The effect of Cu, Zn and Fe chelates on the antioxidative status of thigh meat of broiler chickens.

The studies aimed to verify the effect of Cu, Zn and Fe glycine chelate on the antioxidative status in the thigh meat of broiler chickens. The study assumption was that due to the antioxidative or prooxidative effect of Cu, Zn and Fe, these elements supplemented to chickens in an easily assimilable form would modify the antioxidative status of meat and those having a prooxidative effect could deteriorate the quality of meat. The experiment involved three hundred and fifty Ross 308 chickens divided into seven equipotent experimental groups. Over 42 days of the experiment, the chickens were administered Cu, Zn and Fe glycine chelates in an amount corresponding to 50% of the requirement (experimental factor I) or 25% of the requirement (experimental factor II). The level of oxidative stress indicators such as superoxide dismutase, catalase, glutathione, glutathione peroxidase and malondialdehyde was determined in the muscles and blood. The groups receiving Zn or Cu chelate showed statistically confirmed higher activity of superoxide dismutase, catalase, and a higher level of glutathione in comparison to the group receiving Fe chelate. In order to increase the antioxidative stability of thigh meat, it is sufficient that broiler chickens receive Zn or Cu in the form of glycine chelate in an amount covering 25% of their requirement of such minerals. On the other hand, the use of Fe glycine chelates decreased antioxidative stability due to an increase in the level of malondialdehyde, so it should be considered whether the administration of pro-oxidative Fe chelate to broilers is advisable.

Impact of stress urinary incontinence on female sexual activity.

OBJECTIVE: The study aimed to investigate the impact of SUI (Stress Urinary Incontinence) on the sexual activity of women, to assess their sexual functioning, and to show the extent of the problem that SUI poses to the quality of life of women. PATIENTS AND METHODS: The study involved 70 women aged 20-48 years. The inclusion criteria included the presence of stress urinary incontinence, the sexual activity of the women, and the history of no urogynecological intervention. The authorial questionnaire and the Polish version of the Female Sexual Function Index (FSFI) were used. RESULTS: SUI contributes to reducing the frequency of intercourse and even complete resignation from sexual intercourse. There is a correlation between the occurrence of urinary leakage during intercourse and the occurrence of sexual dysfunction (p=0.023). The most common factors limiting sexual activity are decreased libido, fatigue, lack of desire, and lack of body acceptance. However, age (p=0.070), marital status (p=0.091), Body Mass Index (BMI) (p=0.436), as well as the duration of stress urinary incontinence (p=0.36) have no effect on women's sexual activity. The most common ways of dealing with the loss of urine during intercourse include micturition before intercourse, intercourse only in safe places, restriction of physical activity during intercourse, and reduction of intercourse frequency and duration. CONCLUSIONS: SUI in women has a significant effect on their sexual activity. The cause of this state of affairs is multifactorial. Some women try to cope with the problem and have developed a number of strategies that allow them to be sexually active without unpleasant surprises.

Evaluation of the prognostic value of fibrinolytic elements in invasive breast carcinoma patients.

Breast cancer (BrC) is one of the most serious oncological problems in the world. The aim of the study was to evaluate concentrations of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) and their ratios: t-PA/PAI-1 and PAI-1/t-PA in breast cancer patients and in healthy individuals and to estimate the ability of fibrinolytic parameters in predicting neoplasm disease and disease relapse. One hundred and five women were enrolled in the study, including 60 cases with primary BrC, (M0) and 45 healthy females. Follow-up was completed in all BrC patients with a 16.7% recurrence rate. An immunoassay of t-PA, PAI-1 in all cases was made as well as the immunohistochemistry of estrogen and progesterone receptors, human epidermal growth factor receptor 2, E-cadherin, and Ki-67 was performed in BrC subjects. A significantly higher PAI-1 concentration in breast cancer patients below the age of 55 than in controls was obtained. According to the ROC curve analysis, the PAI-1 concentration demonstrates the most accurate prognostic value with the cut-off point at 33.91 ng/ml, with 90% sensitivity and 36% specificity, which discriminates between controls and cancer patients. However, t-PA presents the highest area under the receiver-operating characteristic curves (AUCROC)=0.634 in predicting disease relapse with the cut-off value of 5.3 ng/ml. According to the Kaplan-Meier curves, a high concentration of t-PA (>5 ng/ml) and a lower PAI-1/t-PA ratio (<7.5) are associated with shorter survival. Evaluation of plasma t-PA and PAI-1 concentrations may deliver relevant prognostic information for breast cancer patients.

