotype in the subclones showing an altered association of the four marker chromosomes. We conclude that two of four marker chromosomes contribute to anchorage independence.

Role of cytochrome P-450 isoenzymes in the bioactivation of hydroxy anthraquinones.

The reductive and the P-450-dependent oxidative bioactivation of various anthraquinones (AQs), 1-hydroxy AQ, 1,2-dihydroxy AQ, 1,4-dihydroxy AQ, 1,8-dihydroxy AQ, 1,2,4-trihydroxy AQ, 1,4-dihydroxy 6-carboxy AQ and 1,8-dihydroxy 3-carboxy AQ, were investigated using purified enzymes, subcellular fractions and four Chinese hamster V79 cell lines lacking and expressing cytochrome P-450 oxidative enzymes. The reduction of AQs performed by NADH-dehydrogenase, NADPH-cytochrome P-450 reductase, homogenates and microsomes of V79 cells, indicated that only the carboxy-containing drugs were fairly good superoxide anion stimulators. The P-450 dependent oxidation of AQs, assayed as NADPH consumption with microsomes and reconstituted enzymic systems, demonstrated that the P-450 1A1 and 1A2 were, as expected, the most active catalysts. However, they appeared to catalyze the formation of polyphenols rather than arene oxides or phenoxy radicals. Further support to the lack of generation of reactive intermediates during the oxidative metabolism of AQs came from the genotoxicity studies. In the three V79 cell lines expressing rat cytochrome P-450 1A1, 1A2 and 2B1, AQs did not significantly enhance the sister chromatid exchange induction above that elicited in the parental V79 line. Thus the present results, collectively taken, suggest that the P-450-mediated oxidation pathway plays a minor role in the bioactivation of AQs.

Superoxide anion production and toxicity in V79 cells of six hydroxy-anthraquinones.

This study investigates the cytotoxic and genotoxic effects of various carboxy AQ, 1,4-dihydroxy 6-carboxy AQ, 1,8-dihydroxy 3-carboxy AQ, 1,4-dihydroxy AQ, 1,5-dihydroxy AQ, 1,8-dihydroxy AQ and 2,6-dihydroxy AQ in V79 Chinese hamster cells. The V79 cells were used since, as they contain flavoproteins but not cytochrome P-450, they can bioactive xenobiotics only through the reductive pathway excluding the oxidative one. In addition, the abilities of AQs to stimulate O2-production using both purified flavoproteins (NADH-dehydrogenase, NADPH-cytochrome P-450 reductase) and V79 subcellular fractions (homogenate and microsomes) were assayed. The NADH and NADPH consumption stimulated by AQs in V79 microsomes was also determined. The results showed that the carboxylic-containing drugs and the 1,4-dihydroxy AQ were weak sister chromatid exchange inducers and the most toxic among the six anthraquinones examined. Dicumarol, a potent inhibitor of DT-diaphorase, reduced, rather than potentiated, both the cytotoxicity and genotoxicity caused by these AQs. Thus, the higher superoxide formation rates stimulated by the carboxylic-containing AQs compared to those of the other quinones with all the in vitro systems used, suggested, except for the 1,4-dihydroxy AQ, a possible relationship between cytotoxicity and O2-production. For the 1,4-dihydroxy AQ toxicity, a specific bioactivation route was hypothesized.

Comparison between plasma concentrations of testosterone, nitric oxide and endothelin 1-2 in penile and brachial venous blood: preliminary results in men with psychogenic impotence.

In the present study venous plasma concentrations of testosterone (T), nitric oxide (NO) and endothelin 1-2 (ET1-2) in the flaccid penis and brachial blood were measured in men with psychogenic impotence. T and NO were significantly lower in the penile venous blood, while ET1-2 showed no statistical difference. These data support the hypothesis of testosterone dependence of penile nitric oxide synthesis (NOS).

Intrachromosomal telomeric repeats and stabilization of truncated chromosomes in V79 Chinese hamster cells.

(TTAGGG)n sequences have been localized on the chromosomes of the Chinese hamster V79 cell line. A correlation between telomeric-like repeats and chromosome breakage has been found. Moreover, the analysis of the truncated chromosomes, typical of this cell line, has suggested that intrachromosomal (TTAGGG)n DNA may be important in the stabilization of the new telomeres.

