Intraoperative saline injection leak test--a simple method to assess mitral valve repair when a simultaneous aortotomy does not allow pressurization of the left ventricle

We describe a simple, safe and reliable intraoperative saline injection leak test for accomplishing and testing the efficacy of mitral valve repair when a simultaneous aortotomy is present.     

HSP90 cross-links branched actin filaments induced by N-WASP and the Arp2/3 complex

N-WASP induces filopodial actin cytoskeleton through activation of the Arp2/3 complex. Here, we show that heat shock protein 90 (HSP90) regulates the structure of actin filaments induced by N-WASP and the Arp2/3 complex. HSP90 binds to N-WASP and to F-actin and bundles actin filaments. Bundling activity of HSP90 does not affect actin filament nucleation induced by N-WASP and the Arp2/3 complex. HSP90 is co-localized with N-WASP at branching points of actin filaments produced by the Arp2/3 complex and thereby bundles branched filaments; this bundled actin structure is inhibited by blocking direct binding between HSP90 and N-WASP. Furthermore, HSP90 converts branched actin filaments on N-WASP-coated beads to filopodia-like star-shaped bundles. These findings indicate that HSP90 promotes the formation of N-WASP/Arp2/3 complex-induced unbranched filopodial actin structures.     

Crystal structure of human DAAM1 formin homology 2 domain

Reorganization of the actin filament is an essential process for cell motility, cell-cell attachment and intracellular transport. Formin proteins promote nucleation and elongation of the actin filament, and thus are key regulators for this process. The formin homology 2 (FH2) domain forms a head-to-tail ring-shaped dimer, and processively moves towards the barbed end. Dishevelled-associated activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. Here, we present the crystal structure of human DAAM1 FH2 dimer at 2.8 A resolution. This is the first dimeric structure of the mammalian formin. The core structure of human DAAM1 is similar to those of mouse mDia1 and yeast Bni1p, whereas the orientations of the FH2 dimeric rings are different between human DAAM1 and yeast Bni1p, despite their similar dimer interactions. This difference supports the previous prediction that the dimer architecture of the formin is highly flexible in the actin-free state. The results of the actin assembly assays using the DAAM1 mutants demonstrated that the length of the linker connecting the N-terminal domain and the core region is crucial for the activity.     

Stimulation of prostaglandin E2 production and induction of specific protein synthesis in rat peritoneal macrophages by a tumor promoter staurosporine

Staurosporine is a microbial anti-fungal alkaloid having potent inhibitory activity on protein kinase C and is a non 12-O-tetradecanoylphorbol-13-acetate-type tumor promoter in two-stage carcinogenesis experiments in mouse skin. Effects of staurosporine and its structurally related compounds K-252a, KT5720 and KT5822 on prostaglandin E₂ production, release of arachidonic acid from membrane phospholipids, and uptake of [³⁵S]methionine into intracellular proteins were examined in rat peritoneal macrophages. Among the four compounds, only staurosporine stimulated the production of prostaglandin E₂ and release of arachidonic acid at concentrations of 1 ng/ml and 10 ng/ml. The uptake of [³⁵S]methionine into cellular proteins, estimated to be 120 kDa and 125 kDa molecular mass, was also stimulated by staurosporine treatment, and the uptake was increased in parallel with the increase in prostaglandin E₂ production. At higher concentrations (100 ng/ml and 1000 ng/ml), staurosporine inhibited prostaglandin E₂ production and did not induce the specific protein synthesis. Other compounds neither stimulated prostaglandin E₂ production nor induced specific protein synthesis. K-252a inhibited prostaglandin E₂ production at concentrations above 10 ng/ml. These results suggest that the staurosporine-induced proteins might participate in the tumor promotion or at least in the staurosporine-induced stimulation of prostaglandin E₂ production.Abbreviation TPA 12-O-tetradecanoylphorbol tetradecanoylphorbol-13-acetate 13-acetate     

Enhancement of Rho/Rho-kinase system in regulation of vascular smooth muscle contraction in tachycardia-induced heart failure

