Preparation of monopodal and bipodal aluminum surface species by selective protonolysis of highly reactive [AlH3(NMe2Et)] on silica.

The synthesis and characterization of silica-grafted monopodal and bipodal aluminum hydrides has been achieved starting from 200 °C- and 700 °C-annealed silica and [AlH3(NMe2Et)]. The mechanism by which aluminum trishydride reacts with isolated and vicinal silanols, assisted by the amine, has been investigated computationally at the ωB97XD-DFT level.

KCa3.1 (IK) modulates pancreatic cancer cell migration, invasion and proliferation: anomalous effects on TRAM-34.

In the recent decades, ion channels became the focus of cancer biologists, as many channels are overexpressed in tumour tissue and functionally they are linked to abnormal cell behaviour with processes including apoptosis, chemo- and radioresistance, proliferation and migration. KCa3.1 is a Ca2+-activated K+ channel that plays a central role in tumour progression in many cancer types. Therefore, the aim of the present study was to investigate KCa3.1 expression in pancreatic cancer cells and assess possible implications to disease progression. Using qPCR technique, we found abundant expression of KCa3.1 in pancreatic cancer cell lines. Patch clamp measurements on MiaPaCa-2 cells revealed a Ca2+-activated K+ current that matched biophysical characteristics as described for KCa3.1. Moreover, the current was sensitive to the commonly used channel modulators TRAM-34, clotrimazole and DC-EBIO, and it was abolished following transient gene knockdown of KCa3.1. We utilized both pharmacology and RNAi to assess a possible role of the channel in tumour cell behaviour. We found that the channel supported MiaPaCa-2 cell proliferation. Using RNAi protocols, we also identified KCa3.1 as important entity in cell invasion. However, TRAM-34 had unexpected stimulatory effects on cell migration and invasion estimated in various assays. Moreover, TRAM-34 increased intracellular Ca2+. In conclusion, we found prominent functional expression of KCa3.1 in pancreatic cancer cells. We provide evidence that the channel has a key role in cell proliferation and for the first time identify KCa3.1 as important entity in PDAC cell migration. We further reveal anomalous effects of TRAM-34.

Early assessment of minimal residual disease in AML by flow cytometry during aplasia identifies patients at increased risk of relapse.

In acute myeloid leukemia (AML), assessment of minimal residual disease (MRD) by flow cytometry (flow MRD) after induction and consolidation therapy has been shown to provide independent prognostic information. However, data on the value of earlier flow MRD assessment are lacking. Therefore, the value of flow MRD detection was determined during aplasia in 178 patients achieving complete remission after treatment according to AMLCG (AML Cooperative Group) induction protocols. Flow MRD positivity during aplasia predicted poor outcome (5-year relapse-free survival (RFS) 16% vs 43%, P<0.001) independently from age and cytogenetic risk group (hazard ratio for MRD positivity 1.71; P=0.009). Importantly, the prognosis of patients without detectable MRD was neither impacted by morphological blast count during aplasia nor by MRD status postinduction. Early flow MRD was also evaluated in the context of existing risk factors. Flow MRD was prognostic within the intermediate cytogenetic risk group (5-year RFS 15% vs 37%, P=0.016) as well as for patients with normal karyotype and NPM1 mutations (5-year RFS 13% vs 49%, P=0.02) or FLT3-ITD (3-year RFS rates 9% vs 44%, P=0.016). Early flow MRD assessment can improve current risk stratification approaches by prediction of RFS in AML and might facilitate adaptation of postremission therapy for patients at high risk of relapse.

ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(-) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(-) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

The role of TMEM16A (ANO1) and TMEM16F (ANO6) in cell migration.

Members of the TMEM16 family have recently been described as Ca(2+)-activated Cl(-) channels. They have been implicated in cancer and appear to be associated with poor patient prognosis. Here, we investigate the role of TMEM16 channels in cell migration, which is largely unknown. We focused on TMEM16A and TMEM16F channels that have the highest expression of TMEM16 channels in Ehrlich Lettre ascites (ELA) cells. Due to the lack of specific pharmacological modulators, we employed a miRNA approach and stably knocked down the expression of TMEM16A and TMEM16F channels, respectively. Migration analysis shows that TMEM16A KD clones are affected in their directional migration, whereas TMEM16F KD clones show a 40 % reduced rate of cell migration. Moreover, TMEM16A KD clones have a smaller projected cell area, and they are rounder than TMEM16F KD clones. The morphological changes are linearly correlated with the directionality of cells. TMEM16A and TMEM16F, thus, have an important function in cell migration-TMEM16A in directional migration, TMEM16F in determination of the speed of migration. We conclude that TMEM16A and TMEM16F channels have a distinct impact on the steering and motor mechanisms of migrating ELA cells.

New aspects of an anti-tumour drug: sorafenib efficiently inhibits HCV replication.

