Acetylcholine and potassium elicit different patterns of exocytosis in chromaffin cells when the intracellular calcium handling is disturbed

During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca2+, pulses of 100 microM ACh or 100 mM K+ of increasing duration (1-5 s) caused similar exocytosis of about 3-4 microC catecholamine. Depletion of endoplasmic reticulum (ER) Ca2+ by pretreatment with 1 microM thapsigargin, 10 mM caffeine and 10 microM ryanodine more than halved the responses to ACh but did not affect the responses to K+ responses. In these ER Ca2+-depleted cells the protonophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20 microM given during the 5 s preceding each pulse) augmented the responses to ACh responses fourfold for all pulse durations applied (1-5 s) whereas responses to K+ were potentiated twofold with 1 to 2 s pulses but were not affected with longer pulse durations. ACh pulses applied to fura-2-loaded cells evoked increases of bulk cytosolic [Ca2+] ([Ca2+](c)) that were substantially smaller than those elicited by K+ pulses. Confocal microscopy of fluo-3-loaded cells showed that ACh pulses elicited discrete and more localized [Ca2+](c) elevations, whereas K+ pulses produced higher [Ca2+](c) transients that spread out quickly throughout the cytosol. These results suggest that mitochondria sense the increase of local [Ca2+](c) elicited by ACh (that evokes firing of action potentials) much better than that induced by K+ (that produces sustained cell depolarisation). This implies that mitochondria are more sensitive to the local [Ca2+](c) changes resulting from the physiological triggering of action potentials by ACh.     

Genetic characterization and structural analysis of VHL Spanish families to define genotype-phenotype correlations

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. This gene, located in the 3p25-26 chromosome, is a tumor suppressor gene associated with the inhibition of angiogenesis and apoptosis, cell cycle exit, fibronectin matrix assembly, and proteolysis. To define the molecular basis of VHL in a Spanish population, we studied 33 patients suspected of suffering familial or de novo VHL disease and two familial pheochromocytoma cases. Sequence analysis of the coding regions of the VHL gene revealed germline sequence variants in 68.7% (24 out of 35) of the patients, and four of them presented with undescribed germline alterations: g.5429-5430insG, p.Leu128Arg, p.Tyr175Cys, and p.Tyr175Asn. For the remaining 11 patients who showed negative for point mutations, we performed Southern blot analysis and detected gross rearrangements in eight cases (22.8% of the index cases). Our results support the relevance of VHL gene analysis in familial pheochromocytoma cases and also in those with no familial history. In order to investigate the relevance of different amino acid changes in the VHL phenotype, we also analyzed the genotype-phenotype correlations using structural analysis to assess protein stability and complexes. The association of clear cell renal carcinoma (CCRC) development with a relatively high loss of structural stability in pVHL missense-mutants was consistent. Structural stability data in the genotype-phenotype correlations therefore provides us with a better understanding of VHL clinical implications. It is also a suitable approach to the evaluation of unknown significance changes.     

Visual object naming in patients with small lesions centered at the left temporopolar region

Naming is considered a left hemisphere function that operates according to a posterior–anterior specificity gradient, with more fine-grained information processed in most anterior regions of the temporal lobe (ATL), including the temporal pole (TP). Word finding difficulties are typically assessed using visual confrontation naming tasks, and have been associated with selective damage to ATL resulting from different aetiologies. Nonetheless, the role of the ATL and, more specifically, of the TP in the naming network is not completely established. Most of the accumulated evidence is based on studies on patients with extensive lesions, often bilateral. Furthermore, there is a considerable variability in the anatomical definition of ATL. To better understand the specific involvement of the left TP in visual object naming, we assessed a group of patients with an epileptogenic lesion centered at the TP, and compared their performance with that of a strictly matched control group. We also administered a battery of verbal and non-verbal semantic tasks that was used as a semantic memory baseline. Patients showed an impaired naming ability, manifesting in a certain degree of anomia and semantically related naming errors, which was influenced by concept familiarity. This pattern took place in a context of mild semantic dysfunction that was evident in different types and modalities of semantic tasks. Therefore, current findings demonstrate that a restricted lesion to the left TP can cause a significant deficit in object naming. Of importance, the observed semantic impairment was far from the devastating degradation observed in semantic dementia and other bilateral conditions.     

Rapid Detection of Staphylococcus aureus in Lower Respiratory Tract Secretions from Patients with Suspected Ventilator-Associated Pneumonia: Evaluation of the Cepheid Xpert MRSA/SA SSTI Assay

A preclinical evaluation was conducted to evaluate the performance of the Cepheid Xpert assay on 135 lower respiratory tract secretions for detection of methicillin-resistant Staphylococcus aureus and S. aureus. Compared with the quantitative culture, the sensitivity, specificity, and positive and negative predictive values were 99.0%, 72.2%, 90.7%, and 96.3%, respectively.     

