Limited understanding of molecular mechanisms of metastasis in melanoma contributes to the absence of effective treatments. Increased knowledge of alterations in genes that underpin critical ...molecular events that lead to metastasis is essential. We have investigated the gene expression profiles of primary melanomas and melanoma metastases in sentinel lymph nodes. A total of 19 samples (10 primary melanomas and 9 sentinel lymph node metastases) were evaluated. Melanoma cells were dissected from tissue blocks. Total mRNA was isolated, amplified, and labeled using an Ambion Recover All Total Nucleic Acid Isolation kit, Nu-GEN WT-Ovation formalin-fixed, paraffin-embedded RNA Amplification System, and FL-Ovation cDNA Biotin Module V2, respectively. Samples were hybridized to the Affymetrix Gene Chip Human U133 Plus 2.0 Array. Data were analyzed using Partek Genomics Suite Version 6.4. Genes selected showed ≥2-fold difference in expression and P<5.00E−2. Validation studies used standard immunohistochemical assays. Hierarchical clustering disclosed two distinct groups: 10 primary melanomas and 9 sentinel lymph node metastases. Gene expression analysis identified 576 genes that showed significant differential expression. Most differences reflected decreased gene expression in metastases relative to primaries. Reduced gene expression in primaries was less frequent and less dramatic. Genes significantly increased or decreased in sentinel lymph node metastases were active in cell adhesion/structural integrity, tumor suppression, cell cycle regulation, and apoptosis. Validation studies indicate that MAGEC1 (melanoma antigen family C1) and FCRL1 (Fc receptor-like 1) are involved in melanoma progression. There are striking differential gene expression patterns between primary and nodally metastatic melanomas. Similar findings were seen with autologous paired primary melanomas and sentinel lymph node metastases, supporting involvement of these gene alterations in evolution of metastases. With further study, it may be possible to determine the exact sequence of molecular events that underlie melanoma metastases.
We describe a novel congenital motor neuron disease with early demise due to respiratory insufficiency with clinical overlap with spinal muscular atrophy with respiratory distress (SMARD) type 1 but ...lacking a mutation in the IGHMBP2 gene.
Exome sequencing was used to identify a de novo mutation in the LAS1L gene in the proband. Pathogenicity of the mutation was validated using a zebrafish model by morpholino-mediated knockdown of las1l.
We identified a de novo mutation in the X-linked LAS1L gene in the proband (p.S477N). The mutation is in a highly conserved region of the LAS1L gene predicted to be deleterious by bioinformatic analysis. Morpholino-based knockdown of las1l, the orthologous gene in zebrafish, results in early lethality and disruption of muscle and peripheral nerve architecture. Coinjection of wild-type but not mutant human RNA results in partial rescue of the phenotype.
We report a patient with a SMARD phenotype due to a mutation in LAS1L, a gene important in coordinating processing of the 45S pre-rRNA and maturation of the large 60S ribosomal subunit. Similarly, the IGHMB2 gene associated with SMARD type 1 has been suggested to have an important role in ribosomal biogenesis from its role in processing the 45S pre-rRNA. We propose that disruption of ribosomal maturation may be a common pathogenic mechanism linking SMARD phenotypes caused by both IGHMBP2 and LAS1L.
Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on ...light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology. To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes. In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the feasibility of using molecular classification as a supplement to standard histology. Our successful use of a standard formalin-fixed and paraffin-embedded tissue further supports the practicability of combining molecular diagnostic testing with histopathology in evaluation of difficult melanocytic lesions.
The LYS7 gene in Saccharomyces cerevisiae encodes a protein (yCCS) that delivers copper to the active site of copper-zinc superoxide dismutase (CuZn-SOD, a product of the SOD1 gene). In yeast lacking ...Lys7 (lys7Delta), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Delta) and lys7Delta yeast show very similar phenotypes, namely poor growth in air and aerobic auxotrophies for lysine and methionine. Here, we demonstrate certain phenotypic differences between these strains: 1) lys7Delta cells are slightly less sensitive to paraquat than sod1Delta cells, 2) EPR-detectable or "free" iron is dramatically elevated in sod1Delta mutants but not in lys7Delta yeast, and 3) although sod1Delta mutants show increased sensitivity to extracellular zinc, the lys7Delta strain is as resistant as wild type. To restore the SOD catalytic activity but not the zinc-binding capability of the SOD1 polypeptide, we overexpressed Mn-SOD from Bacillus stearothermophilus in the cytoplasm of sod1Delta yeast. Paraquat resistance was restored to wild-type levels, but zinc was not. Conversely, expression of a mutant CuZn-SOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but not paraquat resistance. Taken together, these results strongly suggest that CuZn-SOD, in addition to its antioxidant properties, plays a role in zinc homeostasis.