Clastogenicity of anthraquinones in V79 and in three derived cell lines expressing P450 enzymes.

The clastogenicity of seven anthraquinones was investigated in a V79 Chinese hamster cell line expressing only the reductive pathway enzymes and in three derived cell lines transfected and expressing three rat cytochrome P450 (1A1, 1A2, 2B1). The results have shown that chromosomal aberrations are modulated in similar manner both in parental and transfected V79 cell lines, suggesting that the clastogenicity of these compounds is not mediated by cytochrome P450 dependent monooxygenases.

Fragile sites at the centromere of Chinese hamster chromosomes: a possible mechanism of chromosome loss.

On the basis of our previous observations showing that fragile sites (FS) mapped essentially in the centromeric regions of Chinese hamster chromosomes, we consider the possibility that the presence of FS at the centromere might be a source of chromosome loss. In this model a centromeric FS causes a centromeric break giving rise to two chromosome arms which could be lost or maintained with different consequences on the ploidy of daughter cells. To test this hypothesis, Chinese hamster cells have been treated both with N-methyl-N-nitrosourea (MNU), a mutagenic agent which also induces aneuploidy, and vinblastin (VBL), a pure aneugen, used as a control compound, which is supposed not to interact with DNA. The results show that MNU induces the formation of translocated and/or truncated chromosomes, on the contrary VBL is not able to induce chromosome rearrangements. The sites most involved in MNU-induced breaks are the centromeric regions of chromosomes where FS are also present. These breaks cause essentially the loss of one chromosome arm, so that the resulting cells are numerically diploid but presenting partial monosomies. The implications of these results are discussed.

Analysis by BrUdR-labelling technique of induced aneuploidy in mammalian cells in culture.

To study the origin of induced aneuploid cells, the BrUdR-labelling technique was applied to V79/AP4 Chinese hamster cells treated with colcemid or benomyl. In this way we were able to recognize the cells which had undergone one cellular division after the treatment since their chromosomes exhibited sister-chromatid differentiation. The results showed that the induced aneuploid cells can have either a few or numerous additional chromosomes depending on the concentrations of the drug. Moreover, it could be established that aneuploid cells with numerous additional chromosomes were obtained mainly when polyploid cells were also present in the treated population. This strongly suggests that the excess of additional chromosomes found in the aneuploid cells induced by the highest concentrations may be derived by disturbances of the whole mitotic apparatus rather than by a multiplicity of errors affecting individual chromosomes.

Analysis of electroporation-induced genetic damages in V79/AP4 Chinese hamster cells.

Electroporation is a recent technique used to introduce exogenous DNA into eukaryotic cells. It is important to establish that the gene of interest is transferred into a functional, non-mutated recipient cell. V79/AP4 Chinese hamster cells were exposed to high-voltage pulsed electric fields and some biological and genetic effects were measured. The results showed that cytotoxicity was related in a dose-dependent manner to the number of applied pulses. Thioguanine-resistant colony-forming cells as well as chromosomal aberrations were also induced whereas ouabain resistants and sister-chromatid exchanges were not or slightly induced. Spontaneous and electroporation-induced clones that were phenotypically TGR/HATS were used to investigate the hprt locus. Molecular screening of the locus showed that the number of deleted exons was significantly higher in induced than in spontaneous TG-resistant clones, suggesting that the genetic damages induced by electroporation concern the loss of regions well over the size of the hprt locus.

A genetic analysis of the adenine phosphoribosyl transferase locus in Chinese hamster V79-AP4 cells: relevance to mutagenesis studies.

The chromosomal location of the autosomal locus aprt has been investigated in the permanent Chinese hamster cell line V79-AP4 by standard somatic cell genetics methodologies. Aprt is functionally dizygous in V79-AP4 and the 2 alleles map on 2 chromosome 3 homologs, in agreement with the chromosome assignment of the gene in Chinese hamster primary cells. Chromosome G-banding and a Southern blot analysis of V79-AP4 DNA, using as a probe the cloned Chinese hamster aprt gene, have not revealed any structural alteration at either of the 2 aprt alleles. One of the chromosomes 3 has, however, a terminal deletion in its long arm and is therefore morphologically marked. These findings could make V79-AP4 an interesting cell system for the study of mutational mechanisms at the aprt locus in Chinese hamster.