A technical triade for proteomic identification and characterization of cancer biomarkers.
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Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis. (+info)
Characterization of Fxr1 in Danio rerio; a simple vertebrate model to study costamere development.
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The X-linked FMR1 gene, which is involved in the fragile X syndrome, forms a small gene family with its two autosomal homologs, FXR1 and FXR2. Mouse models for the FXR genes have been generated and proved to be valuable in elucidating the function of these genes, particularly in adult mice. Unfortunately, Fxr1 knockout mice die shortly after birth, necessitating an animal model that allows the study of the role of Fxr1p, the gene product of Fxr1, in early embryonic development. For gene function studies during early embryonic development the use of zebrafish as a model organism is highly advantageous. In this paper the suitability of the zebrafish as a model organism to study Fxr1p function during early development is explored. As a first step, we present here the initial characterization of Fxr1p in zebrafish. Fxr1p is present in all the cells from zebrafish embryos from the 2/4-cell stage onward; however, during late development a more tissue-specific distribution is found, with the highest expression in developing muscle. In adult zebrafish, Fxr1p is localized at the myoseptum and in costamere-like granules in skeletal muscle. In the testis, Fxr1p is localized in immature spermatogenic cells and in brain tissue Fxr1p displays a predominantly nuclear staining in neurons throughout the brain. Finally, the different tissue-specific isoforms of Fxr1p are characterized. Since the functional domains and the expression pattern of Fxr1p in zebrafish are comparable to those in higher vertebrates such as mouse and human, we conclude that the zebrafish is a highly suitable model for functional studies of Fxr1p. (+info)
Thin is better!: ultrathin cryosection immunocytochemistry.
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In immunofluorescence microscopy (IFM), the repression of out of focus fluorescence signal is crucial in order to obtain high-resolution images. One option to acquire high vertical resolution (z-axis resolution) is to produce optical sections with a confocal microscope. The z-axis resolution usually obtained with confocal microscopy of biological samples is about 500 nm. Another option is to produce very thin sections with a cryo-ultramicrotome (physical sections). The ultrathin cryosections we employ are about 100 nm in thickness: thus all of the fluorescence must come from within this 100 nm thickness. The use of ultrathin cryosections permits the acquisition of extremely high-quality images and minimizes the possibility for false localization in IFM (Fig. 1). Ultrathin cryosections can be applied to immunoelectron microscopy (IEM) as well as IFM (Fig. 2). We show new methods of ultrathin cryosection immunocytochemistry(1-3). Human full-term placentas were fixed with 4% paraformaldehyde, solidified with 10% gelatin, infiltrated with 2.3 M sucrose, and then frozen in liquid nitrogen. Ultrathin cryosections were cut with a cryo-ultramicrotome and then transferred to glass cover slips for IFM or to nickel grids for IEM. Cryosections were incubated with mouse anti-p230, a trans-Golgi network marker, and subsequently incubated with Alexa 488-labeled goat anti-mouse IgG or with goat anti-mouse 5-nm colloidal gold particles. For visualization and preservation of ultrastructure of cryosections at the electron microscopic level, the sections on grids were postfixed with ferrocyanide-reduced osmium and then stained with uranyl acetate and lead citrate in polyvinyl alcohol(1). Ultrathin cryosection immunocytochemistry should be an important technique for functional genomics research, especially for the analysis of the in situ expression of target molecules(2,3). (+info)
Antimicrobial action of carvacrol at different stages of dual-species biofilm development by Staphylococcus aureus and Salmonella enterica serovar Typhimurium.
