The large GTPase dynamin regulates actin comet formation and movement in living cells.

The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2DeltaPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility.     

Changes of gut barrier function during anesthesia and cardiac surgery

The aim of this clinical study was to investigate the time sequence between intraoperative and postoperative endotoxemia, changes in intramucosal pH(I), mediator release, and acute phase proteins and their relationship to postoperative infections. In 60 patients (median age 61 [33-72] years, male/female: 50/10) plasma levels of endotoxin, endotoxin neutralizing capacity (ENC), leukotriene-C4 (LTC4), 6-ketoprostaglandin-F-1alpha (PGF), thromboxane-B2 (TxB2), interleukin-6 (IL-6), and C-reactive protein (CRP) were measured before, during, and after cardiac surgery. The intraluminal pH(I) of the stomach was assessed as a marker of splanchnic blood circulation. Patients were divided in one group with postoperative infections (group A, n = 8) and another groups without infections (group B, n = 52). Among all measured parameters, endotoxin plasma levels showed the most rapid changes. A significant increase of endotoxin plasma levels and a decrease in ENC appeared after the induction of anesthesia, culminating in a peak after reperfusion. Endotoxin showed a significantly higher increase in group A (14fold) compared to group B (sixfold, p<0.001), whereas ENC decreased by eightfold in both groups. The parameters of the arachidonic cascade increased and pH(I) decreased, however, there were no significant differences between both groups. The latest increase was observed for the acute phase proteins IL-6 and CRP. IL-6 levels peaked 6 hours postoperatively with a 20fold (group B) and 30fold (group A) increase (p < 0.001 vs baseline; no differences between groups), whereas CRP rose at the first postoperative day with a 21 fold (group B) and 25fold (group A) increase at day 2 (p<0.001 vs baseline, no difference between groups). Differences between both groups appeared at the second postoperative day for IL-6 (median values group A/B: 421/219 pg/mL; p <0.05) and at the fifth postoperative day for CRP (median values group A/B: 321/81 mg/L; p < 0.05). In conclusion, endotoxin seems to be the earliest trigger of the mediator cascade in acute phase response and may indicate infections in the postoperative course.     

RSD1235 blocks late INa and suppresses early afterdepolarizations and torsades de pointes induced by class III agents

OBJECTIVE: RSD1235 is a novel antiarrhythmic drug with atria-selective electrophysiological actions on Na(+) and K(+) currents. The mechanism for its protection of ventricular repolarization was assessed by its action on Purkinje fibers, and by block of late sodium current active during repolarization. Further, RSD1235's ability to reverse the pro-arrhythmic actions of the class III agents dofetilide and clofilium was assessed in isolated Purkinje fibers and an in vivo model of torsades de pointes (TdP). METHODS: Action potential and early after-depolarization (EAD) recordings were made from in situ and isolated rabbit Purkinje fibers at 37 degrees C using floating sharp microelectrodes; late I(Na) was recorded using a whole-cell patch clamp technique of Nav1.5 expressed in HEK cells at 22 degrees C; In vivo, anesthetized methoxamine-sensitized rabbits were used to test the ability of RSD1235 to suppress clofilium-induced TdP. RESULTS: RSD1235 (0.5-30 microM) had minor dose-dependent effects on action potential duration (APD) at 50% and 90% repolarization in Purkinje fibers, but pre-treatment significantly attenuated the APD-prolonging effects of dofetilide (300 nM). EADs induced by 300 nM dofetilide were terminated by 30 microM RSD1235 in all experiments (n=7). RSD1235 blocked a late component of Na current (I(Na)), which can produce inward currents contributing to EAD formation. RSD1235 pre-treatment (1 micromol/kg/min) or acute infusions prevented/terminated TdP induced by clofilium in 8 of 9 rabbits, and reduced the duration of TdP episodes from 71 +/- 23 s in control to 17 +/- 7 and 14 +/- 14 s at infusion rates of 0.3 and 1.0 micromol/kg/min, respectively (n = 9, p < 0.001). CONCLUSION: RSD1235 itself has minor actions on repolarization in Purkinje fibers, but can reverse the AP-prolonging actions of class III agents and terminate arrhythmias in a model of TdP. We suggest that these protective actions of RSD1235 may result, at least in part, from its ability to inhibit late I(Na) during action potential repolarization.     

