Proteome analysis of Leishmania (Viannia) braziliensis by two-dimensional gel electrophoresis and mass spectrometry

Leishmania (Viannia) braziliensis, a protozoan parasite widespread in the New World, is responsible for the infection of different mammal orders, including humans. This species is considered to be a major etiological agent of American cutaneous leishmaniasis. A proteomic study was carried out to identify proteins expressed by L. (V.) braziliensis. One hundred and one spots representing 75 protein entries were identified by MALDI-TOF-TOF. Isoelectric point values estimated by gel electrophoresis matched closely with predicted values, although some discrepancies existed suggesting that post-translational protein modifications may be common in L. braziliensis. Moreover, 20 hypothetical proteins were experimentally identified. Identified proteins were classified into 15 groups according to biological process. Among the proteins identified, approximately 40% have not been previously reported in a proteomic map of Leishmania. In addition, a number of potential virulence factors and drug targets were identified in this protein map, including some proteins associated with the metastatic phenotype. This study describes the first compilation of a proteomic reference map for L. braziliensis (pI 4-7, M(r) 10-130 kDa) and provides a very useful tool for comparative studies of strains isolated from patients presenting different clinical manifestations of leishmaniasis as well as a potential tool to identify markers for clinical diagnosis, therapeutics, and prognosis.     

Perceptions and attitudes toward the menopause among middle aged women from Guayaquil, Ecuador

BACKGROUND: Studies reporting the perspective of Latin American women, Ecuador, included, regarding the menopausal phenomena are scarce or lacking. OBJECTIVES: Obtain information regarding the perception and attitudes toward the menopause among middle aged women of Guayaquil, Ecuador. METHODS: Women aged 40 or more, nursing staff members of two major associated teaching hospitals of the Universidad Católica de Santiago de Guayaquil, Ecuador, were surveyed with a structured questionnaire containing items intended to assess women's perception and attitudes toward the menopause. Secondarily level of information related to the menopause was explored. RESULTS: During the study period, 349 women were surveyed of which mean age was 48+/-6.8 years. A 41.3% were postmenopausal, 55% premenopausal and 3.7% had a history of hysterectomy with conserved ovaries. Women more frequently perceived the menopause as a positive event as they agreed that it is a normal (93.7%) and important event (73.6%), that it gives more confidence and maturity (78.8%), that they may fully enjoy sexual relations (74.8%), that there is a relief as the risk of becoming pregnant is null (65.3%) and that life becomes easier and calmer (60.7%). A relatively high rate demonstrated a preoccupying attitude toward the menopause as 79.4% responded to be concerned about it indicating that seeking medical attention was important, moreover, 77.9% responded that health during this phase be taken care of and life styles changed. Less than 50% of surveyed women considered having enough information regarding the menopause whereas a high rate indicated wanting to receive educational sessions related to the menopause. Married and lower educated women were not concerned about the menopause in a higher rate than their counterparts. CONCLUSIONS: Despite the fact that women perceived the menopause as a positive event, displaying a concerned attitude toward it, their related knowledge was low.     

Bilateral Leydig cell tumour of the ovary: a rare cause of virilization in postmenopausal patient

BACKGROUND: Leydig cell tumours of the ovary are very rare benign neoplasms, frequently associated with symptoms of virilisation, in postmenopausal patients. Only four cases of bilateral Leydig tumours have been reported in the literature. CASE REPORT: We report an additional case of bilateral presentation. A 77-year-old postmenopausal patient was referred from the Endocrinology Service due to a biochemical diagnosis of hyperandrogenism during hospital admission with unbalanced diabetes mellitus. The patient had suffered from alopecia and hirsutism for many years. Biological data confirmed an increment of androgen production. Ultrasonography showed a well-defined hyperechoic lesion of 12 mm in the right ovary. A CT scan demonstrated adrenal glands and ovaries without tumour. Bilateral adnexectomy by laparoscopy was carried out. RESULT: The pathological finding was a bilateral Leydig cell tumour, measuring 15 mm in the right ovary and 3 mm in the left ovary. Following treatment the patient experienced regression of the hirsutism and the plasma testosterone dropped to a normal level. CONCLUSION: In spite of its low incidence, it is a possibility that must be considered in all postmenopausal women with hyperandrogenism, and bilateral adnexectomy by laparoscopy is recommended as a diagnostic test and definitive treatment.     

