Microwave histoprocessing of bone marrow trephine biopsies

Summary In recent years, the microwave oven has been increasingly used in the pathology laboratory for processing of tissue for diagnostic purposes with a remarkable reduction in processing time and also reports of excellent morphology and immunohistochemistry. We evaluated some of these processes on post mortem bone marrow trephine biopsies and describe a novel way of processing these biopsies in the microwave oven.     

Ultrarapid endocytotic uptake of large molecules inDunaliella species

Summary This paper describes the uptake of Lucifer Yellow carbohydrazide and fluorescent dextrans labeled with fluorescein isothiocyanate or Sodium Green (molecular masses ranging from 522 to 2 × 10⁶ Da) byDunaliella spp. halotolerant unicellular green algae isolated from salt pools in the Sinai peninsula. The fluorescent dyes were taken up into a set of vesicles around the nucleus and just above the chloroplast. It proved impossible to inhibit uptake of the fluorescent compounds in cells treated with a large variety of metabolic and other inhibitors. Cell labeling was complete within half a minute of addition of fluorescent compounds to the outside medium; efflux was equally rapid. The results are interpreted in terms of an endocytotic process whereby the outside medium, together with any substance dissolved in it, remains within vesicles enclosed within the cell body but cycles rapidly between the plasma membrane and the interior of the cell. The outside medium does not pass across the vesicular membrane, nor enters the cytosol.Abbreviations LYCH Lucifer Yellow carbohydrazide - FITC fluorescein-5-isothiocyanate - TCA trichloroacetic acid - DMSO dimethylsulfoxide - NEM N-ethyl maleimide - DNP dinitrophenol - CCCP m-chlorocarbonyl-cyanide phenylhydrazone - APM amiprophos-methyl     

Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2-5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of approximately 150 micros, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.     

Ultrarapid caspase-3 dependent apoptosis induction by serine/threonine phosphatase inhibitors

The protein phosphatase (PP) inhibitors nodularin and microcystin-LR induced apoptosis with unprecedented rapidity, more than 50% of primary hepatocytes showing extensive surface budding and shrinkage of cytoplasm and nucleoplasm within 2 min. The apoptosis was retarded by the general caspase inhibitor Z-VAD.fmk. To circumvent the inefficient uptake of microcystin and nodularin into nonhepatocytes, toxins were microinjected into 293 cells, Swiss 3T3 fibroblasts, promyelocytic IPC-81 cells, and NRK cells. All cells started to undergo budding typical of apoptosis within 0.5 - 3 min after injection. This was accompanied by cytoplasmic and nuclear shrinkage and externalization of phosphatidylserine. Overexpression of Bcl-2 did not delay apoptosis. Apoptosis induction was slower and Z-VAD.fmk independent in caspase-3 deficient MCF-7 cells. MCF-7 cells stably transfected with caspase-3 showed a more rapid and Z-VAD.fmk dependent apoptotic response to nodularin. Rapid apoptosis induction required inhibition of both PP1 and PP2A, and the apoptosis was preceded by increased phosphorylation of several proteins, including myosin light chain. The protein phosphorylation occurred even in the presence of apoptosis-blocking concentrations of Z-VAD.fmk, indicating that it occurred upstream of caspase activation. It is suggested that phosphatase-inhibiting toxins can induce caspase-3 dependent apoptosis in an ultrarapid manner by altering protein phosphorylation.     

Evidence for a Shared Mechanism in the Formation of Urea-Induced Kinetic and Equilibrium Intermediates of Horse Apomyoglobin from Ultrarapid Mixing Experiments

In this study, the equivalence of the kinetic mechanisms of the formation of urea-induced kinetic folding intermediates and non-native equilibrium states was investigated in apomyoglobin. Despite having similar structural properties, equilibrium and kinetic intermediates accumulate under different conditions and via different mechanisms, and it remains unknown whether their formation involves shared or distinct kinetic mechanisms. To investigate the potential mechanisms of formation, the refolding and unfolding kinetics of horse apomyoglobin were measured by continuous- and stopped-flow fluorescence over a time range from approximately 100 μs to 10 s, along with equilibrium unfolding transitions, as a function of urea concentration at pH 6.0 and 8°C. The formation of a kinetic intermediate was observed over a wider range of urea concentrations (0–2.2 M) than the formation of the native state (0–1.6 M). Additionally, the kinetic intermediate remained populated as the predominant equilibrium state under conditions where the native and unfolded states were unstable (at ~0.7–2 M urea). A continuous shift from the kinetic to the equilibrium intermediate was observed as urea concentrations increased from 0 M to ~2 M, which indicates that these states share a common kinetic folding mechanism. This finding supports the conclusion that these intermediates are equivalent. Our results in turn suggest that the regions of the protein that resist denaturant perturbations form during the earlier stages of folding, which further supports the structural equivalence of transient and equilibrium intermediates. An additional folding intermediate accumulated within ~140 μs of refolding and an unfolding intermediate accumulated in <1 ms of unfolding. Finally, by using quantitative modeling, we showed that a five-state sequential scheme appropriately describes the folding mechanism of horse apomyoglobin.