Septic arthritis of the knee caused by Edwardsiella tarda after a catfish puncture wound.

Anglers are wary of catfish as their sharp fin spines may cause injury and, in some species, envenomation. We describe another complication of catfish spine injury--septic arthritis caused by Edwardsiella tarda. We believe this is the first report of this organism causing septic arthritis.

Ultrastructural study of the epithelium of the normal human vulva.

An ultrastructural study of the pathology of vulvar vestibulitis syndrome (Friedrich, 1987) necessitated a detailed knowledge of the epithelial surface of the vulva. As this has previously been characterised only at the light microscopy level, a comprehensive electron microscopical study of the vulva was considered critical to the study of the pathological tissue. A comparison between vulvar, vaginal and keratinised perineal epithelia, confirmed the resemblance of vulvar epithelium to partially keratinised mucosal-like epithelia. No conclusive evidence of keratinisation was observed and the characteristic granular and spiny cell layers found in skin were absent. The prominent cell type was the glycogenated intermediate cell, which formed a structurally homogeneous layer of cells. Other cell types present were: superficial flattened cells, many having nuclei, traces of organelles and large glycogen deposits; suprabasal cells; and basal cells, some of which appeared mitotically active rich in organelles. All cell layers were characterised by the presence of numerous interdigitating cytoplasmic processes, small numbers of infrequent desmosomal junctions and pale staining cytokeratin filaments.

The imidazole antimycotics econazole and miconazole reduce agonist-evoked protein-tyrosine phosphorylation and evoke membrane depolarisation in human platelets: cautions for their use in studying Ca2+ signalling pathways.

In many cell types depletion of the intracellular Ca2+ stores promotes divalent cation entry across the plasma membrane, although the mechanism of such store-regulated Ca2+ entry remains unclear. It has been suggested that cytochrome P-450 plays a role in the communication from intracellular stores to plasma membrane in human platelets and other cells. These studies involved the use of the imidazole antimycotics, econazole and miconazole, which are inhibitors of cytochrome P-450. Here we report additional effects of the imadazole antimycotics, which we show to inhibit agonist-evoked protein-tyrosine phosphorylation and to evoke plasma membrane depolarisation in human platelets. Both of these effects might be expected to influence agonist-evoked Ca2+ entry in these and other cells. These data suggest that great caution is required in interpreting the results from studies using imadazole antimycotics and in particular imply that the effects of these inhibitors cannot be taken as evidence for a role for cytochrome P-450 in the Ca2+ entry pathway.

The tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, inhibit the release of (3H)arachidonate from human platelets stimulated by thrombin or collagen.

We have investigated the effects of the tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, on [3H]arachidonic acid release from human platelets. Both tyrosine kinase inhibitors blocked, in a dose-dependent manner, the release of arachidonic acid stimulated by thrombin or suspensions of collagen fibres. Blockade by the tyrosine kinase inhibitors occurred early in the arachidonate release time course. Both genistein and methyl 2,5-dihydroxycinnamate also inhibited tyrosine phosphorylation in platelets. The inhibitors were specific in that they did not affect protein kinase C activity, ATP levels or mobilization of Ca2+ from internal stores. These findings suggest a role for tyrosine kinase activity in the regulation of phospholipase A2 in platelets stimulated by the physiological ligands, thrombin and collagen.

Calcium store depletion in dimethyl BAPTA-loaded human platelets increases protein tyrosine phosphorylation in the absence of a rise in cytosolic calcium.

The endomembrane Ca(2+)-ATPase inhibitor, thapsigargin, was used to deplete the intracellular Ca2+ stores of fura-2-loaded human platelets. In control cells, thapsigargin evoked a rise in cytosolic [Ca2+] and a substantial increase in protein tyrosine phosphorylation. Thapsigargin also evoked an increase in tyrosine phosphorylation in cells co-loaded with fura-2 and the Ca2+ chelator dimethyl BAPTA, such that the rise in cytosolic [Ca2+] was abolished. These data support the existence of a tyrosine phosphatase regulated by the Ca2+ content of the intracellular store, a requirement of the putative model for reciprocal control of Ca2+ entry by cytosolic and store [Ca2+] via protein tyrosine phosphorylation.

