Idiopathic linear pustular acantholytic dermatosis in a young Brittany Spaniel dog.

A unilateral, linear, papular-pustular dermatosis is described in a young Brittany Spaniel dog. The dermatosis appeared to follow Blaschko's lines and extended from the left inguinal region to the medial aspect of the left metatarsal area. The predominant histological finding was an eosinophilic and neutrophilic pustular mural folliculitis with prominent acantholysis of infundibular epithelium. There was a rapid and long-lasting (> 15 months) resolution after oral administration of methylprednisolone (1.6 mg kg(-1)).

Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts.

Fibroblast migration involves complex mechanical interactions with the underlying substrate. Although tight substrate contact at focal adhesions has been studied for decades, the role of focal adhesions in force transduction remains unclear. To address this question, we have mapped traction stress generated by fibroblasts expressing green fluorescent protein (GFP)-zyxin. Surprisingly, the overall distribution of focal adhesions only partially resembles the distribution of traction stress. In addition, detailed analysis reveals that the faint, small adhesions near the leading edge transmit strong propulsive tractions, whereas large, bright, mature focal adhesions exert weaker forces. This inverse relationship is unique to the leading edge of motile cells, and is not observed in the trailing edge or in stationary cells. Furthermore, time-lapse analysis indicates that traction forces decrease soon after the appearance of focal adhesions, whereas the size and zyxin concentration increase. As focal adhesions mature, changes in structure, protein content, or phosphorylation may cause the focal adhesion to change its function from the transmission of strong propulsive forces, to a passive anchorage device for maintaining a spread cell morphology.

Enforced polarisation and locomotion of fibroblasts lacking microtubules.

The polarisation and locomotion of fibroblasts requires an intact microtubule cytoskeleton [1]. This has been attributed to an influence of microtubule-mediated signals on actin cytoskeleton dynamics, either through the generation of active Rac to promote protrusion of lamellipodia [2], or through the modulation of substrate adhesion via microtubule targeting events [3] [4]. We show here that the polarizing role of microtubules can be mimicked by externally imposing an asymmetric gradient of contractility by local application of the contractility inhibitor ML-7. Apolar fibroblasts lacking microtubules could be induced to polarize and to move by application of ML-7 by micropipette to one side of the cell and then to the trailing vertices that developed. The release and retraction of trailing adhesions could be correlated with a relaxation of traction on the substrate and a differential shortening of stress-fibre bundles, with their distal tips relaxed. Although retraction and protrusion in these conditions resembled control cell locomotion, the normal turnover of adhesion sites that form behind the protruding cell front was blocked. These findings show that microtubules are dispensable for fibroblast protrusion, but are required for the turnover of substrate adhesions that normally occurs during cell locomotion. We conclude that regional contractility is modulated by the interfacing of microtubule-linked events with focal adhesions and that microtubules determine cell polarity via this route.

The yeast kinesin-related protein Smy1p exerts its effects on the class V myosin Myo2p via a physical interaction.

We have discovered evidence for a physical interaction between a class V myosin, Myo2p, and a kinesin-related protein, Smy1p, in budding yeast. These proteins had previously been linked by genetic and colocalization studies, but we had been unable to determine the nature of their association. We now show by two-hybrid analysis that a 69-amino acid region of the Smy1p tail interacts with the globular portion of the Myo2p tail. Deletion of this myosin-binding region of Smy1p eliminates its ability to colocalize with Myo2p and to overcome the myo2-66 mutant defects, suggesting that the interaction is necessary for these functions. Further insights about the Smy1p-Myo2p interaction have come from studies of a new mutant allele, myo2-2, which causes a loss of Myo2p localization. We report that Smy1p localization is also lost in the myo2-2 mutant, demonstrating that Smy1p localization is dependent on Myo2p. We also found that overexpression of Smy1p partially restores myo2-2p localization in a myosin-binding region-dependent manner. Thus, overexpression of Smy1p can overcome defects in both the head and tail domains of Myo2p (caused by the myo2-66 and myo2-2 alleles, respectively). We propose that Smy1p enhances some aspect of Myo2p function, perhaps delivery or docking of vesicles at the bud tip.

Observations on the use of tetracycline and niacinamide as antipruritic agents in atopic dogs.

Tetracycline and niacinamide were administered in combination to 19 atopic dogs to determine their effectiveness in controlling pruritus. The pruritus was controlled successfully in only one dog. One dog experienced diarrhea that was severe enough to warrant stopping the medication.

A contractile activity that closes phagosomes in macrophages.

Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface.

Efficacy of clindamycin hydrochloride capsules for the treatment of deep pyoderma due to Staphylococcus intermedius infection in dogs.