BACKGROUND AND AIMS: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and is associated with significant morbidity and mortality. Since there is evidence for an interaction of NS5A with c-Raf we studied whether the c-Raf inhibitor sorafenib affects HCV replication. METHODS: HCV replicating HuH7.5 cells were treated with sorafenib and examined for HCV RNA titres by northern blotting or real time polymerase chain reaction (PCR), for core, NS3 and NS5A expression by immunostaining, and for replication by luciferase reporter assays. RESULTS: Here we demonstrate that in cells replicating infectious HCV particles, NS5A recruits c-Raf to the replicon complex resulting in the activation of c-Raf. Therefore, we studied the effect of inhibition of c-Raf on HCV replication using the anti-tumour drug sorafenib that is known to inhibit c-Raf with high specificity. Sorafenib efficiently blocks HCV replication and viral gene expression. In addition, in HCV-replicating cells sorafenib decreased the hyperphosphorylated form of NS5A and resulted in the formation of additional hypophosphorylated forms. Further, sorafenib caused a rapid dissociation of lipid droplets. We provide evidence that the antiviral effect of sorafenib indeed is caused by inhibition of c-Raf. By contrast, inhibition of targets downstream of c-Raf or inhibition of tyrosine kinases by sunitinib did not affect HCV replication. CONCLUSION: Our data demonstrate that the well-characterised anti-tumour drug sorafenib efficiently blocks HCV replication in vitro. This novel effect of sorafenib should be further explored as an antiviral strategy for patients with chronic HCV infection.

[Nonsymptomatic leukocytosis]. / Leukozytose ohne weitere Symptome. Im Zweifelsfall ein Differenzialblutbild.

Leukocytosis is a condition often met with in the clinical and ambulatory setting. Although it is usually caused by an increase in the numbers of neutrophilic granulocytes, an increase in other leukocytes populations may also account for leukocytosis. Etiologically, both primary pathological conditions affecting the white blood cells, such as various forms of leukemia and lymphomas, and also rare genetic disorders must be considered. Decidedly more common, however, are reactive changes caused by infections, cigarette smoking, chronic inflammation, necrotic tissue or certain drugs. Although moderate leukocytosis in the absence of a clinical correlate and/or an apparent trigger, requires no diagnostic clarification, it should be kept under observation. If the etiology is uncertain, or a treatment-requiring disorder is suspected, the differential blood count is at the focus of the further diagnostic work-up. Depending upon the findings, this is supplemented by additional laboratory parameters, bone marrow examination, microbiological investigations and imaging procedures. Leukostasis resulting from vasoocclusion in the presence of very high numbers of leukocytes represents an emergency situation, and is an indication for leukapheresis.

Effects of dietary fish oil on the induction of experimental membranous nephropathy in the rat.

We examined the effect of a fish oil-enriched diet on the development of experimental membranous nephropathy in the rat induced by administration of cationic bovine gamma globulin (CBGG). Rats were placed on a fish oil-enriched diet and control rats received a diet containing an equivalent amount of beef tallow. After 6 weeks on either diet, rats were pre-immunized and injected with CBGG. Proteinuria was significantly reduced in the fish oil-fed group as compared to the control group (160 +/- 40 mg/24 hours, n = 15, versus 280 +/- 36 mg/24 hours, n = 17, p less than 0.02). Glomerular filtration rate was also significantly higher in the fish oil-fed rats than in controls (0.91 +/- 0.07 ml/minute, n = 11, versus 0.60 +/- 0.05 ml/minute, n = 10, p less than 0.005). Glomerular production of prostaglandin E2 and thromboxane B2 the stable product of thromboxane A2, were inhibited by 68% and 70%, respectively, by the fish oil-enriched diet (n = 8, p less than 0.01 versus control). Glomerular leukotriene B4 was also inhibited by 50% in the fish oil-treated rats (n = 6, p less than 0.01), but inhibition of leukotriene B4 by the specific inhibitor L-663,536 in control rats did not ameliorate proteinuria. There was no difference in the amount of distribution of glomerular immune deposits as demonstrated by immunofluorescence and electron microscopy between the experimental and control groups. Moreover, comparable amounts of glomerular IgG deposits were present in the two groups. The specific immune response, assessed by measuring anti-BGG antibody levels, was not different between the two dietary groups, while more than 85% suppression of the splenic T- and B-cell mitogenic response to concanavalin-A and lipopolysaccharide was noted in rats fed the fish oil-enriched diet. We conclude that a fish oil-enriched diet reduces proteinuria and preserves the glomerular filtration rate in rats with CBGG-induced membranous nephropathy. Its mechanism of action remains to be established.

Hematologic aspects of toxicology.

Exposure to a number of toxins may result in a variety of hematologic abnormalities. Derangements of the synthesis of cellular elements, a shortened red cell half life, abnormalities of coagulation, and interference in oxygen-carrying capacity are some of the pathologic effects. Pharmacologic agents, heavy metals, and animal and insect venoms are among the toxins implicated in the production of hematologic abnormalities. This article focuses on some of these issues.