Calcium signalling in platelets and other nonexcitable cells.

By virtue of their biological simplicity and widespread availability, platelets frequently have been used as a model system to study signal transduction. Such studies have revealed that changes in intracellular free calcium concentration are central to platelet functioning. The following article reviews current concepts of platelet structure and function, with particular emphasis on the mechanisms involved in platelet Ca2+ signalling.

ADP- and thapsigargin-evoked Ca2+ entry and protein-tyrosine phosphorylation are inhibited by the tyrosine kinase inhibitors genistein and methyl-2,5-dihydroxycinnamate in fura-2-loaded human platelets.

We have investigated the mechanism of Ca2+ entry in fura-2-loaded human platelets using the inhibitors of tyrosine kinases, genistein, and methyl-2,5-dihydroxycinnamate. Genistein (100 microM; 30 min) or methyl-2,5-dihydroxycinnamate (1 microgram/ml; 30 min) reduced ADP-evoked protein-tyrosine phosphorylation at specific bands as assessed by gel electrophoresis and Western blotting with a specific antiphosphotyrosine antibody. Both compounds also reduced ADP-evoked [Ca2+]i rises in the presence, but not the absence, of external Ca2+, suggesting a relatively selective inhibition of Ca2+ entry over internal release. The inactive analogue of genistein, daidzein, was without effect on protein-tyrosine phosphorylation or ADP-evoked Ca2+ elevation in the presence or absence of external Ca2+. Methyl-2,5-dihydroxycinnamate (1 microgram/ml; 5 min) significantly reduced the Ca2+ influx evoked by depletion of the intracellular Ca2+ stores using the inhibitor of the endomembranous Ca(2+)-ATPase, thapsigargin. These results with tyrosine kinase inhibitors are unlikely to be the result of the inhibition of other protein kinases since kinases A, C, and G all inhibit agonist-evoked rises in [Ca2+]i in platelets. These data support a role for tyrosine kinases in the control of Ca2+ entry in human platelets.

The tyrosine kinase inhibitors methyl 2,5-dihydroxycinnamate and genistein reduce thrombin-evoked tyrosine phosphorylation and Ca2+ entry in human platelets.

Platelet activation is associated with the phosphorylation of a number of platelet proteins at tyrosine residues. The significance of this is unknown. Here we have investigated the effects of two tyrosine kinase inhibitors, methyl 2,5-dihydroxycinnamate and genistein, on thrombin-evoked protein tyrosine phosphorylation and Ca2+ signal generation in fura-2-loaded human platelets. Both compounds inhibited thrombin-evoked tyrosine phosphorylation and reduced the elevation of [Ca2+]i in the presence, but not the absence, of external Ca2+. This suggested a selective inhibition of thrombin-evoked Ca2+ entry but not release from internal stores. Both compounds also reduced thrombin-evoked Mn2+ entry. In contrast, selective blockade of protein kinase C with Ro 31/8220-002 potentiated the thrombin-evoked Ca2+ signal. These data are compatible with a role for protein tyrosine phosphorylation contributing to thrombin-evoked Ca2+ entry in human platelets.

Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry.

We have previously reported that a component of ADP-evoked Ca2+ entry in human platelets appears to be promoted following the release of Ca2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca2+ stores and plasma membrane Ca2+ permeability in Fura-2-loaded human platelets. Ca2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn2+. It has been suggested that cytochrome P-450 may couple Ca2+ store depletion to an increased plasma membrane Ca2+ permeability. In apparent agreement with this, Mn2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca(2+)-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn2+ influx was only partially inhibited by these compounds at the same concentration (3 microM). Econazole (3 microM) reduced the Mn2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn2+ entry or rises in [Ca2+]i. These data confirm the existence of a Ca2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca2+ content, rather than in promoting Ca2+ influx in the initial stages of platelet Ca2+ signal generation.