Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Five haplotypes account for fifty-five percent of ATM mutations in Brazilian patients with ataxia telangiectasia: seven new mutations

We have studied the molecular genetics of 27 Brazilian families with ataxia telangiectasia (AT). Five founder effect haplotypes accounted for 55.5% of the families. AT is an autosomal recessive disorder of childhood onset characterized by progressive cerebellar ataxia, ocular apraxia, telangiectasia, immunodeficiency, radiation sensitivity, chromosomal instability, and predisposition to cancer. The ATM gene spans more than 150 kb on chromosome region 11q23.1 and encodes a product of 3056 amino acids. The ATM protein is a member of the phosphatidylinositol 3-kinase (PI-3K) family of proteins and is involved in cell cycle control and DNA repair pathways. DNA was isolated from lymphoblastoid cell lines and haplotyped using four STR markers (D11S1818, NS22, D11S2179, D11S1819) within and flanking the ATM gene; all allele sizes were standardized in advance. In addition to the STR haplotypes, SNP haplotypes were determined using 10 critical polymorphisms. The entire gene was screened sequentially by protein truncation testing (PTT), single strand conformation polymorphism (SSCP), and then denaturing high performance liquid chromatography (dHPLC) to identify the disease-causing mutations. Of the expected 54 mutations, 50 were identified. All mutations but one, led to a truncated or null form of the ATM protein (nonsense, splice site, or frameshift). Five families (18.5%) carried a deletion of 3450nt (from IVS28 to Ex31), making this one of the two most common Brazilian mutations. Mutations were located throughout the entire gene, with no clustering or hotspots. Standardized STR haplotype analysis greatly enhanced the efficiency of mutation screening.     

Association Between Maternal Work and Exclusive Breastfeeding in Countries of Latin America and Caribbean

Maternal and Child Health Journal - The aim of this study was to estimate the association between individual and contextual characteristics related to maternal work and exclusive breastfeeding...     

Cytogenetics of benign breast lesions

This review summarizes the cytogenetic information on benign breast lesions of various histologies, i.e., fibrocystic lesions from women with and without a known hereditary predisposition to breast cancer, fibroadenomas, phyllodes tumors, and papillomas, and relate the chromosomal features with those in breast carcinoma. In general, the frequency of chromosome abnormalities is lower in benign lesions than in breast cancer, and seems to correlate with the histologic features of the tissue, and the corresponding risk of developing invasive mammary carcinoma; aberrations are more common in proliferative than in nonproliferative lesions. The karyotypes are generally less complex than those detected in invasive carcinoma, and more often involve balanced rearrangements. No lesion-specific aberration has so far been detected; on the contrary, changes repeatedly encountered in breast cancer samples can be found in benign lesions as well, e.g., gain of 1q, interstitial deletion of 3p, and trisomies 7, 18, and 20. Especially intriguing is the prevalence of rearrangements of the short arm of chromosome 3, with the minimally deleted bands 3p13–14, in proliferative lesions from prophylactic mastectomies in breast cancer families. The potential tumor suppressor gene(s) in this region remains, however, to be identified.     

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A(2).

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A(2), and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H(2)O(2)) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 microg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcgamma, complement, mannose and beta-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and beta-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H(2)O(2) by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H(2)O(2) only at a concentration of 3.2 microg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5-2.5 microg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages up regulating phagocytosis, mainly via mannose and beta-glucan receptors, and the respiratory burst.     

Effect of chronic exposure to aluminium on isoform expression and activity of rat (Na+/K+)ATPase.

The ability of aluminum to inhibit the (Na(+)/K(+))ATPase activity has been observed by several investigators. The (Na(+)/K(+))ATPase is characterized by a complex molecular heterogeneity that results from the expression and differential association of multiple isoforms of both catalytic (alpha) and regulatory (beta) subunits. For instance, three main alpha (alpha(1), alpha(2) and alpha(3)) and three beta (beta(1), beta(2) and beta(3)) subunit isoforms exist in vertebrate nervous tissue, whereas only alpha(1) and beta(1) have been identified in kidney. However, no studies have focused on determining the change in (Na(+)/K(+))ATPase isoforms caused by chronic exposure to aluminum and its relation with aluminum toxicity. In this study, adult male Wistar rats were submitted to chronic dietary AlCl(3) exposure (0.03 g/day of AlCl(3) for 4 months), and the activity and protein expression of (Na(+)/K(+))ATPase isozymes were studied in brain cortex synaptosomes and in kidney homogenates. The intracellular levels of adenine nucleotides, plasma membrane integrity, and aluminum accumulation were also studied in brain synaptosomes. Aluminum accumulation upon chronic dietary AlCl(3) administration significantly decreased the (Na(+)/K(+))ATPase activity measured in the presence of nonlimiting Mg-ATP concentrations, without compromising protein expression of alpha-subunit isoforms in brain and kidney. Aluminum-induced synaptosomal (Na(+)/K(+))ATPase inhibition was due to a reduction in the activity of isozymes containing alpha(1)-alpha(2) and alpha(3)-subunits. The onset of enzyme inhibition was accompanied by a decrease of the (Na(+)/K(+))ATPase sensitivity to submicromolar concentrations of ouabain, and it preceded major damage in plasma membrane integrity and energy supply, as revealed by the analysis of lactate dehydrogenase leakage and endogenous adenine nucleotides. The data suggest that, during chronic dietary exposure to AlCl(3), brain (Na(+)/K(+))ATPase activity drops, even if no significant alterations of catalytic subunit protein expression, cellular energy depletion, and changes in cell membrane integrity are observed. Implications regarding underlying mechanisms of aluminum neurotoxicity are discussed.     

