Silencing the expression of mitochondrial acyl-CoA thioesterase I and acyl-CoA synthetase 4 inhibits hormone-induced steroidogenesis

Arachidonic acid and its lypoxygenated metabolites play a fundamental role in the hormonal regulation of steroidogenesis. Reduction in the expression of the mitochondrial acyl-CoA thioesterase (MTE-I) by antisense or small interfering RNA (siRNA) and of the arachidonic acid-preferring acyl-CoA synthetase (ACS4) by siRNA produced a marked reduction in steroid output of cAMP-stimulated Leydig cells. This effect was blunted by a permeable analog of cholesterol that bypasses the rate-limiting step in steroidogenesis, the transport of cholesterol from the outer to the inner mitochondrial membrane. The inhibition of steroidogenesis was overcome by addition of exogenous arachidonic acid, indicating that the enzymes are part of the mechanism responsible for arachidonic acid release involved in steroidogenesis. Knocking down the expression of MTE-I leads to a significant reduction in the expression of steroidogenic acute regulatory protein. This protein is induced by arachidonic acid and controls the rate-limiting step. Overexpression of MTE-I resulted in an increase in cAMP-induced steroidogenesis. In summary, our results demonstrate a critical role for ACS4 and MTE-I in the hormonal regulation of steroidogenesis as a new pathway of arachidonic acid release different from the classical phospholipase A2 cascade.     

Neonatal TSH levels as an index of iodine sufficiency: differences related to time of screening sampling and methodology

INTRODUCTION: Current WHO guidelines consider that under adequate iodine intake <3% of newborns should have neonatal TSH levels of >5 mU/l blood when screening is performed in cord blood or at 3 days to 3 weeks of age. OBJECTIVE: To estimate whether this absolute criterion when applied to newborns older than 48 h of age and native to Buenos Aires coincides with the traditional ones (goiter and urinary iodine in school-age children (SAC)), and if the evaluation varies with either the methodology used for TSH measurements and/or the time of specimen sampling. POPULATION AND METHODS: TSH was measured by an immunofluorometric assay (IFMA) on filter paper blood spots of 186 cord blood samples, 112 babies <48 h of age and 1,500 newborns >48 h of age, and by immunoradiometric assay (IRMA) in 238 newborns. The WHO ICCIDD absolute criteria were applied to each population. Thyroid volume was assessed by direct palpation in 500 SAC, and in 100 of them urinary iodine levels were measured. RESULTS: TSH levels were >5 mU/l blood in 11.3% of the cord blood samples and in 3.6% of the samples from babies <48 h of age, suggesting mild iodine deficiency. TSH was >5 mU/l in 2.7% of newborns >48 h of age tested by IFMA (iodine sufficient) and in 30% measured by IRMA (moderate iodine insufficiency). Median urinary iodine and goiter prevalence in SAC were 143 mug/l and 4.5%, respectively, as expected in an iodine-sufficient area. conclusion: The TSH levels in Buenos Aires conform with the WHO criterion that defines iodine sufficiency. Application of this criterion, however, to cord blood samples or samples from babies <48 h old and the use of different methodologies may lead to erroneous conclusions.     

Sugar cane buds as an efficient explant for plantlet regeneration

An efficient and reproducible protocol for regeneration of plantlets at a high frequency was developed by using sugar cane buds. Disinfected buds were firstly submerged in ethanol sodium hypochlorite solution with 0.1 % polyvinylpyrrolidone, 1.5 % ascorbic acid and 1.75 % citric acid as antioxidants and subsequently treated with solution of agrimicin:captan (1:1). The upper stalk segment was better to obtain bud in vitro culture compared to lower segments. The medium for induction of multiple shoots consisted of Murashige and Skoog basal medium (MS) supplemented with 2 mg dm⁻³ thidiazuron and 1 mg dm⁻³ naphthalene acetic acid. An average of 24 shoots per bud was obtained for cv. Mex 68-P23 within four weeks and 29 shoots for cv. MY 55-14 within six weeks. Indole-3-butyric acid induced more roots in both cultivars compared to the untreated plantlets. Plantlets transferred to soil showed normal growth with up to four axilliary buds in each node. It was concluded that the germplasm obtained through the above mentioned technique generated stalks with more buds in each node which would give farmers more vegetative material for plantations in field with 100 % germination.This research was funded by Fundacion Produce Chiapas A.C. (Mexico).     

Heat-shock proteins associated with base excision repair enzymes in HeLa cells

Two enzymes of base excision repair (BER), uracil DNA glycosylase (UDG) and DNA polymerase beta (beta pol), from HeLa cells co-eluted from Superose 12 FPLC columns. The UDG was completely displaced from 150-180-kDa fractions to 30- 70-kDa fractions by brief treatment with 0.5 N NaCl, pH 3.0, as expected when protein-protein associations are disrupted, but beta pol was not displaced by this treatment. UDG was not essential to the presence of beta pol in the 150-180-kDa enzyme complex. beta pol and UDG apparently reside in separate but co-eluting structures. Immunoaffinity chromatography showed that the association of UDG and beta pol was accounted for by attachment in common to DNA and that the association was abolished by eliminating DNA. Evidence for base excision repairosomes containing UDG and beta pol in protein-protein assemblies was not found. However, UDG and human AP endonuclease (HAP1) were associated with HSP70 and HSP27, which are present in 150-180-kDa and 30-70-kDa proteins of cell sonicates. The association of HSPs with BER enzymes was confirmed by hydroxyl radical protein-protein footprinting and immunoaffinity tests. The association of HSPs and BER enzymes is a novel finding. HSP binding may account for the presence of BER enzymes in the two large size class fractions and HSPs may have functional roles in BER.     