The clustered Fcgamma receptor II is recruited to Lyn-containing membrane domains and undergoes phosphorylation in a cholesterol-dependent manner.

Phosphorylation of clustered Fcgamma receptor II (FcgammaRII) by Src family tyrosine kinases is the earliest event in the receptor signaling cascade. However, the molecular mechanisms for the interaction between FcgammaRII and these kinases are not elucidated. To asses this problem we isolated high molecular weight complexes of cross-linked FcgammaRII from non-ionic detergent lysates of U937 monocytic cells. CD55, a glycosylphosphatidylinositol-anchored protein, a ganglioside GM1 and Lyn, a Src family tyrosine kinase, were also located in these complexes. Gradient centrifugation demonstrated that the complexes containing cross-linked FcgammaRII displayed a low buoyant density. The FcgammaRII present in the complexes underwent tyrosine phosphorylation. Cross-linked FcgammaRII and Lyn occupied common 100-200 nm detergent-resistant membrane fragments, as demonstrated by immunoprecipitation and microscopy studies. Pretreatment of the cells with beta-cyclodextrin, a cholesterol acceptor, depleted membrane cholesterol and released CD55, GM1 and Lyn from the detergent-resistant complexes. In parallel, the association of Lyn with cross-linked FcgammaRII was disrupted and phosphorylation of the receptor inhibited. Reincorporation of cholesterol evoked the relocation of Lyn into the detergent-resistant membrane fraction and restored both Lyn association with cross-linked FcgammaRII and tyrosine phosphorylation of the receptor. Our data demonstrate that cholesterol-enriched membrane rafts can facilitate tyrosine phosphorylation of clustered FcgammaRII by Lyn kinase.

The role of cholesterol and sphigomyelin in tyrosine phosphorylation of proteins and capping of Fcgamma receptor II.

Cross-linking of cell surface receptors by multivalent ligands, e.g. by antibodies, evokes their clustering -- patching. Subsequently, these clusters can be translocated by the acto-myosin machinery toward one pole of the cell and assembly cap. Patching of FcgammaRII in U937 cells correlates with tyrosine phosphorylation of several proteins while cap assembly correlates with their dephosphorylation. To study the mechanism of activation of tyrosine kinases during FcgammaRII activation we disturbed the organization of the putative plasma membrane microdomains by depletion of membrane cholesterol and sphingomyelin. Cholesterol was removed with the use of beta-cyclodextrin while sphingomyelin was decomposed by exogenous sphingomyelinase. Cyclodextrin at 5-10 mM removed about 70% of cholesterol from the cells and abolished the assembly of FcgammaRII caps thereby arresting the receptors at the patching stage. Similarly, 70 mU/ml sphingomyelinase inhibited cap formation by 60%. Cholesterol and sphingomyelin depletion also suppressed the tyrosine phosphorylation of proteins which accompanied cross-linking of FcgammaRII. The observations indicate that cholesterol and sphingomyelin can control the interactions of tyrosine kinases with clustered FcgammaRII.

Engagement of spectrin and actin in capping of FcgammaRII revealed by studies on permeabilized U937 cells.

Plasma membrane receptors can undergo translocation in the plane of plasma membrane after binding of polyvalent ligands. Ligand/receptor clusters, named patches, can collect into a polar cap, presumably due to their association with the submembrane actin-based cytoskeleton. We found that the assembly of Fcgamma receptor II caps in human monocytic U937 cells was accompanied by the accumulation of spectrin and actin in the cap region. Permeabilization of cells with streptolysin O rendered capping sensitive to inhibition by phalloidin, an actin filament stabilizing agent. A rabbit antibody directed against the chicken erythrocyte alpha-subunit of spectrin, an actin- and membrane-binding protein, also blocked the capping in a dose dependent manner. The inhibition reached approximately 50% after 20 minutes of cell treatment with the antibody. Anti-alpha-spectrin targeted specifically its submembrane antigen, in contrast to unspecific antibodies which remained dispersed in the cell interior and had no influence on the cap assembly. Our results indicate an active engagement of spectrin and actin filaments in the capping of Fcgamma receptor II.

Signaling pathways in phagocytosis.