Inflammation induced by Bothrops asper venom: release of proinflammatory cytokines and eicosanoids, and role of adhesion molecules in leukocyte infiltration.

Bothrops asper venom (BaV) causes systemic and local effects characterized by an acute inflammatory reaction with accumulation of leukocytes and release of endogenous mediators. In this study, the effects of BaV on the release of the cytokines IL-1, IL-6 and TNF-alpha and the eicosanoids LTB4 and TXA2 in the peritoneal cavity of mice were analyzed. We also investigated the participation of beta2 integrin chain, l-selectin, LFA-1, ICAM-1 and PECAM-1 adhesion molecules in the BaV-induced leukocyte accumulation. Levels of proinflammatory cytokines IL-6 and TNF-alpha, as well as eicosanoids LTB4 and TXA2 were significantly increased after BaV injection (250 microg/kg), whereas no increment in IL-1 was observed. Anti-mouse l-selectin, LFA-1, ICAM-1, PECAM-1 and beta2 integrin chain monoclonal antibodies resulted in a reduction of neutrophil accumulation induced by BaV injection compared with isotype-matched control injected animals. These data suggest that BaV is able to induce the activation of leukocytes and endothelium to express adhesion molecules involved in the recruitment of neutrophils into the inflammed site. Furthermore, these results showed that BaV induces the release of cytokines and eicosanoids in the local of the venom injection; these inflammatory mediators may be important for the initiation and amplification of the inflammatory reaction characteristic from Bothrops sp envenomation.     

Risk management strategies in the postmarketing period : safety experience with the US and European bosentan surveillance programmes.

In view of the shortcomings of the current system for postmarketing drug surveillance that is based on voluntary spontaneous adverse drug reaction (ADR) reporting, new approaches are needed.We describe an approach involving a combination of limited distribution, patient and physician education, as well as a novel pharmaco-vigilance system that is capable of promoting the safe and adequate use of a new drug. Importantly, it provides the possibility of calculating true ADR occurrence rates, as the exposed population (denominator) and the number of patients with events (numerator) are known. These measures were taken for the oral dual endothelin ET(A)/ET(B) antagonist bosentan (Tracleer). In recent guidelines issued by the European Society of Cardiology, American College of Chest Physicians and the WHO, this drug is considered as first-line oral treatment for the treatment of pulmonary arterial hypertension, a devastating orphan disease associated with a poor prognosis. Bosentan was approved in 2001/2 on the basis of two pivotal studies that showed improved exercise capacity and haemodynamic parameters while delaying time to clinical worsening. Elevations in serum liver aminotransferase levels of >3 times the upper limit of normal were noted in 10.2% of patients (placebo-subtracted incidence). Therefore, liver function tests have to be performed on a regular basis. In addition, bosentan has potential as a teratogen.In the US, a controlled distribution network for bosentan (Tracleer) Access Program [T.A.P.]) and the development of a patient database to follow patients was set up. Accompanied by comprehensive physician and patient education programmes, T.A.P. was developed to provide a mechanism to assist with the primary risk management goals for bosentan therapy, namely pregnancy prevention and liver enzyme monitoring and prevention of hepatic injury.In Europe, the Tracleer) Excellence (TRAX PMS) database is a novel European non-interventional, prospective, internet-based surveillance system initiated by the manufacturer in cooperation with the European Medicines Agency. It collected potential safety signals associated with bosentan use including adverse events, elevations of liver aminotransferase levels, other abnormal laboratory values, death and hospitalisation. TRAX PMS has accrued 79% of all known patients in the EU and the data provide supportive 'real-life' evidence on the long-term safety of bosentan.The two different systems had similar goals and outcomes. The data received concerning thousands of patient-years of use have confirmed the clinical trial results regarding product safety and the favourable benefit/risk ratio of bosentan, especially with regard to known type A adverse events. The clinical monitoring algorithm has also been confirmed. In addition, no rare type B events were uncovered despite the increased reporting rate. These systems might serve as templates for future pharmaco-vigilance efforts regarding drugs that require particular safety attention.     

A case of esophageal complication in epidermolysis bulhosa simplex

It is well known the involvement of the gastrointestinal tract in Epidermolysis Bulhosa (EB) and its consequences on both nutrition and growth. We present a case of a young girl with EB diagnosed soon after birth, admitted to our emergency room with food impactation. The endoscopy revealed a double barrel esophagus. No medical or surgical therapy was started and after 2 months of enteric feeding, spontaneous resolution was observed at endoscopy. Skin biopsy was compatible with EB Simplex. According to the literature EB Simplex is the subtype the least associated with GI tract involvement, presenting with dysphagia in only 2% of the patients, and with the lowest cumulative risk of esophageal stricturing.     

Evaluation of Border Entry Screening for Infectious Diseases in Humans

In response to the severe acute respiratory syndrome (SARS) pandemic of 2003 and the influenza pandemic of 2009, many countries instituted border measures as a means of stopping or slowing the spread of disease. The measures, usually consisting of a combination of border entry/exit screening, quarantine, isolation, and communications, were resource intensive, and modeling and observational studies indicate that border screening is not effective at detecting infectious persons. Moreover, border screening has high opportunity costs, financially and in terms of the use of scarce public health staff resources during a time of high need. We discuss the border-screening experiences with SARS and influenza and propose an approach to decision-making for future pandemics. We conclude that outbreak-associated communications for travelers at border entry points, together with effective communication with clinicians and more effective disease control measures in the community, may be a more effective approach to the international control of communicable diseases.