Los primeros pobladores de América y sus relaciones con poblaciones del Océano Pacífico según los genes HLA

HLA allele frequencies were compared with those of other First American Natives and also those of other worldwide populations in order to clarify the still unclear peopling of the Americas and the origins of Amerindians. All possible HLA data already obtained on early Native American populations are used. Genetic distances and N-J dendrogram methods are applied. Results and discussion have led to the following conclusions: 1) North West Canadian Athabaskans have had gene flow with close neighbouring populations, Amerindians, Pacific Islanders, including East Australians, and Siberians, since they share DRB1-DQB1 haplotypes with these populations (i.e.: DRB1*14:01-DQB1*05:03, DRB1*09:01-DQB1*03:03); 2) Amerindians entrance to America may have been different to that of Athabaskans, Aleuts and Eskimos; Amerindians may have been in their lands long before Athabaskans and Eskimos as they present an altogether different set of HLA-DRB1 allele frequencies; 3) Amerindians show very few “particular” single-locus alleles (i.e.: DRB1*04:11, DRB1*04:17), but have unique extended haplotypes (i.e.: A*02-B*35-DRB1*04:07-DQB1*03:02, A*02-B*35-DRB1*08:02-DQB1*04:02); 4) Our results do not support the three-wave model of American peopling but another model, where the Pacific Coast is also an entrance point. Pacific Ocean sea voyages may have contributed to the HLA genetic American profile. Reverse migration (America to Asia) is not discarded, and different movements of people in either direction in different times are supported by the Athabaskan population admixture with Asian-Pacific population and with Amerindians.     

A distinct cellular profile is seen in the human endocervix during Chlamydia trachomatis infection

Problem The endocervix is a major target of Chlamydia trachomatis infection, but little is known about the immune repertoire in this tissue, or its response to these common bacteria. Method of study Using a cytobrush, we isolated cells from the endocervix of 20 women during C. trachomatis infection, and post‐antibiotic treatment. Endocervical swabs and blood were taken in parallel. Endocervical cells were enumerated, and endocervical and blood T cells immunophenotyped. Chlamydia trachomatis was genotyped by sequence analysis of the OmpA gene, and quantified by culture. Results Chlamydia trachomatis genotypes were D, E, F and Ia, and infectious burden varied considerably. Endocervical T cell and neutrophil numbers were highly elevated during infection, with both CD4 and CD8 T‐cell subsets accumulating. Regardless of the presence or absence of infection, the endocervical cell infiltrate was dominated by effector memory T cells, and the numbers of CCR5 and CD103 expressing T cells was significantly higher than in the blood. Human leukocyte antigen (HLA‐DR) expression by endocervical T cells was significantly increased during infection. Conclusion The human endocervix exhibits a distinct cellular response to C. trachomatis infection that can be longitudinally evaluated by cytobrush sampling. Infecting organisms can be sampled and analyzed in parallel.     

Primary lymphomas of the breast: a report on 5 cases studied in a period of 5 years at the Hospital General de México

Breast lymphomas can be primary or secondary. Among the primary lymphomas, the most common histologic types are the large B-cell diffuse lymphomas and the extranodal B mucosa-associated lymphatic tissue lymphomas. We studied 5 cases of primary breast lymphoma in female patients. The criteria for the diagnosis were based on the proposal of Wiseman and Liao: (1) in the biopsy or surgical specimen, the lymphoma involves the breast parenchyma, and (2) nonsystemic disease at diagnosis. Clinical data, histologic findings, immunohistochemical reactions, treatment, and clinical follow-up were reviewed. The 5 patients were young women with average age of 27 years; the youngest was 20 years old, and the oldest was 44 years old. The right breast was the most affected, and 1 patient was HIV positive. The most common symptoms were the presence of nodes, progressive increase of volume, collateral venous network, and hemorrhagic discharge from the nipple. The clinical course was of 1 to 14 months before diagnosis. Three patients died because of central nervous system infiltration, one is still alive, and the other was lost during follow-up. Histologically, all primary breast lymphomas were large B-cell lymphomas; one had focal starry sky pattern, and the other 3 were centroblastic. All were positive to CD20 and CD79^a, 3 expressed bcl2, and 2 expressed bcl6. The proliferation index was between 60% and 80%. Primary breast lymphomas are rare. The average age of our patients was 27 years, and their clinical course was aggressive with central nervous system infiltration. The most common histologic type was the large B-cell diffuse lymphoma. Differential diagnosis must be established in the presence of poorly differentiated lobules and ductal carcinoma.     

Molecular characterization of a t(9;12)(p21;q13) balanced chromosome translocation in combination with integrative genomics analysis identifies C9orf14 as a candidate tumor-suppressor

A large number of nevi (LNN) is a high risk phenotypic trait for developing cutaneous malignant melanoma (CMM). In this study, the breakpoints of a t(9;12)(p21;q13) balanced chromosome translocation were finely mapped in a family with LNN and CMM. Molecular characterization of the 9p21 breakpoint identified a novel gene C9orf14 expressed in melanocytes disrupted by the translocation. Integrative analysis of functional genomics data was applied to determine the role of C9orf14 in CMM development. An analysis of genome-wide DNA copy number alterations in melanoma tumors revealed the loss of the C9orf14 locus, located proximal to CDKN2A, in approximately one-fourth of tumors. Analysis of gene expression data in cancer cell lines and melanoma tumors suggests a loss of C9orf14 expression in melanoma tumorigenesis. Taken together, our results indicate that C9orf14 is a candidate tumor-suppressor for nevus development and late stage melanoma at 9p21, a region frequently deleted in different types of human cancers.