Leptin Signaling in the Arcuate Nucleus Reduces Insulin’s Capacity to Suppress Hepatic Glucose Production in Obese Mice

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Validation of Zebrafish （Danio redo） Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR （qRT-PCR） is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or housekeeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The fl-actin, EFlot and Rpll3ot genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1 or, Rpll3α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

GABA(A) receptor alpha-subunit proteins in human chronic alcoholics

Antibodies were raised against specific peptides from N-terminal regions of the alpha1 and alpha3 isoforms of the GABA(A) receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post-mortem delay. The regression of expression on post-mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of alpha1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on alpha3 expression. The alpha1 and alpha3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, alpha1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, alpha3 expression was significantly lower in superior frontal than in motor cortex. Expression of alpha1 was significantly different from that of alpha3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.     

BMPs as mediators of roof plate repulsion of commissural neurons

During spinal cord development, commissural (C) neurons, located near the dorsal midline, send axons ventrally and across the floor plate (FP). The trajectory of these axons toward the FP is guided in part by netrins. The mechanisms that guide the early phase of C axon extension, however, have not been resolved. We show that the roof plate (RP) expresses a diffusible activity that repels C axons and orients their growth within the dorsal spinal cord. Bone morphogenetic proteins (BMPs) appear to act as RP-derived chemorepellents that guide the early trajectory of the axons of C neurons in the developing spinal cord: BMP7 mimics the RP repellent activity for C axons in vitro, can act directly to collapse C growth cones, and appears to serve an essential function in RP repulsion of C axons.     

Logistic regression analysis of low grade spindle cell lesions. A cytologic study

OBJECTIVE: To identify key diagnostic cytologic criteria for various low grade spindle cell lesions. STUDY DESIGN: We reviewed 20 synovial sarcomas, 18 benign neural tumors, 10 reparative lesions, 24 other benign and 27 additional malignant low grade spindle cell lesions. All specimens were coded as to the presence or absence of the following variables: high cellularity, tissue fragments, tissue culture appearance, epithelial fragments, vessel fragments, vascular arcades, fibrillar ground substance, myxoid background, microcystic areas, parallel arrangement of nuclei, naked nuclei, single cells, binucleate cells, multinucleate cells, long filamentous cells, short spindle cells, stellate cells, lipoblasts, nuclear pleomorphism, nuclei with pointed ends, comma/fishhook nuclei, cigar-shaped nuclei, ovoid/round nuclei, small nucleoli, large nucleoli, mitotic figures, intranuclear vacuoles and background histiocytes. A logistic regression analysis was performed to identify the variables predictive of malignant lesions, specifically synovial sarcomas, benign neural tumors and reparative lesions. RESULTS: Statistical analysis selected high cellularity, short spindle cells, small nucleoli and absence of tissue culture appearance as the main criteria for malignant neoplasms. Tissue fragments and high cellularity were selected as the primary criteria and absence of long filamentous cells and of myxoid background as the secondary criteria for synovial sarcomas. It selected fibrillar ground substance and absence of ovoid/round nuclei as the key criteria for benign neural tumors. The presence of a tissue culture appearance was the major criterion for reparative lesions. CONCLUSION: There are many previously described cytologic criteria, but we found that when subjected to statistical analysis, only a few features were significant in the evaluation of low grade spindle cell lesions.     

Cytologic findings in vitreous fluids. Analysis of 74 specimens

OBJECTIVE: To review the cytologic findings of vitreous specimens and propose a simplified approach to them. STUDY DESIGN: Seventy-four vitreous specimens from 60 patients obtained either during a pars plana vitrectomy or by vitreous aspiration were reviewed. Clinical correlation was obtained on all patients. RESULTS: Findings suggestive of a specific disorder were present in 30 specimens (41%); cytologic examination of the remaining 44 showed nonspecific changes. A lymphoproliferative disorder, the intraocular malignancy suspected most often in this series, was identified in eight specimens (11%). Large cell lymphomas were evident in 5 specimens, 2 specimens were suspicious for lymphoma, and 1 specimen was consistent with plasmacytoma. Twelve specimens (16%) contained hemorrhage. In rare instances, specific infectious agents, such as parasites (5%), bacteria (1%) and fungi (3%), could be identified. The diagnosis of viral infections required ancillary studies. Lens fragments were identified in four cases (5%), and a diagnosis of lens-induced endophthalmitis could be rendered in one case (1%). Changes consistent with sarcoidosis were present in 3% of cases. CONCLUSION: Based on this experience with vitreous specimens submitted for clinical reasons, we found that they could be divided into three broad diagnostic categories: inflammation/infection (54 specimens/41 patients), hemorrhage (12 specimens/12 patients) and malignancy (8 specimens/7 patients).     

Signet ring adenocarcinoma metastatic to the bronchus and mimicking goblet cell hyperplasia. A case report

BACKGROUND: Goblet cells in the lower respiratory tract are metaplastic bronchial epithelial cells usually associated with asthma or chronic bronchitis. Goblet cells acquire their name by a tendency to distend with mucus, with subsequent distortion in cell shape. Due to similarity of shape, metaplastic goblet cells and signet ring cells can be easily confused in cytologic samples. CASE: A 55-year-old male with a history of gastrointestinal adenocarcinoma underwent brushing, washing and biopsy of a bronchial lesion. The bronchial wash and brush samples showed a very cellular specimen, with large aggregates of distended columnar cells. These were arranged in long strips, thick bundles and occasional three-dimensional aggregates. Some aggregates contained numerous rounded cells with markedly distended cytoplasm. The rounded cells were slightly larger than the distended columnar cells. These cells had a relatively large but innocuous-appearing nucleus displaced to the periphery of the cell. The corresponding bronchial biopsy revealed signet ring adenocarcinoma, presumably metastatic from the gastrointestinal primary. CONCLUSION: Signet ring adenocarcinoma, either primary or metastatic, can be difficult to diagnose in cytologic and histologic specimens. There are numerous mimics of signet ring cells, both benign and neoplastic. In respiratory cytologic specimens, one of the benign imposters is goblet cell metaplasia.     

The gene encoding a periplasmic deoxyribonuclease from Aeromonas hydrophila

A gene encoding a deoxyribonuclease, dnsH, was cloned from Aeromonas hydrophila JMP636. The predicted mature protein was very similar to the previously described extracellular Dns from this organism and an N-terminal region corresponding to a large putative signal sequence was predicted for the JMP636 protein. Inactivation of dnsII demonstrated that the DnsH protein was not present extracellularly in this strain. As DnsH degraded plasmid DNA and was believed to have a periplasmic location, a dnsH mutant was constructed to determine whether electroporation of A. hydrophila with plasmid DNA could be achieved. No transformants were detected. From SDS-PAGE studies, at least two additional DNases remain to be characterised from A. hydrophila JMP636.