Phagocytosis is an uptake of large particles governed by the actin-based cytoskeleton. Binding of particles to specific cell surface receptors is the first step of phagocytosis. In higher Eucaryota, the receptors able to mediate phagocytosis are expressed almost exclusively in macrophages, neutrophils, and monocytes, conferring immunodefence properties to these cells. Receptor clustering is thought to occur upon particle binding, that in turn generates a phagocytic signal. Several pathways of phagocytic signal transduction have been identified, including the activation of tyrosine kinases and (or) serine/threonine kinase C in pivotal roles. Kinase activation leads to phosphorylation of the receptors and other proteins, recruited at the sites of phagocytosis. Monomeric GTPases of the Rho and ARF families are likely to be engaged downstream of activated receptors. The GTPases, in cooperation with phosphatidylinositol 4-phosphate 5-kinase and phosphatidylinositol 3-kinase lipid modifying enzymes, can modulate locally the assembly of the submembranous actin filament system leading to particle internalization.

Tyrosine phosphorylation/dephosphorylation controls capping of Fcgamma receptor II in U937 cells.

In the capping of cell-surface receptors two stages can be distinguished: 1) clustering of the receptors (patching) induced by cross-linking with specific antibodies and 2) subsequent assembly of patches into a cap which is driven by the actin-based cytoskeleton. We found that patching of Fcgamma receptor II in U937 cells was correlated with tyrosine phosphorylation of certain proteins, most prominently those of 130, 110, 75 and 28 kDa. The phosphotyrosine-bearing proteins were accumulated at the receptor patches. Formation of the receptor caps was coincident with dephosphorylation of these proteins. Inhibition of protein tyrosine kinases with herbimycin A and genistein attenuated the protein tyrosine hyperphosphorylation and blocked capping in a dose-dependent manner. Phenylarsine oxide and pervanadate, inhibitors of protein tyrosine phosphatases, also suppressed capping of Fcgamma receptor II in a concentration-dependent fashion. Simultaneously, tyrosine hyperphosphorylation of proteins occurred. In the presence of the tyrosine kinase and phosphatase inhibitors the receptors were arrested at the patching stage. In contrast, okadaic acid, a serine/threonine phosphatase blocker, did not affect assembly of the receptor caps. The inhibitory effect of phenylarsine oxide was rapidly reversed by dithiols, 2,3-dimercapto-1-propanoldithiol and dithiotreitol, and was coincident with dephosphorylation of protein tyrosine residues. Extensive washing of pervanadate-exposed cells also resulted in progressive restoration of the cap assembly. Using streptolysin O-permeabilized cells we confirmed regulatory function played by dephosphorylation of tyrosine residues in capping of Fcgamma receptor II. Exogenous phosphatases, applied to permeabilized cells in which activity of endogenous tyrosine phosphatases was blocked, evoked dephosphorylation of protein tyrosine residues that was accompanied by recovery of capping ability in the cells.

[Approach to acute non-inflammatory increase in the circumference of the neck]. / Problem naglego niezapalnego powiekszania sie obwodu szyi.

We describe a case of spontaneous rupture of a left common carotid artery aneurysm during coughing which had lead to the demise of the patient despite a surgical intervention. This case is correlated with three cases of cervical haematomas which also presented with an acute increase in the neck circumference. Based on the literature and our own experience, we discuss the diagnostic and surgical approach to patients suspected of having aneurysms of the great vessels of the neck.

Tyrosine phosphorylation and Fcgamma receptor-mediated phagocytosis.

Phagocytosis of IgG-opsonized particulate material in hematopoietic cells is mediated by Fcgamma receptors (FcgammaRs). Interaction of the receptors with Fc domains of IgG triggers transduction of phagocytic signal in which a key role is played by phosphorylation of tyrosine residues of the receptors. These residues are arranged into a specific motif (immunoreceptor tyrosine-based activation motif; ITAM) which is located either in the cytoplasmic part of FcgammaRIIA or in gamma chains associated with FcgammaRI and FcgammaRIIIA. The conserved tyrosine residues are phosphorylated by, and associate with, tyrosine kinases of Src and Syk families. Coordinated action of these components initiates numerous intracellular events leading finally to local rearrangement of the actin-based cytoskeleton and internalization of the particles.

Syk kinase, tyrosine-phosphorylated proteins and actin filaments accumulate at forming phagosomes during Fcgamma receptor-mediated phagocytosis.

