Selective localization and regulated release of calcitonin gene-related peptide from dense-core vesicles in engineered PC12 cells

Introduction of the gene for calcitonin into the neuroendocrine PC12 cell line resulted in the expression of the neuronal-specific splice product, calcitonin gene-related peptide (CGRP). Expression of this neuropeptide did not require treatment of the PC12 cells with NGF. By all available criteria, including biochemical, immunological, and morphological analysis, we have determined that the CGRP in stably transfected PC12 cells is sorted selectively into the large, dense-core catecholamine-containing secretory vesicles. Conversely, the CGRP is excluded from the small, synaptophysin-rich vesicles present in the same cells. Stimulation conditions that trigger the release of catecholamines cause a parallel burst in the release of CGRP. In all these respects, the engineered PC12 cells process the foreign CGRP in a manner similar to that seen in spinal motor neurons in vivo. These results indicate that this small (37 amino acids) peptide contains sorting information sufficient for targeting to large, dense-core vesicles in heterologous cells, placing very narrow constraints on the possible location of sorting signals. In addition, this CGRP-expressing cell line opens the possibility of studying the physiological role of CGRP in the establishment and maintenance of neuromuscular contacts. © 1996 Wiley-Liss, Inc.     

Rolling Circle Amplification : A New Approach to Increase Sensitivity for Immunohistochemistry and Flow Cytometry

Immunohistochemistry is a method that can provide complementary diagnostic and prognostic information to morphological observations and soluble assays. Sensitivity, specificity, or requirements for arduous sample preparation or signal amplification procedures often limit the application of this approach to routine clinical specimens. Rolling circle amplification (RCA) generates a localized signal via an isothermal amplification of an oligonucleotide circle. The application of this approach to immunohistochemistry could extend the utility of these methods to include a more complete set of immunological and molecular probes. RCA-mediated signal amplification was successfully applied to the sensitive and specific detection of a variety of cell surface antigens (CD3, CD20, and epithelial membrane antigen) and intracellular molecules (vimentin and prostate-specific antigen) within a variety of routinely fixed specimens, as well as samples prepared for flow cytometry. RCA technology, which has an intrinsically wide dynamic range, is a robust and simple procedure that can provide a universal platform for the localization of a wide variety of molecules as a function of either antigenicity or nucleic acid sequence. The use of RCA in this way could enhance the use of markers of current interest as well as permit the integration of emerging information from genomics and proteomics into cell- and tissue-based analyses.     

The salivary microbiota as a diagnostic indicator of oral cancer: A descriptive,non-randomized study of cancer-free and oral squamous cell carcinoma subjects

Background  

The purpose of the present investigation was to determine if the salivary counts of 40 common oral bacteria in subjects with an oral squamous cell carcinoma (OSCC) lesion would differ from those found in cancer-free (OSCC-free) controls.     

NRP-1/CD304 expression in acute leukemia: a potential marker for minimal residual disease detection in precursor B-cell acute lymphoblastic leukemia

Neuropilin-1 (NRP-1)/CD304 is a marker for plasmacytoid dendritic cells. We determined the distribution of NRP-1/CD304 expression on normal hematopoietic cells and in 167 acute leukemias by flow cytometry. NRP-1/CD304 surface expression was frequent in precursor B-cell acute lymphoblastic leukemia (36/51 [71%]) and uncommon in acute myeloid leukemia (22.9%). In acute myeloid leukemia, expression was noted in all (4/4) acute myeloid leukemias with the M4eo subtype and in 50% of specimens (6/12) with complex cytogenetics. On hematopoietic cells, NRP-1/CD304 was expressed on normal erythroid progenitors, plasma cells, and B-cell progenitors, as well as plasmacytoid dendritic cells. Expression was not consistently detected on other hematopoietic cell types. Owing to this distribution of expression, the detection of NRP-1/CD304 alone on a hematopoietic cell cannot be used to determine plasmacytoid dendritic cell differentiation. Finally, we show that NRP-1/CD304 is overexpressed in 30% of precursor B-cell acute lymphoblastic leukemia samples compared with normal B-cell progenitors, allowing for its potential use as a marker for the detection of minimal residual disease.     