Membrane insertion and topology of the p7B movement protein of Melon Necrotic Spot Virus (MNSV)

Cell-to-cell movement of the Melon Necrotic Spot Virus (MNSV) is controlled by two small proteins working in trans, an RNA-binding protein (p7A) and an integral membrane protein (p7B) separated by an amber stop codon. p7B contains a single hydrophobic region. Membrane integration of this region was observed when inserted into model proteins in the presence of microsomal membranes. Furthermore, we explored the topology and targeting mechanisms of full-length p7B. Here we present evidence that p7B integrates in vitro into the ER membrane cotranslationally and with an Nt-cytoplasmic/Ct-luminal orientation. The observed topology was monitored in vivo by fusing GFP to the Ct of p7B, enabling the overexpression in Escherichia coli cultures. Finally, the topology of a putative p14 movement protein was established by replacing the amber stop codon located between p7A and p7B.     

HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration

Human Immunodeficiency Virus Type 1 (HIV-1) establishes a latent reservoir early in infection that is resistant to the host immune response and treatment with highly active antiretroviral therapy (HAART). The best understood of these reservoirs forms in resting CD4(+) T cells. While it remains unclear how reservoirs form, a popular model holds that the virus can only integrate in activated CD4(+) T cells. Contrary to this model, our previous results suggest that HIV-1 can integrate directly into the genomes of resting CD4(+) T cells. However, a limitation of our previous studies was that they were conducted at high viral inoculum and these conditions may lead to cellular activation or saturation of restriction factors. In the present study, we tested if our previous findings were an artifact of high inoculum. To do this, we enhanced the sensitivity of our integration assay by incorporating a repetitive sampling technique that allowed us to capture rare integration events that occur near an Alu repeat. The new technique represents a significant advance as it enabled us to measure integration accurately down to 1 provirus/well in 15,000 genomes--a 40-fold enhancement over our prior assay. Using this assay, we demonstrate that HIV can integrate into resting CD4(+) T cells in vitro even at low viral inoculum. These findings suggest there is no threshold number of virions required for HIV to integrate into resting CD4(+) T cells.     

The intracellular sites of early replication and budding of SARS-coronavirus

In this study, we analyzed the replication and budding sites of severe acute respiratory syndrome coronavirus (SARS-CoV) at early time points of infection. We detected cytoplasmic accumulations containing the viral nucleocapsid protein, viral RNA and the non-structural protein nsp3. Using EM techniques, we found that these putative viral replication sites were associated with characteristic membrane tubules and double membrane vesicles that most probably originated from ER cisternae. In addition to its presence at the replication sites, N also accumulated in the Golgi region and colocalized with the viral spike protein. Immuno-EM revealed that budding occurred at membranes of the ERGIC (ER-Golgi intermediate compartment) and the Golgi region as early as 3 h post infection, demonstrating that SARS-CoV replicates surprisingly fast. Our data suggest that SARS-CoV establishes replication complexes at ER-derived membranes. Later on, viral nucleocapsids have to be transported to the budding sites in the Golgi region where the viral glycoproteins accumulate and particle formation occurs.     

Extracellular aminoacids in the amygdala and nucleus accumbens in the rat during acute pain

In the present experiments extracellular arginine, glutamate and aspartate were studied in the basolateral nucleus of the amygdala and core of the nucleus accumbens during the formalin test (phase I). A combination of capillary zone electrophoresis with laser induced fluorescence detection and microdialysis in freely moving rats was used. Glutamate and arginine significantly increased in the nucleus accumbens after formalin injection; glutamate, arginine and aspartate significantly increased in the basolateral nucleus of the amygdala, after formalin injection. These experiments suggest that rapid neurotransmitters changes observed in the nucleus accumbens and amygdala, are possibly related to immobility and emotional states such as anxiety, aversion and/or depression caused by pain.     