Spontaneous coronary artery dissection is a rare cause of acute myocardial infarction and sudden death. It typically, but not always, occurs in healthy postpartum women without traditional risk ...factors for atherosclerosis. Moreover, the site of dissection usually involves the proximal, major coronary arteries: left main coronary artery and/or the left anterior descending artery, and in men, more often the right coronary artery. We report a case of sudden death caused by dissection of the obtuse marginal branch of the left circumflex artery, in a 49-year-old man, a very rare site of fatal coronary dissection.
Sigma factors in the sigma70 family can be classified into the primary and alternative sigma factors according to their physiological functions and amino acid sequence similarities. The primary sigma ...factors are composed of four conserved regions, with the conserved region 1 being divided into two subregions. Region 1.1, which is absent from the alternative sigma factor, is poor in conservation; however, region 1.2 is well conserved. We investigated the importance of these two subregions to the function of Bacillus subtilis sigmaA, which belongs to a subgroup of the primary sigma factor lacking a 254-amino-acid spacer between regions 1 and 2. We found that deletion of not more than 100 amino acid residues from the N terminus of sigmaA, which removed part or all region 1.1, did not affect the overall transcription activity of the truncated sigmaA-RNA polymerase in vitro, indicating that region 1.1 is not required for the functioning of sigmaA in RNA polymerase holoenzyme. This finding is consistent with the complementation data obtained in vivo. However, region 1.1 is able to negatively modulate the promoter DNA-binding activity of the sigmaA-RNA polymerase. Further deletion of the conserved Arg-103 at the N terminus of region 1.2 increased the content of stable secondary structures of the truncated sigmaA and greatly reduced the transcription activity of the truncated sigmaA-RNA polymerase by lowering the efficiency of transcription initiation after core binding of sigmaA. More importantly, the conserved Arg-103 was also demonstrated to be critical for the functioning of the full-length sigmaA in RNA polymerase. PUBLICATION ABSTRACT
Characterizations of mutant Saccharomyces cerevisiae deficient in the SOD1 locus (encoding copper-zinc superoxide dismutase, or CuZnSOD) have provided invaluable insight into processes involved in ...cellular metabolism of reactive oxygen species (ROS). Interestingly, these scientific endeavors have also uncovered an intimate connection between transition metal homeostasis and cellular redox status. The LYS7 gene in S. cerevisiae encodes a protein that mediates copper incorporation into CuZnSOD. In yeast lacking LYS7 (lys7Δ), the SOD1 polypeptide is present but inactive; thus, yeast cells with a null mutation at either the LYS7 or SOD1 locus (i.e. lys7Δ or sod1Δ) display similar phenotypes, with a few exceptions. The most striking difference is their capacity to tolerate zinc. While lys7Δ yeast can withstand highly elevated zinc concentrations in the growth medium, sod1Δ mutants are exquisitely sensitive to excess zinc. Overexpression of a bacterial Mn-SOD in the cytoplasm of sod1Δ yeast restored its resistance to paraquat, a superoxide-generating chemical, but not to zinc. Conversely, expression of a mutant CuZnSOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but remained sensitive to paraquat. Collectively, these results suggest that CuZnSOD plays a role in zinc homeostasis, probably serving as a temporary depot for zinc. Further studies into the effect of excess zinc on wild-type, lys7Δ, and sod1Δ yeast strains have implicated mitochondrial dysfunction and general disruption to cellular metal metabolism as putative mechanisms of zinc toxicity in this organism. Under physiological conditions, zinc is oxidatively inactive, and a significant portion of the published literature has focused on the role of CuZnSOD in the cellular metabolism of copper and other redox active metal ions. However, according to the results depicted in these studies, CuZnSOD appears to participate in the regulation of zinc homeostasis inside the cells, and zinc overload can impose strains on certain cellular processes.
Idiopathic pulmonary fibrosis (IPF) may present comorbid obstructive lung diseases with small airway dysfunction (SAD). Existing guidelines suggest that inhaled bronchodilators should be used if the ...ratio of forced expiratory volume in the 1st second and forced vital capacity (FEV
/FVC) < 0.7 in IPF. However, most IPF patients have FEV
/FVC > 0.7 even with coexisting emphysema. We retrospectively enrolled IPF patients who were registered at our outpatient clinic. At baseline, 63 patients completed computed tomography (CT) scans, lung function measurements, and symptom questionnaires. Among these patients, 54 (85.71%) underwent antifibrotic treatment and 38 (60.32%) underwent long-acting bronchodilator treatment. The median FEV
/FVC was 0.86. Not all patients treated with bronchodilators showed significant changes in lung function. IPF patients with SAD, determined by IOS parameters, showed significant improvement in FEV
, FEF
, and symptom scores after bronchodilator treatment. Bronchodilator efficacy was not observed in patients without SAD. CT-confirmed emphysema was seen in 34.92% of patients. There were no changes in lung function or symptom scores after bronchodilator treatment in patients with emphysema. In conclusion, FEV
/FVC cannot reflect the airflow limitation in IPF. Emphysema in IPF is not a deciding factor in whether patients should receive bronchodilator treatment. IOS parameters may be useful to guide bronchodilator therapy in patients with IPF coexisting with SAD.
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