Abnormal sex-duct development in female moles: the role of anti-Müllerian hormone and testosterone

We have performed a morphological, hormonal and molecular study of the development of the sex ducts in the mole Talpa occidentalis. Females develop bilateral ovotestes with a functional ovarian portion and disgenic testicular tissue. The Müllerian ducts develop normally in females and their regression is very fast in males, suggesting a powerful action of the anti-Müllerian hormone in the mole. RT-PCR demonstrated that the gene governing this hormone begins to be expressed in males coinciding with testis differentiation, and expression continues until shortly after birth. Immunohistochemical studies showed that expression occurs in the Sertoli cells of testes. No expression was detected in females. Wolffian duct development was normal in males and degenerate in prenatal females, but developmental recovery after birth gave rise to the formation of rudimentary epididymides. This event coincides in time with increasing serum testosterone levels and Leydig cell differentiation in the female gonad, thus suggesting that testosterone produced by the ovotestes is responsible for masculinisation of female moles. During postnatal development, serum testosterone concentrations decreased in males but increased in females, thus approaching the levels that adult males and females have during the non-breeding season.     

Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti-excitotoxic potency of guanine nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.     

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy

Alzheimer''s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.     

Oxidized LDL and its correlation with lipid profile and oxidative stress biomarkers in young healthy Spanish subjects

The biological effects of oxidized LDL (oxLDL) may contribute to initiation and progression of the atherosclerotic process, and the association between cardiovascular disease and oxidation of LDL has been largely demonstrated. The objectives of this study were to establish the reference values of oxidative stress biomarkers in a young healthy Spanish population to determine the concentration of oxLDL and its relationship with lipid profile and with these biomarkers. oxLDL, F₂-isoprostanes, protein carbonyls (PC), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were determinate by ELISA in 72 healthy subjects. Antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR) were carried out on a Hitachi 912 analyzer; lipid profile were assayed using automated systems (Cobas 711, Roche Diagnostics). All statistics were analyzed by using SPSS for Windows 15.0. SPSS Inc, Chicago, IL, USA. (Normal mean reference values): oxLDL (63.23 ± 16.23 U/L), (Male/Female 68.06 ± 17.69/58.39 ± 13.6 U/L), F₂-isoprostanes (2.26 ± 0.9 μg/g creatinine), PC (0.34 ± 0.15 nmol/mg), 8-OHdG (23.27 ± 10.58 ng/ml), SOD (931.97 ± 271.09 U/g Hb), GR (46.56 ± 11.68 U/L), GPx (27.58 ± 6.89 U/gHb (Male/Female 25.91 ± 5.03/29.2 ± 8.07 U/L)). OxLDL (63.23 U/L) was significantly (p < 0.05) positively correlated with BMI (22.53 Kg/m²), total cholesterol (175.79 mg/dl), triglycerides (87.58 mg/dl), LDL cholesterol (96.25 mg/dl), and uric acid (4.78 mg/dl), while negatively correlated with HDL-cholesterol (62.25 mg/dl). We have found different correlation between oxLDL and isoprostanes by gender with the rest of parameters. Normal reference values have been found significantly different for oxLDL and GPx by gender. Oxidized LDL is correlated with lipid profile but not with the oxidative stress biomarkers. Urinary isoprostanes are positively correlated with triglycerides and negatively with GR and GPx.     

Comparative study of enzymes in testes and ovaries from adult Dipetalogaster maximus (Uhler) and triatoma infestans (Klug) (Hemiptera: Reduviidae). correlation with fine structural organization.

Activities of hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), fructose-6-phosphate kinase (F6PK), glutamate dehydrogenase (GlutDH), aspartate aminotransferase (AAT), malate dehydrogenase (MDH) and glycerol-3-phosphate dehydrogenase (GPDH) were determined in tissue extracts of testes and ovaries of adult Dipetalogaster maximus (Uhler) and Triatoma infestans (Klug) (Hemiptera: Reduviidae), insect vectors of Chagas disease. The fine structure organization of the same organs were studied by electron microscopy. Results allow the following inferences: in testes from both species, most of the glucose would be utilized through the glycolytic pathway. Amino acid catabolism for energy purposes appears to be unimportant. The number of mitochondria and the development of the rough endoplasmic reticulum in cells of the spermatogenic line indicate the occurrence of active oxidative metabolism and protein synthesis; in ovaries, levels of G6PDH indicate the existence of an active pentose pathway which would supply the NADPH required for fat and ecdysteroid synthesis. Amino acid catabolism appears to be relatively more important in ovary than in testis. Fat and glycogen are stored in follicular cells of D. maximus; oocytes of both species contain numerous fat droplets. Abundant mitocondria are present in follicular cells and oocytes. A well developed rough endoplasmic reticulum and free ribosomes are also conspicuous in these cells. The malate/aspartate H-transfer system seemed to be relatively more important than the glycerophosphate shuttle in ovaries as well in testes.     