Re-expression of a developmentally restricted potassium channel in autoimmune demyelination: Kv1.4 is implicated in oligodendroglial proliferation

Mechanisms of lesion repair in multiple sclerosis are incompletely understood. To some degree, remyelination can occur, associated with an increase of proliferating oligodendroglial cells. Recently, the expression of potassium channels has been implicated in the control of oligodendrocyte precursor cell proliferation in vitro. We investigated the expression of Kv1.4 potassium channels in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Confocal microscopy revealed expression of Kv1.4 in AN2-positive oligodendrocyte precursor cells and premyelinating oligodendrocytes in vitro but neither in mature oligodendrocytes nor in the spinal cords of healthy adult mice. After induction of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, Kv1.4 immunoreactivity was detected in or around lesions already during disease onset with a peak early and a subsequent decrease in the late phase of the disease. Kv1.4 expression was confined to 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendroglial cells, which were actively proliferating and ensheathed naked axons. After a demyelinating episode, the number of Kv1.4 and 2',3'-cyclic nucleotide 3'-phosphodiesterase double-positive cells was greatly reduced in ciliary neurotrophic factor knockout mice, a model with impaired lesion repair. In summary, the re-expression of an oligodendroglial potassium channel may have a functional implication on oligodendroglial cell cycle progression, thus influencing tissue repair in experimental autoimmune encephalomyelitis and multiple sclerosis.     

Effects of JC virus infection on anti-apoptotic protein survivin in progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system resulting from the productive infection of oligodendrocytes by the opportunistic polyomavirus JC virus (JCV). Apoptosis is a host defense mechanism to dispose of damaged cells; however, certain viruses have the ability to deregulate apoptotic pathways to complete their life cycles. One such pathway involves survivin, a member of the inhibitor of apoptosis family, which is abundantly expressed during development in proliferating tissues but should be absent in normal, terminally differentiated cells. Immunohistochemistry performed in 20 cases of PML revealed the presence of survivin in JCV-infected oligodendrocytes and bizarre astrocytes within demyelinated plaques. Survivin up-regulation was also found in oligodendroglial and astrocytic cultures infected with JCV. Cell cycle analysis and DNA laddering demonstrated a significantly lower number of cells undergoing apoptosis on JCV infection compared with noninfected cultures; small interfering RNA inhibition of survivin resulted in a dramatic increase in apoptotic cells in JCV-infected cultures. This is the first report describing the activation of survivin by JCV infection in vitro and in PML clinical cases. These observations provide new insights into the anti-apoptotic mechanisms used by JCV to complete its lytic cycle and may suggest new therapeutic targets for PML.     

Long-term potentiation (LTP) in the central amygdala (CeA) is enhanced after prolonged withdrawal from chronic cocaine and requires CRF1 receptors

The amygdala is part of the brain reward circuitry that plays a role in cocaine-seeking and abstinence in animals and cocaine craving and relapse in humans. Cocaine-seeking is elicited by cocaine-associated cues, and the basolateral amygdala (BLA) and CeA are essential in forming and communicating drug-related associations that are thought to be critical in long-lasting relapse risk associated with drug addiction. Here we simulated a cue stimulus with high-frequency stimulation (HFS) of the BLA-CeA pathway to examine mechanisms that may contribute to drug-related associations. We found enhanced long-term potentiation (LTP) after 14-day but not 1-day withdrawal from 7-day cocaine treatment mediated through N-methyl-d-aspartate (NMDA) receptors (NRs), L-type voltage-gated calcium channels (L-VGCCs), and corticotropin-releasing factor (CRF)(1) receptors; this was accompanied by increased phosphorylated NR1 and CRF(1) protein not associated with changes in NMDA/AMPA ratios in amygdalae from cocaine-treated animals. We suggest that these signaling mechanisms may provide therapeutic targets for the treatment of cocaine cravings.