OBJECTIVE: The Rho/Rho-kinase system regulates Ca(2+) sensitivity in vascular smooth muscle. A new drug, Y-27632, specifically inhibits Rho-kinase and hence decreases the phosphorylation of myosin light chain, thus reducing contraction. Here, we compare the effects of Y-27632 and nifedipine on the vasoconstrictor response of the femoral artery in heart failure. METHODS: Heart failure (HF) was produced by chronic rapid RV pacing (250 bpm, 28 days, six dogs). Indo1-AM was loaded into endothelium-denuded femoral artery segments for measuring intracellular [Ca(2+)]. Tension and changes in intracellular [Ca(2+)] [the change in the ratio (418 nm/468 nm) of Indo1 fluorescence (F(ratio))] were simultaneously measured in Krebs-Ringer solution. RESULTS: In HF: (i) norepinephrine (10 microM) produced greater tension (784+/-52 g/cm(2)) than in control (502+/-64 g/cm(2)) despite a similar increase in F(ratio), indicating increased Ca(2+) sensitivity in vascular smooth muscle; (ii) nifedipine attenuated this enhanced response by only a maximum of 27% at 1 micromol/l with a 56% reduction in F(ratio); (iii) Y-27632 attenuated it by a maximum of 80% at 100 micromol/l without a significant change in F(ratio); (iv) RhoA protein and mRNA expression levels in the femoral artery were up-regulated by +110% and +56%, respectively, while those of Rho-kinase were unchanged. CONCLUSIONS: The Ca(2+)-sensitizing mechanism involving the Rho/Rho-kinase system may be deeply involved in the enhanced arterial vasoconstriction seen in HF. Since Y-27632 attenuated this response in small arteries, it shows potential as a novel, potent vasodilator for the treatment of HF.     

Influence of Helicobacter pylori infection and cetraxate on gastric mucosal blood flow during healing of endoscopic mucosal resection-induced ulcers

BACKGROUND AND AIM: Helicobacter pylori (H. pylori) infection is known to affect the gastric microcirculation, and cetraxate is reported to accelerate gastric ulcer healing, possibly by augmenting gastric mucosal blood flow (MBF). The aim of this study is to clarify the effect of H. pylori infection and cetraxate on MBF during gastric ulcer healing. METHODS: Forty-two patients who had undergone endoscopic mucosal resection (EMR) were studied. Mucosal blood flow was measured by the use of a laser Doppler flowmeter in the surrounding mucosa and at the ulcer margin, before, 1 day, 1 week and 4 weeks after EMR. Helicobacter pylori infection was confirmed by the use of bacterial culture and histology. After EMR, patients were randomly assigned to receive 30 mg lansoprazole (u.i.d; L-regimen) or 30 mg lansoprazole (u.i.d.) with 200 mg cetraxate (q.i.d; LC-regimen) for 4 weeks. RESULTS: The MBF ratio (MBF at ulcer margin/MBF in surrounding mucosa) 1 week after EMR was significantly lower than that before or 4 weeks after EMR only in H. pylori-positive patients treated with the L-regimen. No such decrease in MBF was observed after 1 week in H. pylori-positive patients treated with the LC-regimen or in H. pylori-negative patients. CONCLUSION: A transient decrease in MBF was detected at the ulcer margin during healing of EMR-induced ulcers in H. pylori-infected patients. Cetraxate seemed to prevent this decrease in MBF.     

Aeromonas hydrophila septicemia with necrotizing myofascitis in a patient with hypoplastic leukemia

A 54-year-old male was admitted to Kawasaki Medical School Hospital with the complaint of fever. His diagnosis of hypoplastic leukemia had been made one year ago. After the admission, cecal mass with pain and high fever were noted. Four days later, he suddenly lost consciousness and died. Aeromonas hydrophila was isolated from blood cultures and also from the myofascitis specimen. Autopsy specimen of the iliopsoas muscle showed necrotizing myofascitis. The specimen obtained from the cecum showed submucosal hemorrhage with edema and these findings were compatible to ischemic colitis. This pathogen is widely distributed in nature, especially in water fields. Therefore, it would be advised to consider the Aeromonas hydrophila as one of the pathological organisms pathognomonic for the septicemia, when one may see febrile and gastrointestinal symptoms in a patient with hematological malignancies.     

Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP

All WASP family proteins share a common C terminus that consists of the verprolin homology domain (V), cofilin homology domain (C), and acidic region (A), through which they activate Arp2/3 complex-induced actin polymerization. In this study, we characterized the Arp2/3 complex-mediated actin polymerization activity of VCA fragments of all of the WASP family proteins: WASP, N-WASP, WAVE1, WAVE2, and WAVE3. All of the VCA fragments stimulated the nucleating activity of Arp2/3 complex. Among them, N-WASP VCA, which possesses two tandem V motifs, had a more potent activity than other VCA proteins. The chimeric protein experiments revealed that the V motif was more important to the activation potency than the CA region; two V motifs were required for full activity of N-WASP. COS7 cells overexpressing N-WASP form microspikes in response to epidermal growth factor. However, when a chimeric protein in which the VCA region of N-WASP is replaced with WAVE1 VCA was overexpressed, microspike formation was suppressed. Interestingly, when the N-WASP VCA region was replaced with WAVE1 VCA, having two V motifs, this chimeric protein could induce microspike formation. These results indicate that strong activation of Arp2/3 complex by N-WASP is mainly caused by its two tandem V motifs, which are essential for actin microspike formation.