Ribose 5-phosphate isomerase type B from Trypanosoma cruzi: kinetic properties and site-directed mutagenesis reveal information about the reaction mechanism

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phosphate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P). 4-phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competitively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.     

Direct molecular profiling of minicircle signatures and lineages of Trypanosoma cruzi bloodstream populations causing congenital Chagas disease

Congenital transmission of Trypanosoma cruzi may occur in some or all the gestations from a T. cruzi-infected mother. Variable rates of congenital transmission have been reported in different geographical areas where different parasitic strains predominate, suggesting that parasitic genotypes might play a role in the risk of congenital transmission. Moreover, in cases of transmission it is unknown if the whole maternal T. cruzi population or certain clones are preferentially transmitted by the transplacental route. In this study, bloodstream T. cruzi lineages were identified in blood samples from congenitally infected children, transmitting and non-transmitting mothers and unrelated Chagas disease patients, using improved PCR strategies targeted to nuclear genomic markers. T. cruzi IId was the prevalent genotype among 36/38 PCR-positive congenitally infected infants, 5/5 mothers who transmitted congenital Chagas disease, 12/13 mothers who delivered non-infected children and 28/34 unrelated Chagas disease patients, all coming from endemic localities of Argentina and Bolivia. These figures indicate no association between a particular genotype and vertical transmission. Furthermore, minicircle signatures from the maternal and infants' bloodstream trypanosomes were profiled by restriction fragment length polymorphism of the 330-bp PCR-amplified variable regions in seven cases of mothers and congenitally infected infants. Minicircle signatures were nearly identical between each mother and her infant/s and unique to each mother-infant/s case, a feature that was also observed in twin deliveries. Moreover, allelic size polymorphism analysis of microsatellite loci from populations transmitted to twins showed that all clones from the maternal polyclonal population were equally infective to both siblings.     

Canonical interaction of cyclin G associated kinase with adaptor protein 1 regulates lysosomal enzyme sorting

The adaptor protein 1 (AP1) complex is a heterotetramer that participates in cargo sorting into clathrin-coated vesicles at the trans-Golgi network (TGN) and endosomes. The gamma subunit of AP1 possesses a C-terminal "ear" domain that recruits a cohort of accessory proteins through recognition of a shared canonical motif, PsiG[PDE][PsiLM] (where Psi is an aromatic residue). The physiological relevance of these ear-motif interactions, however, remains to be demonstrated. Here we report that the cyclin G-associated kinase (GAK) has two sequences fitting this motif, FGPL and FGEF, which mediate binding to the AP1-gamma-ear domain in vitro. Mutation of both gamma-ear-binding sequences or depletion of AP1-gamma by RNA interference (RNAi) decreases the association of GAK with the TGN in vivo. Depletion of GAK by RNAi impairs the sorting of the acid hydrolase, cathepsin D, to lysosomes. Importantly, expression of RNAi-resistant GAK restores the lysosomal sorting of cathepsin D in cells depleted of endogenous GAK, whereas expression of a similar construct bearing mutations in both gamma-ear-binding sequences fails to correct the sorting defect. Thus, interactions between the PsiG[PDE][PsiLM]-motif sequences in GAK and the AP1-gamma-ear domain are critical for the recruitment of GAK to the TGN and the function of GAK in lysosomal enzyme sorting.     

Neural-network simulations of two context-dependence phenomena

This paper describes simulations of two context-dependence phenomena in Pavlovian conditioning, using a neural-network model that draws on knowledge from neuroscience and makes no distinction between operant and respondent learning mechanisms. One phenomenon is context specificity or the context-shift effect, the decrease of conditioned responding (CR) when the conditioned stimulus (CS) is tested in a context different from the one in which it had been paired with the unconditioned stimulus (US). The other effect is renewal, the recovery of CR in the training context after extinction in another context. For specificity (simulation 1), two neural networks were first given 200 CS-US pairings in a context. Then, the CS was tested either in the training context or a new context. Output activations in the new context were substantially lower. For renewal (simulation 2), two networks were first given 200 CS-US pairings in a context, then 100 extinction trials in either the same context or a new one, and then tested back in the training context. Output activations during the test phase were substantially higher after extinction in a new context. The results are interpreted in terms of the dynamics of activations and weights.