Phagocytosis mediated by Fcgamma receptors (FcgammaRs) is thought to be regulated by a cascade of tyrosine phosphorylation events that finally leads to the rearrangement of submembranous actin-based cytoskeleton and internalization of particles. Suggestions concerning the functional relationship between protein tyrosine kinases, their substrates, and actin filament reorganization prompted us to determine cellular distribution of these elements during uptake of IgG-coated particles in murine thio-macrophages. We found that the onset of uptake of the particles was accompanied by tyrosine phosphorylation of several proteins, among which 90, 50, 40, 30, and 25 kDa polypeptides were distinguished. In most of the proteins the tyrosine hyperphosphorylation persisted up to 3 min of the uptake; however, kinetics of the phosphorylation of individual proteins varied. Immunofluorescence data showed that the phosphotyrosine-bearing proteins were localized in regions of the particle uptake, being concentrated at phagocytic cups and nascent phagosomes. The local enrichment in tyrosine phosphorylated proteins was correlated with accumulation of actin filaments at these early stages of phagosome formation. During phagosome maturation, both tyrosine phosphorylated proteins and microfilaments disappeared from the periphagosomal regions. Syk, one of the tyrosine kinases, was translocated to the regions where FcgammaR-mediated phagocytosis had started. On the contrary, no enrichment in phosphatidylinositol 3-kinase was detected in these places.

Local accumulation of alpha-spectrin-related protein under plasma membrane during capping and phagocytosis in Acanthamoeba.

During capping and phagocytosis the interaction between cluster cell surface receptors and the submembraneous actin-based skeleton may be mediated by spectrin-like proteins. To test this possibility we examined the localization of an alpha-spectrin immunoanalogue, that had been previously identified in whole extracts of Acanthamoeba, during capping of Con A receptors and during phagocytosis of Con A-coated yeast. During capping alpha-spectrin and filamentous actin co-migrated with the Con A receptors and accumulated in the region of cap formation, as demonstrated by double immunofluorescence studies. Immunoelectron microscopy revealed submembraneous location of alpha-spectrin in cells exposed to Con A, both at the time of initial cross-linking and during accumulation of alpha-spectrin in the region of the cap. Phagocytosis studies showed that alpha-spectrin and actin filaments were concentrated around phagocytic cups that enclosed ConA-coated yeast upon internalization. The proteins also surrounded nascent phagosomes present in the vicinity of the plasma membrane but were absent at the later time point of phagosome maturation. These data demonstrate a correlation between clustering of cell surface receptors and submembraneous localization of alpha-spectrin, suggesting an involvement of spectrin-like proteins in mediating the interaction of receptor clusters with the actin cytoskeleton.

beta-Thymosins are not simple actin monomer buffering proteins. Insights from overexpression studies.

beta-Thymosins are the currently favored candidates for maintaining the large actin monomer (G-actin) pool in living cells. To determine if beta-thymosin behaves like a simple G-actin buffering agent in the complex environment of a cell, we overexpressed thymosin beta10 (Tbeta 10) in NIH3T3 cells and determined the effect on the monomer/polymer equilibrium. Tbeta 10 is the predominant beta-thymosin isoform in the NIH3T3 cell line, and it is present in approximately equal molar ratio to profilin and cofilin/actin depolymerizing factor, two other well characterized actin monomer binding proteins. Clonal cell lines that overexpressed three times more Tbeta 10 had 23-33% more polymerized actin than control cells, and the filaments appeared thicker after staining with fluorescent phalloidin. There was no change in total actin, profilin, and cofilin/actin depolymerizing factor content. The overexpressing cells were more motile; they spread faster and had higher chemotactic and wound healing activity. Assuming that there is no compensatory inactivation of the other classes of monomer binding proteins, our paradoxical observation can be accounted for quantitatively by a parallel in vitro study (Carlier, M.-F., Didry, D., Erk, I., Lepault, J., Van Troys, L., Vanderkekove, J., Perelroizen, I., Yin, H. L., Doi, Y., and Pantaloni, D., (1996) J. Biol. Chem. 271, 9231-9239). beta-Thymosin at levels comparable with that found in the overexpressing cells binds actin filaments and decreases the critical concentration (C(c)) for actin polymerization. This reduces the monomer buffering ability of beta-thymosin, so that above a certain threshold an incremental increase in thymosin does not lead to a corresponding increase in G-actin. Furthermore, the decrease in C(c) reduces the buffering capacity of the other actin monomer binding proteins. As a consequence, an increase in beta-thymosin does not necessarily result in a proportionate increase in actin monomer content in a complex environment containing other actin monomer binding proteins. The outcome depends on the level of beta-thymosin expression relative to the composition of the other actin monomer binding protein. Our results suggest that beta-thymosins are not simple actin buffering proteins and that their biphasic action may have physiological significance.

Effects of CapG overexpression on agonist-induced motility and second messenger generation.

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control-transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.

Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells.

