In Vitro Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study.

Intermediate filament (IF) elongation proceeds via full-width "mini-filaments", referred to as "unit-length" filaments (ULFs), which instantaneously form by lateral association of extended coiled-coil complexes after assembly is initiated. In a comparatively much slower process, ULFs longitudinally interact end-to-end with other ULFs to form short filaments, which further anneal with ULFs and with each other to increasingly longer filaments. This assembly concept was derived from time-lapse electron and atomic force microscopy data. We previously have quantitatively verified this concept through the generation of time-dependent filament length-profiles and an analytical model that describes assembly kinetics well for about the first ten minutes. In this time frame, filaments are shorter than one persistence length, i.e. ~1 µm, and thus filaments were treated as stiff rods associating via their ends. However, when filaments grow several µm in length over hours, their flexibility becomes a significant factor for the kinetics of the longitudinal annealing process. Incorporating now additional filament length distributions that we have recorded after extended assembly times by total internal reflection fluorescence microscopy (TIRFM), we developed a Monte Carlo simulation procedure that accurately describes the underlying assembly kinetics for large time scales.

The effect of retail packaging method on objective and consumer assessment of beef quality traits.

This study evaluated the effect of 7days of modified atmosphere packaging (MAP: 80% O2, 20% CO2) or skin packaging (no oxygen) of beef M. longissimus steaks after 1 or 7days of ageing in vacuum on objective and sensory meat quality traits and degradation of desmin. Shear force was negatively affected by MAP after both 1 and 7days of ageing in vacuum (P<0.005). Sensory evaluation of grilled steaks revealed significantly negative effects of MAP on sensory traits, resulting in an overall decrease of 8 points in the Meat Standards Australia (MSA) eating quality score (MQ4). Desmin degradation was not affected by packaging method, suggesting that the toughening effect of high-oxygen MAP is not due to inhibition of postmortem proteolysis. The results of this study and others suggest that packaging method should be incorporated as a variable in the MSA grading system. Further research to quantify the impact of oxidative cross-linking of proteins on tenderness appears warranted.

Prognostic assessment of gastrointestinal stromal tumor.

The term "gastrointestinal stromal tumor" (GIST) has been applied to a collection of distinctive mesenchymal tumors occurring within the human gastrointestinal tract. As new drug therapy becomes available, data regarding the natural history of these unusual tumors are necessary to provide selection factors for treatment. Ninety-eight patients had light microscopy compatible with GIST at a single institution from 1989 to 2000. After immunostaining with c-kit and histopathologic review, 69 were judged to be GIST. All prognostic indicators were determined for gastric GIST, intestinal GIST, and all locations combined. The location of the GIST did not have a significant impact on survival. Clinically, tumor size, peritoneal cancer index, and completeness of cytoreduction had a significant impact on prognosis for GIST at all locations. Pathologically, cytologic atypia, necrosis, invasion and number of mitoses were significant prognostic indicators for GIST. Criteria to separate three pathologic groups of GIST according to the tumor size and the mitotic count were useful to evaluate the tumor behavior; in the borderline pathologic group invasion and cytologic atypia were statistically significant prognostic criteria. The cell phenotypes, as determined by immunostains, correlated with the prognosis of gastric GIST but not intestinal GIST. A correlation between the immunostain Ki-67 but not CD-34 or desmin and the prognosis was observed. It is possible to select clinical and pathologic parameters of GIST that impact on prognosis. Invasion and necrosis help to determine the prognosis with borderline tumors. The immunostain Ki-67 correlated with the prognosis and may be helpful to assess prognosis when dealing with small biopsy specimens.

Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tunel technique enhanced with counterstains.

Apoptosis is a distinct form of cell death, induced, for example, by ischaemia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique to detect apoptosis is terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining which labels the ends of DNA strand breaks characteristic of the apoptotic process. However, without the employment of additional staining, TUNEL is only a qualitative procedure that gives no information about the proportion of negative cells nor the cell type undergoing apoptosis. We have utilised propidium iodide (PI) as a counterstain to visualise TUNEL negative nuclei together with anti-desmin antibody in order to assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjected to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraffin. Serial sections (5 microns) were cut, dewaxed and pretreated by incubation with trypsin at 37 degrees C for 30 min. After the employment of the TUNEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in control hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only significantly higher in vascular cells (44+/-5%) and cardiac myocytes (6+/-2%). This simple method therefore allows quantification of apoptosis in myocytic and non-myocytic cells in tissue sections. Use of alternative immunohistochemical markers would permit adaptation of the method to the quantitative assessment of apoptosis in other tissues.

Alveolar soft part sarcoma. Assessment of immunohistochemical demonstration of desmin using paraffin sections and frozen sections.

The many different theories on the histogenesis of alveolar soft part sarcoma (ASPS) have caused great confusion. Owing to the recent rapid advance in immunohistochemical studies, two major hypotheses have been proposed. One group of researchers supports the idea that ASPS shows myogenic differentiation, while the other group opposes the idea. This confrontation is essentially one between a group that believes in the immunohistochemically demonstrated presence of desmin in ASPS and a group that denies it. In the present study we detected desmin in 6 of 10 formalin-fixed paraffin sections (although there were differences due to the use of five commercially available types of anti-desmin antibodies). When acetone-fixed paraffin sections and periodate-lysin-paraformaldehyde (PLP)-fixed frozen sections were used in one and three cases, respectively, they were found to be desmin positive, regardless of the type of antibody. The consistent positivity for all anti-desmin antibodies in the cases treated with acetone or PLP is very suggestive of a myogenous origin of ASPS. It is important to take into consideration the fact that formalin-fixed paraffin sections are not very suitable for immunohistochemical study of desmin.