Evaluation of the Vitek-2 extended-spectrum β-lactamase test against non-duplicate strains of Enterobacteriaceae producing a broad diversity of well-characterised β-lactamases

The Vitek-2 extended-spectrum β-lactamase (ESBL) test was assessed using a collection of 94 ESBL-positive and 71 ESBL-negative non-duplicate isolates of Enterobacteriaceae. These isolates produced a wide diversity of well-characterised β-lactamases, including 61 different ESBLs, two class A carbapenemases and various species-specific β-lactamases. ESBL detection was performed using (i) the conventional synergy test as recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie, (ii) the CLSI phenotypic confirmatory test for ESBLs, and (iii) the Vitek-2 ESBL test. For Escherichia coli and klebsiellae, the sensitivity/specificity values were 97.3%/96.9% for the synergy test, 91.8%/100% for the CLSI phenotypic confirmatory test, and 91.8%/100% for the Vitek-2 ESBL test. For other organisms, the sensitivity/specificity values were 100%/97.4% for the synergy test, 90.5%/100% for the CLSI phenotypic confirmatory test, and 90.5%/100% for the Vitek-2 ESBL test. The Vitek-2 ESBL test seemed to be an efficient method for routine detection of ESBL-producing isolates of Enterobacteriaceae, including isolates producing AmpC-type enzymes.     

The effect of gravity on the horizontal and vertical vestibulo-ocular reflex in the rat

Horizontal and vertical eye movements were recorded in alert pigmented rats using chronically implanted scleral search coils or temporary glue-on coils to test the dependence of the vestibulo-ocular reflex (VOR) upon rotation axis and body orientation. The contributions of semicircular-canal versus otolith-organ signals to the VOR were investigated by providing canal-only (vertical axis) and canal plus otolith (horizontal axis) stimulation conditions. Rotations that stimulated canals only (upright yaw and nose-up roll) produced an accurate VOR during middle- and high-frequency rotations (0.2-2 Hz). However, at frequencies below 0.2 Hz, the canal-only rotations elicited a phase-advanced VOR. The addition of a changing gravity stimulus, and thus dynamic otolith stimulation, to the canal signal (nose-up yaw, on-side yaw, and upright roll) produced a VOR response with accurate phase down to the lowest frequency tested (0.02 Hz). In order to further test the dependence of the VOR on gravitational signals, we tested vertical VOR with the head in an inverted posture (inverted roll). The VOR in this condition was advanced in phase across all frequencies tested. At low frequencies, the VOR during inverted roll was anticompensatory, characterized by slow-phase eye movement in the same direction as head movement. The substantial differences between canalonly VOR and canal plus otolith VOR suggest an important role of otolith organs in rat VOR. Anticompensatory VOR during inverted roll suggests that part of the otolith contribution arises from static tilt signals that are inverted when the head is inverted.     

红细胞保存期影响早产儿疗效的随机对照试验:研究设计

尽管目前趋于逐渐降低输血阅值,并开发技术避免输注异体血液,但异体红细胞输注仍是罹患疾病和贫血的新生儿重症监护患者一个重要的支持和挽救生命的方法.     

BAK1 gene variation and abdominal aortic aneurysms

We sought to examine the role of genetics in the multifactorial disease, abdominal aortic aneurysm (AAA), by studying sequence variation in the BAK1 gene (BAK1) that codes for an apoptotic‐promoting protein, as chronic apoptosis activation has been linked to AAA development and progression. BAK1 abdominal aorta cDNA from AAA patients and nondiseased individuals were compared with each other, as well as to the BAK1 genomic sequence obtained from matching blood samples. We found specific BAK1 single nucleotide polymorphism (SNP) containing alleles in both aneurysmic (31 cases) and healthy aortic tissue (5 cases) without seeing them in the matching blood samples. These same BAK1 SNPs have been reported, although rarely (average frequency <0.06%), in reference BAK1 DNA sequences. Based on this and other similar observations, we propose a novel hypothesis postulating that multiple variants of genes may preexist in “minority” forms within specific nondiseased tissues and be selected for, when intra‐ and/or extracellular conditions change. Therefore, the fact that different BAK1 variants can exist in both diseased and nondiseased AA tissues compared to matching blood samples, together with the rare occurrence of these same SNPs in reference sequences, suggests that selection may be a significant factor in AAA ontogeny. Hum Mutat 30:1–5, 2009. © 2009 Wiley‐Liss, Inc.