TheDictyostelium discoideum 30,000 dalton protein contributes to phagocytosis

Summary TheDictyostelium discoideum 30 kDa actin-bundling protein cross-links actin filaments into bundles in vitro, and is present in filopodia and pseudopodia in living cells. Monoclonal antibodies reactive with this protein have been isolated, and employed as specific probes for the function of this protein. The monoclonal antibody B2C blocks the interaction of the 30 kDa protein with F-actin in vitro, and decreases phagocytosis ofE. coli when introduced into livingDictyostelium cells by controlled sonication. Use of this monoclonal antibody for visualization of the 30 kDa protein by immunofluorescence microscopy reveals striking localization around food particles during the process of phagocytosis. Double staining with rhodamine-labelled phalloidin and the monoclonal antibody documents the co-localization of the 30 kDa protein and actin during formation of phagocytic cups. The dissociation of the 30 kDa protein occurs during the process of maturation to form phagolysosomes. These results support the hypothesis that this actin cross-linking protein participates in dynamic rearrangements of actin filaments accompanying phagocytosis.Abbreviations ATP adenosine 5-triphosphate - DEAE diethyl aminoethyl - EDTA (ethylenedinitrilo)-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - PAGE polyacrylamide gel electrophoresis - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - SDS sodium dodecyl sulfate - Sorensen's buffer 2mM Na₂HPO₄+15mM KH₂PO₄, pH6.1 - Tris tris-(hydroxymethyl) aminomethane     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Inhibition of interferon secretion by vinblastine

The plant alkaloids vinblastine and colchicine are known to arrest cells in mitosis by virtue of their binding to spindle protein. These drugs are also capable of binding to microtubule protein and causing these structures to disaggregate into nonfunctional subunits (1, 2). Microtubular structures are thought to be involved in the secretory process of a number of proteins including insulin (7), collagen (4), and thyroid hormone (12). In this report we present our findings on the effects of these two drugs on the synthesis and secretion of interferon in a high producing human foreskin fibroblast strain (FS-4) (11).     

In vitro collagen fibril assembly in glycerol solution: evidence for a helical cooperative mechanism involving microfibrils

Glycerol inhibits the in vitro self-association of monomeric collagen into fibrils and induces the dissociation of fibrils preassembled from NaBH4-reduced collagen. These effects were investigated in an effort to understand the mechanism of fibril assembly of the protein. In PS buffer (0.03 M NaPi and 0.1 M NaCl, pH 7.0) containing 0.1-1.0 M glycerol, the self-association of type I collagen from calf skin took place only if the protein concentration was above a critical value. This critical protein concentration increased with increasing glycerol concentration. Velocity sedimentation studies showed that below the critical protein concentration and under fibril assembly conditions, the collagen was predominantly in a monomeric state. Electron microscopic examinations revealed that the collagen aggregates formed above the critical concentration consisted mostly of microfibrils of 3-5-nm diameter along with some banded fibrils were found. Collagen treated with pepsin to remove its nonhelical telopeptides also self-associated into microfibrils and fibrils in the presence of glycerol, but the reaction did not exhibit any critical concentration. These results are consistent with a mechanism of in vitro collagen fibril assembly which involves the initial formation of microfibrils through a helical cooperative mechanism. They also suggest that contacts of the nonhelical telopeptides of each collagen with its neighboring molecules provide the necessary negative free energy change for the cooperativity and that subsequent lateral association of the microfibrils leads to banded fibrils.     

Prestress as an optimal biomechanical parameter for needle penetration

Drug delivery requires precise intradermal and subcutaneous injections of formulations to clinically relevant penetration depths. However, penetration depth is confounded by skin deflection, which occurs prior to and during penetration as the skin surface deforms axially with the needle, and which varies profoundly due to differing intrinsic mechanical (e.g. viscoelastic) tissue properties, disease state, aging, and ethnicity. Herein, an ex vivo model was utilized to study factors that affect skin deflection and the efficacy of injection, including prestress applied at the tissue surface, needle gauge, velocity, and actuation depth. The application of prestress minimized skin deflection during needle penetration and allowed for needle actuation to the targeted penetration depths with minimum variability. The force required to achieve target penetration depths was found to increase with prestress and decrease with needle gauge. Our findings emphasize the need for prestress applied to the skin surface to minimize variation in skin properties and administer formulations for intradermal and subcutaneous treatments with maximum precision.     

NF-kappaB independent inhibition of lipopolysaccharide-induced cyclooxygenase by a conjugated linoleic acid cognate, conjugated nonadecadienoic acid

10t, 12c-CLA was shown to inhibit COX-2 expression through the NF-kappaB pathway. In the current study, conjugated nonadecadienoic acid (CNA) was shown to decrease inducible COX-2 protein and mRNA and PGE(2) release to the similar extent as 10t, 12c-CLA in Raw264.7 macrophage. However, unlike 10t, 12c-CLA, inhibition of COX-2 mRNA/protein by CNA was independent of the NF-kappaB pathway. The data indicate the regulation of COX-2 by select conjugated fatty acids and hence their anti-inflammatory actions could operate through different signal transduction pathways.     

Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.