Clindamycin hydrochloride capsules (11 mg/kg body weight, q24 h) were administered orally to 20 dogs with deep staphylococcal pyoderma. Response to therapy was excellent in 100% of the dogs. Duration of therapy varied from 21 to 91 d, with an average duration of 45 d. Relapses occurred in 25% of the dogs within a 3-month period. One dog vomited when the clindamycin was given on an empty stomach. Under the conditions of the study, clindamycin was an effective, safe, and convenient antibiotic for the treatment of deep staphylococcal pyoderma in dogs.

Observations on the use of cyproheptadine hydrochloride as an antipruritic agent in allergic cats.

Cyproheptadine hydrochloride was administered to 20 presumed or proven allergic cats to determine its efficacy in controlling pruritus. Each cat received 2 mg, orally, every 12 h. The pruritus was satisfactorily controlled in 9 cats. Side effects were seen in 8 cats, and included polyphagia, sedation, vocalization, affectionate behavior, and vomiting.

A permanent human cell line (EA.hy926) preserves the characteristics of endothelin converting enzyme from primary human umbilical vein endothelial cells.

Purification of endothelin converting enzyme (ECE) from endothelial cells has been hindered by the difficulty in obtaining primary endothelial cells in large quantity. We therefore tested transformed human umbilical vein endothelial cells (EA.hy926) for ECE activity. Our data clearly demonstrate that this transformed cell line preserves the ECE properties of the primary cell line. These include: (i) one sharp activity optimum at neutral pH; (ii) characteristics typical of a metalloprotease; (iii) IC50 value for phosphoramidon of 1.8 microM (2.7 microM for HUVEC); (iv) no inhibition by captopril and thiorphan, inhibitors of angiotensin converting enzyme and neutral endopeptidase 24.11. The enzyme showed a substrate specificity for big ET-1:big ET-2:big ET-3 in a ratio of 40:2.5:1. This report presents evidence that a permanent human endothelial cell line, EA.hy926, preserves the ECE activity of HUVEC and is useful for the study of ECE and its regulation of ET-1 production.

The endothelin-converting enzyme from human umbilical vein is a membrane-bound metalloprotease similar to that from bovine aortic endothelial cells.

A phosphoramidon-sensitive, membrane-bound metalloprotease that cleaves big endothelin 1 (big-ET-1) to ET-1 was obtained from human umbilical vein endothelial cells and also from bovine aortic endothelial cells by isolation of plasma-membrane vesicles free of lysosomes. The enzyme was characterized by RIA with an antibody specific for ET-1 and also by reverse-phase HPLC. For both sources, the pH rate profile of the membrane fraction had a very sharp maximum at pH 7.0; little or no activity was seen at more acidic pH values. In contrast, the cytosolic fraction had a major peak at acidic pH values, as well as a broad peak in the neutral region. The activity at pH 7.0 in the membrane fraction was shown by reverse-phase HPLC to produce ET-1 and C-terminal fragment as products. This activity was abolished by phosphoramidon, EDTA, and 1,10-phenanthroline but was not inhibited by pepstatin A, phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, or E-64--consistent with the characteristics of a metalloprotease. These results suggest that this activity is from the physiologically relevant, phosphoramidon-inhibitable, endothelin-converting enzyme. The activity found at neutral pH values in the cytosolic fraction was only partially inhibited by EDTA and 1,10-phenanthroline but was not inhibited by phosphoramidon. The membrane-bound endothelin-converting enzyme from human umbilical vein endothelial cells and bovine aortic endothelial cells showed marked similarities, including IC50 values for phosphoramidon of 2.7 and 1.8 microM and Km values for big-ET-1 of 45.4 and 20.9 microM, respectively. The apparent molecular mass by gel filtration was approximately 300-350 kDa for the enzyme from either source. This report characterizes human endothelin-converting enzyme, which may be an important therapeutic target for cardiovascular disease.

Pharmacologic/pharmacokinetic evaluation of emesis induced by analogs of RSU 1069 and its control by antiemetic agents.

RB 6145, the ring-opened analog of RSU 1069, and PD 130908, the desoxy ring-opened analog of RSU 1069, were compared to RSU 1069 for their emetic potential in dogs. When RB 6145 and PD 130908 were administered intravenously at doses ranging from 20% to 50% of the mouse equivalent maximum tolerated dose (MTD), both analogs were less emetic than RSU 1069 on a molar basis. Furthermore, the 5HT3 antagonist ondansetron prevented emesis at doses as high as 75% of the MTD. The reactivity, and hence the emetic liability of these compounds, is thought to be mediated by formation of the corresponding aziridine intermediate. In mouse plasma, both analogs rapidly converted to two products, the reactive aziridine and a stable oxiazolidinone species formed upon reaction with bicarbonate in the blood. A positive correlation exists between the amounts of aziridine formed by these analogs and their emetic potential.