Sinonasal tumors and inflammatory tissues: differentiation with MR imaging

The magnetic resonance (MR) and computed tomographic (CT) images of 53 patients with sinonasal tumors were analyzed and compared for accuracy in tumor mapping. the findings were confirmed by means of either surgery or biopsy. The MR images of 60 patients with inflammatory disease were also studied, and the findings were confirmed at surgery. Forty-seven additional MR images were also examined of patients with tumors showing histologic characteristics similar to those found in the sinonasal tumor group but occurring elsewhere in the head and neck, excluding the orbit. This study concludes that nearly 95% of sinonasal tumors have an intermediate T2 signal, while only 5% have bright T2 signals. This small latter group is composed almost exclusively of some minor salivary gland tumors and some neuromas. The inflammatory tissues all had bright T2 signals. The distinction between sinonasal tumors and inflammatory tissues was best accomplished with T2-weighted studies, and MR imaging was more accurate than CT.     

Association between the activation of macrophages, changes of iron metabolism and the degree of anaemia in patients with malignant disorders.

In this study, we investigated a possible association between the degree of macrophage activation - as measured by serum neopterin concentrations - and disturbances of iron metabolism, determined by the concentrations of ferritin and serum iron, in patients with malignant disorders. Additionally we evaluated correlations between these factors and the degree and type of anaemia. Seventy-three patients, who suffered from non-Hodgkin's lymphoma (NHL) (n = 43), Hodgkin's disease (n = 11), myeloma or monoclonal gammopathy of unknown significance (n = 9), myelodysplastic syndrome (n = 1), and solid tumours (n = 9), were examined. Mean neopterin levels were raised in all groups, patients with NHL showing the highest concentrations. Ferritin but not neopterin concentrations were higher in males than in females. A significant correlation was found between neopterin and ferritin concentrations (p less than 0.01). Considering only female patients the strength of the correlation was the same (p less than 0.02). In addition, we found inverse correlations of neopterin with haemoglobin and iron concentrations (all p less than 0.01). Similar relationships existed in patients during follow-up. Our results support the hypothesis of an association between the degree of activation of macrophages and the development of anaemia by a shift or iron towards the storage sites.     

Molecular basis for the lack of T cell proliferation induced by an altered peptide ligand

In this report, we explore the mechanisms underlying cell cycle progression in T cells stimulated with an altered peptide ligand (APL) versus wild-type peptide. APL stimulation did not induce proliferation compared to wild-type peptide stimulation. To determine the point at which cell cycle progression is blocked, we have examined molecules responsible for regulating the retinoblastoma tumor suppressor gene product, pRb, which in its active state prevents G1/S progression. The majority of cells stimulated with an APL did not progress beyond G1; however, a small population did make the G1/S transition. These few cells passed the late G1 restriction point, divided and subsequently arrested at the next G1 phase. The lack of sustained signaling events following stimulation with an APL failed to induce cyclin E:cdk2 activity, a regulator which hyper-phosphorylates and inactivates pRb. Exogenous IL-2 addition did not compensate for the lack of proliferation following APL stimulation. Furthermore, the inability of the cells to enter S phase during partial T cell activation cannot be accounted for by p27Kip1 inhibition of cyclin E:cdk2 complexes. Upon APL stimulation, an increase in association of p27Kip1 with cyclin E:cdk2 complex was not observed, suggesting that instead, decreased cyclin E:cdk complex formation might contribute to the failure to progress from G1/S. Therefore, while for a majority of cells, wild-type stimulation results in cell cycle progression, APL stimulation is not sufficient to drive cells beyond G1.