MS-222 (tricaine methane sulfonate) does not kill the amphibian chytrid fungus Batrachochytrium dendrobatidis

MS-222 (tricaine methane sulfonate) is an agent commonly used to anaesthetise or euthanize amphibians used in experiments. It is administered by immersing the animal to allow absorption through the skin. Chytridiomycosis is an important disease of amphibians and research involves experiments with live animals. Batrachochytrium dendrobatidis, the fungus which causes chytridiomycosis, is located in the skin and therefore the organism should come into contact with MS-222 when it is used. B. dendrobatidis is a sensitive organism which could possibly be killed by MS-222. Hence, results of chytridiomycosis studies in which MS-222 is used could be unreliable. A concentration of 2 g l(-1) and an exposure duration of 1 h is at the high end of the range at which MS-222 would be most commonly used. Exposure to 2 g l(-1) MS-222 for 1 h does not kill B. dendrobatidis cultures, suggesting that MS-222 is safe to use in chytridiomycosis studies.     

Mass rearing history negatively affects mating success of male Anastrepha ludens (Diptera: Tephritidae) reared for sterile insect technique programs

Mating competitiveness and sterility induction into cohorts of wild Anastrepha ludens (Loew) (Diptera: Tephritidae) was compared among wild and laboratory flies reared for use in the sterile insect technique Mexican program. Laboratory flies stemming from an 11-yr-old bisexual strain were either not irradiated, irradiated at 3 krad (low dose), or irradiated at 8 krad. In 30 by 30 by 30-cm Plexiglas cages, where a cohort of laboratory flies (male and female) irradiated at different doses (0, 3, and 8 krad) was introduced with a cohort of wild flies, males and females of each type mated randomly among themselves. Compared with nonirradiated laboratory and wild males, irradiated males, irrespective of dose (3 or 8 krad), induced shorter refractory periods and greater mating frequency in wild females. Nevertheless, laboratory flies irradiated at a low dose induced greater sterility into cohorts of wild flies than laboratory flies irradiated at a high dose. In a 3 by 3 by 3-m walk-in cage, wild males gained significantly more matings with wild females than nonirradiated and irradiated laboratory males a finding that revealed a strong effect of strain on mating performance. Mating incompatibility of the laboratory strain might have obscured the effect of reduced irradiation doses on male mating performance in the walk-in cage. Our results highlight an urgent need to replace the A. ludens strain currently used by the Mexican fruit fly eradication campaign and at least suggest that reducing irradiation doses result in an increase in sterility induction in wild populations.     

Nonhealing infection despite Th1 polarization produced by a strain of Leishmania major in C57BL/6 mice

Experimental Leishmania major infection in mice has been of immense interest because it was among the first models to demonstrate the importance of the Th1/Th2 balance to infection outcome in vivo. However, the Th2 polarization that promotes the development of nonhealing cutaneous lesions in BALB/c mice has failed to adequately explain the mechanisms underlying nonhealing forms of leishmaniasis in humans. We have studied a L. major strain from a patient with nonhealing lesions that also produces nonhealing lesions with ulcerations and high parasite burden in conventionally resistant C57BL/6 mice. Surprisingly, these mice develop a strong, polarized, and sustained Th1 response, as evidenced by high levels of IFN-gamma produced by Leishmania-specific cells in the draining lymph node and in the ear lesion, and an absence of IL-4 or IL-13. The parasites fail to be effectively cleared despite high level induction of inducible NO synthase in the lesion, and despite their sensitivity to killing by IFN-gamma-activated macrophages in vitro. Infection of IL-10(-/-) mice, blockade of the IL-10R, or depletion of CD25(+) cells during the chronic phase promotes parasite killing, indicating that IL-10 and regulatory T cells play a role in rendering the Th1 responses ineffective at controlling infection in the skin. Mice with nonhealing primary lesions are nonetheless resistant to reinfection in the other ear. We suggest that nonhealing infections in animal models that are explained not by aberrant Th2 development, but by overactivation of homeostatic pathways designed to control inflammation, provide better models to understand nonhealing or reactivation forms of leishmaniasis in humans.     

Monocyte differentiation and HIV replication after prolonged culture of peripheral blood mononuclear cells from HIV-infected individuals

Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.     

Functional diversity across space and time: trends in wader communities on British estuaries

Aim British estuarine ecosystems support large populations of protected migratory waders. Understanding how wader communities vary spatially and how they may be changing temporally can greatly improve the understanding of these dynamic ecosystems. Here, we explore the variation in functional diversity (using a range of morphological and ecological traits) in order to identify the processes shaping wader communities on British estuaries and how these processes may be changing. Location England, Wales and Scotland. Methods We use national survey data (Wetland Bird Survey) from 1980/1981 to 2006/2007 winter to calculate functional diversity (FD) – an index that measures trait dispersion – in wader communities on 100 estuaries. We test for evidence of non‐random patterns of diversity and explore the relative importance of two key processes, environmental filtering and competition, in shaping these communities. Results The observed FD was significantly and positively associated with species richness and to a lesser extent estuary area, followed by longitude. An increase in observed FD was observed since 1980, supported by a small but significant slope. In the majority of cases, changes in FD were mirrored by changes in species richness. Observed FD was on average lower than expected by chance, as indicated by a negative value of observed minus expected FD. However, this difference became less negative over time, with observed minus expected FD values increasing slightly, but significantly, over the study period. Main conclusions Wader FD varies across British estuaries, and the relative influence of the processes by which communities are structured appears to be changing through time. We discuss the potential drivers underlying these patterns and the importance of identifying such drivers for the protection of wader communities.