Although the actin cytoskeleton has been implicated in vesicle trafficking, docking and fusion, its site of action and relation to the Ca(2+)-mediated activation of the docking and fusion machinery have not been elucidated. In this study, we examined the role of actin filaments in regulated exocytosis by introducing highly specific actin monomer-binding proteins, the beta-thymosins or a gelsolin fragment, into streptolysin O-permeabilized pancreatic acinar cells. These proteins had stimulatory and inhibitory effects. Low concentrations elicited rapid and robust exocytosis with a profile comparable to the initial phase of regulated exocytosis, but without raising [Ca2+], and even when [Ca2+] was clamped at low levels by EGTA. No additional cofactors were required. Direct visualization and quantitation of actin filaments showed that beta-thymosin, like agonists, induced actin depolymerization at the apical membrane where exocytosis occurs. Blocking actin depolymerization by phalloidin or neutralizing beta-thymosin by complexing with exogenous actin prevented exocytosis. These findings show that the cortical actin network acts as a dominant negative clamp which blocks constitutive exocytosis. In addition, actin filaments also have a positive role. High concentrations of the actin depolymerizing proteins inhibited all phases of exocytosis. The inhibition overrides stimulation by agonists and all downstream effectors tested, suggesting that exocytosis cannot occur without a minimal actin cytoskeletal structure.

Actin monomer binding proteins.

Small actin monomer binding proteins are essential components of the actin polymerization machinery. Originally thought of as passive buffers that prevent polymerization of actin monomers, recent discoveries elucidate how some actin monomer binding proteins can promote as well as inhibit polymerization, and how they cooperate to regulate actin assembly.

Actin-binding proteins involved in the capping of epidermal growth factor receptors in A431 cells.

A capping process of epidermal growth factor receptors (EGF-Rs) was used for the study of the relation between the receptors and the actin-binding proteins (spectrin, vinculin, annexin I) that may be involved in EGF-R-cytoskeleton interaction. In intact, adherent A431 cells, EGF-Rs were diffusively distributed on the cell surface. Spectrin, vinculin, and annexin I were located beneath the plasma membrane. An abundance of EGF-Rs as well as submembrane proteins was observed in regions of membrane ruffles and cell-cell contacts. Annexin I was localized also in cytoplasm being attached to filamentous structures surrounding the nucleus and extending to the cell periphery. Under polyvalent ligand treatment, EGF-Rs of adherent cells were aggregated on one side of the cell. Spectrin, vinculin, and annexin I dislocated together with EGF-Rs and were concentrated under plasma membrane at regions where cap formation took place. In suspended A431 cells only spectrin was located under the plasma membrane whereas annexin I and vinculin were diffusively distributed through the cells. During cap formation only spectrin was colocalized with EGF-Rs. The results confirmed the major role of spectrin as a receptor-microfilament linking protein.

The role of microfilaments in the capping of epidermal growth factor receptor in A431 cells.

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.

Participation of membrane skeleton proteins in aggregation of epidermal growth factor receptors in A431 cells.

Polyclonal antibody against alpha-spectrin of chicken erythrocytes was prepared. This antibody as well as anti-vinculin and anti-annexin I and II, were used for localization of the antigens in A431 cells during translocation of epidermal growth factor receptors (EGF-Rs) on cell surface. During aggregation of EGF-Rs only spectrin and actin aggregates colocalized with the "capped" receptors in adherent as well as in suspended cells. Physiological implication of spectrin involvement in EGF-Rs redistribution in A431 cells is discussed.

Alpha-spectrin immunoanalog in Acanthamoeba cells.

A monospecific, affinity purified antibody was prepared against chicken erythrocyte alpha-spectrin. The antibody cross-reacted with only one high molecular weight polypeptide (235 kDa) from whole Acanthamoeba cells. The localization of alpha-spectrin-related antigen in Acanthamoeba cells was examined using immunofluorescence and postembedding cytochemical techniques. Three patterns of distribution of alpha-spectrin immunoanalog were distinguished: as submembranous layer, cytoplasmic aggregates and uniform dispersion through the cytoplasm. Immunoelectron microscopic studies showed that the colloidal gold label was located in the cytoplasm in the vicinity of the plasma membrane. The gold particles were also aggregated around unidentified cytoplasmic filamentous structures. The presence of spectrin-related protein in protozoan cells of Acanthamoeba is in accordance with previous assumptions of the widespread occurrence of spectrin-related proteins. The heterogenous distribution of the immunoanalog of alpha-spectrin protein in Acanthamoeba cells is discussed.