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The effects of carvacrol, a natural biocide, on dual-species biofilms formed by Staphylococcus aureus and Salmonella enterica serovar Typhimurium were investigated with a constant-depth film fermentor. Biofilm development reached a quasi-steady state in 12 days at 25 degrees C with S. aureus predominance ( approximately 99%). Cryosectional analysis detected viable S. aureus and S. enterica serovar Typhimurium at depths of 320 and 180 mum from the film surface, respectively. Carvacrol pulses (1.0 mmol/h) inhibited S. aureus by 2.5 log CFU/biofilm during the early stages of film formation, ultimately causing a significant reduction (P < 0.001) of the staphylococcal population at quasi-steady state. Initial carvacrol pulsing elicited a 3 log CFU/biofilm reduction in viable S. enterica serovar Typhimurium, and additional periodic carvacrol pulses instigated significant inhibition of salmonellae (1 to 2 log CFU/biofilm) during biofilm development. Carvacrol pulsing reduced protein levels fivefold (P < 0.001) during initial biofilm development. Comparative studies with a peroxide-based commercial sanitizer (Spor-Klenz RTU) revealed that this commercial sanitizer was more biocidal than carvacrol during early biofilm development. When the biofilm reached quasi-steady state, however, periodic pulses with 1 mmol of carvacrol per h (P = 0.021) elicited a significantly higher inhibition than Spor-Klenz RTU (P = 0.772). Dual-species microcolonies formed under the influence of continuously fed low carvacrol concentrations (1.0 mmol/h) but failed to develop into a mature quasi-steady-state biofilm and did not reach any stage of film formation in the presence of high concentrations (5.0 mmol/h). These data show that carvacrol is an effective natural intervention to control dual-species biofilm formation. (+info)
The inhibitor of differentiation or inhibitor of DNA binding (Id) family are members of the helix-loop-helix (HLH) group of transcription factors that play important roles in cell proliferation, differentiation, cell cycle control, and apoptosis. They modulate the formation of active class A-class B basic HLH (bHLH) complexes. Ids lack the amino-terminal associated basic region necessary for DNA binding, thus sequestering the class A factors, inhibiting the formation of active class A-class B heterodimers and, therefore, are considered to act as dominant-negative regulators of differentiation pathways. We isolated zebrafish id2, and its expression during development was characterized. id2, in addition to regions of expression detected in Xenopus and mice, is also expressed in the tegmentum; midbrain-hindbrain boundary; cerebellum; rhombomeres 2,3,4,6; notochord; and corpuscles of Stannius. Furthermore, we show that expression of id2 is repressed in mind bomb mutants, suggesting a role of Notch upstream of Id2. (+info)
Frozen sectioning of the pancreatic cut surface during resection of intraductal papillary mucinous neoplasms of the pancreas is useful and reliable: a prospective evaluation.
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OBJECTIVE: To prospectively evaluate the accuracy of frozen sectioning (FS) of the pancreatic transection margin and its influence on surgery during resection of intraductal papillary and mucinous neoplasms (IPMNs). SUMMARY BACKGROUND DATA: Preoperative assessment of IPMN extension is difficult and transection margin is frequently tumoral on the surgical specimen. PATIENTS AND METHODS: FS was performed in 127 patients who underwent partial pancreatectomy for IPMN from 1996 to 2004, corresponding to 90 pancreaticoduodenectomies (1-4 successive FS; total = 132), 25 distal pancreatectomies (1-2 FS; total = 27), and 12 medial pancreatectomies (2-4 FS; total = 29). Dysplasia was graded in both main (MD) and branch ducts (BD), and pancreatectomy was extended if FS revealed at least IPMN adenoma on the MD or borderline IPMN on BD (defined as "significant" lesions). RESULTS: The 188 FS revealed that MD and BD epithelium comprised significant noninvasive lesions in 49 and 13 cases, respectively, and infiltrating carcinoma in 4 other ones. Definitive examination corroborated FS in 176 of 188 cases (94%). Altogether, 54 of 188 (29%) FS comprised significant lesions that resulted in 46 additional resections in 38 patients (30%). Eight patients did not have additional resection because of either high operative risk or preoperative diagnosis of noncurable infiltrating carcinoma. The 134 FS without significant lesions were associated with 7 additional resections mainly because of macroscopic suspicion of another tumor location. Conflicting results between FS and definitive examination resulted in inadequate extent of pancreatectomy in 4 patients (3%). CONCLUSIONS: Results of FS of the transection margin are confirmed by definitive examination in 94% of cases. According to our protocol, FS changes the extent of resection in 30% of patients and allows adequate resection in 97% of patients. (+info)
Establishment of cardiac cytoarchitecture in the developing mouse heart.
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Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level. (+info)
In vitro decapsidation of turnip yellow mosaic virus investigated by cryo-electron microscopy: a model for the decapsidation of a small isometric virus.
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The in vitro decapsidation of a small isometric plant virus, turnip yellow mosaic virus (TYMV), was investigated by cryo-electron microscopy. Cryo-electron micrographs of TYMV and empty shells show that rapidly frozen virions still contain their RNA. Images of vitrified virions resemble closely those previously obtained by negative staining. Rapidly frozen virions decapsidate upon thawing although they remain well dispersed on the grid. The escape of the RNA through a hole at the periphery of the capsid could be visualized. The results suggest a model for the in situ decapsidation of small icosahedral viruses. (+info)