DMSO Induces Dehydration near Lipid Membrane Surfaces

Dimethyl sulfoxide (DMSO) has been broadly used in biology as a cosolvent, a cryoprotectant, and an enhancer of membrane permeability, leading to the general assumption that DMSO-induced structural changes in cell membranes and their hydration water play important functional roles. Although the effects of DMSO on the membrane structure and the headgroup dehydration have been extensively studied, the mechanism by which DMSO invokes its effect on lipid membranes and the direct role of water in this process are unresolved. By directly probing the translational water diffusivity near unconfined lipid vesicle surfaces, the lipid headgroup mobility, and the repeat distances in multilamellar vesicles, we found that DMSO exclusively weakens the surface water network near the lipid membrane at a bulk DMSO mole fraction (X_DMSO) of <0.1, regardless of the lipid composition and the lipid phase. Specifically, DMSO was found to effectively destabilize the hydration water structure at the lipid membrane surface at X_DMSO <0.1, lower the energetic barrier to dehydrate this surface water, whose displacement otherwise requires a higher activation energy, consequently yielding compressed interbilayer distances in multilamellar vesicles at equilibrium with unaltered bilayer thicknesses. At X_DMSO >0.1, DMSO enters the lipid interface and restricts the lipid headgroup motion. We postulate that DMSO acts as an efficient cryoprotectant even at low concentrations by exclusively disrupting the water network near the lipid membrane surface, weakening the cohesion between water and adhesion of water to the lipid headgroups, and so mitigating the stress induced by the volume change of water during freeze-thaw.     

Antitumoral effects of cyclin-dependent kinases inhibitors CR8 and MR4 on chronic myeloid leukemia cell lines

Background

Although Imatinib mesylate has revolutionized the treatment of chronic myeloid leukemia, some patients develop resistance with progression of leukemia. Alternative or additional targeting of signalling pathways deregulated in Bcr-Abl-driven chronic myeloid leukemia may provide a feasible option for improving clinical response and overcoming resistance.

Results

In this study, we investigate ability of CR8 isomers (R-CR8 and S-CR8) and MR4, three derivatives of the cyclin-dependent kinases (CDKs) inhibitor Roscovitine, to exert anti-leukemic activities against chronic myeloid leukemia in vitro and then, we decipher their mechanisms of action. We show that these CDKs inhibitors are potent inducers of growth arrest and apoptosis of both Imatinib-sensitive and –resistant chronic myeloid leukemia cell lines. CR8 and MR4 induce dose-dependent apoptosis through mitochondrial pathway and further caspases 8/10 and 9 activation via down-regulation of short-lived survival and anti-apoptotic factors Mcl-1, XIAP and survivin which are strongly implicated in survival of Bcr-Abl transformed cells.

Conclusions

These results suggest that CDK inhibitors may constitute a complementary approach to treat chronic myeloid leukemia.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0163-x) contains supplementary material, which is available to authorized users.     

Increased diagnostic accuracy of atypical glandular cells in cervical liquid‐based cytology using cell blocks

E. K. J. Risse, J. P. Holierhoek, E. M. Meijer‐Marres, E. Ouwerkerk‐Noordam and M. E. Boon Increased diagnostic accuracy of atypical glandular cells in cervical liquid‐based cytology using cell blocks Objective: The purpose of this study was to reduce the number of diagnoses of atypical glandular cells (AGC). Residual material from the cervical ThinPrep® samples (Hologic, Marlboruogh, MA, USA) was used for cell blocks (CB) and immunohistochemistry (IHC). Methods: In 2007 there were 87 patients (0.12% of tests) with AGC on liquid‐based cytology (LBC) in the Leiden Cytology and Pathology Laboratory (LCPL) using the Bethesda System 2001 (TBS). CB with IHC was used for 26 of these cases. The vials still containing the brush (Cervex‐Brush^® Combi) were placed in a shaker for 10 minutes to dislodge the material trapped between the bristles. The residual sampling fluid was used to prepare paraffin sections (Shandon Cytoblock^®) stained with Papanicolaou and immunostaining. Results: Four of five cases with AGC not otherwise specified (NOS) were diagnosed with CB/IHC as benign mimics (endometrium, tubal metaplasia, follicular cervicitis, microglandular hyperplasia) and one of four with AGC‐favour neoplasia (FN) (endocervical polyp). In one of five cases with AGC‐NOS and in two of seven with AGC‐FN, CIN3 was found on subsequent histological biopsy. Of six cases diagnosed as adenocarcinoma in situ (AIS) on LBC with CB/IHC the diagnosis was confirmed in four; one was adenocarcinoma and one glandular atypia. Of eight cases diagnosed as adenocarcinoma on cytology and CB/IHC, the diagnosis was confirmed in three. The other five cases were found to be one each of AIS, squamous cell carcinoma, CIN3, CIN2 with glandular atypia, and cervical endometriosis. Conclusions: By reducing the number of benign mimics of AGC, we achieved a high proportion (16/26; 61.5%) of neoplastic or preneoplastic lesions (glandular or squamous) on histological outcome potentially avoiding colposcopy. Histological biopsy verification by the gynaecologist is needed for final diagnosis of AGC‐FN, AIS and adenocarcinoma.     

Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor

The epidermal growth factor receptor (EGFR) is frequently dysregulated in human malignancies and a validated target for cancer therapy. Two monoclonal anti-EGFR antibodies (cetuximab and panitumumab) are approved for clinical use. However, the percentage of patients responding to treatment is low and many patients experiencing an initial response eventually relapse. Thus, the need for more efficacious treatments remains. Previous studies have reported that mixtures of antibodies targeting multiple distinct epitopes are more effective than single mAbs at inhibiting growth of human cancer cells in vitro and in vivo. The current work describes the rational approach that led to discovery and selection of a novel anti-EGFR antibody mixture Sym004, which is currently in Phase 2 clinical testing. Twenty-four selected anti-EGFR antibodies were systematically tested in dual and triple mixtures for their ability to inhibit cancer cells in vitro and tumor growth in vivo. The results show that targeting EGFR dependent cancer cells with mixtures of antibodies is superior at inhibiting their growth both in vitro and in vivo. In particular, antibody mixtures targeting non-overlapping epitopes on domain III are efficient and indeed Sym004 is composed of two monoclonal antibodies targeting this domain. The superior growth inhibitory activity of mixtures correlated with their ability to induce efficient EGFR degradation.Key words: EGFR, antibody synergy, functional screening, epitope binning, antibody combinations     

Ischemic preconditioning, insulin, and morphine all cause hexokinase redistribution

Association of hexokinase (HK) with mitochondria preserves mitochondrial integrity and is an important mechanism by which cancer cells are protected against hypoxic conditions. Maintenance of mitochondrial integrity also figures prominently as a major characteristic of many cardioprotective manipulations. In this study, we provide evidence that cardioprotective interventions may promote HK redistribution from the cytosol to the mitochondria in the heart. Isolated Langendorff-perfused rat hearts (n = 6/group) were subjected to normoxic perfusion (control, Con), three 5-min ischemia-reperfusion periods (ischemic preconditioning, IPC), 1 U/l insulin (Ins), or 1 microM morphine (Mor). Hearts were immediately homogenized and centrifuged to obtain whole cell, cytosolic, and mitochondrial fractions. HK, lactate dehydrogenase (LDH), and citrate synthase (CS) enzyme activities were determined. No change in LDH or CS present in the cytosol fraction relative to whole cell activity was observed with any of the cardioprotective interventions. By contrast, HK present in the cytosol fraction relative to whole cell activity decreased significantly (P < 0.05) with all cardioprotective interventions, from 0.58 +/- 0.03 (Con) to 0.46 +/- 0.04 (IPC), 0.41 +/- 0.01 (Ins), and 0.45 +/- 0.02 (Mor). In addition, HK relative to CS activity in the mitochondrial fraction increased significantly with cardioprotection, from 0.15 +/- 0.001 (Con) to 0.21 +/- 0.002 (IPC), 0.18 +/- 0.003 (Ins), and 0.21 +/- 0.005 (Mor). Our novel data suggest that well-known cardioprotective interventions share a common end-effector mechanism of cytosolic HK translocation. Association of HK with mitochondria may promote inhibition of the mitochondrial permeability transition pore and thereby reduce cell death and apoptosis.     

Identification, cloning, and characterization of a Lactococcus lactis branched-chain alpha-keto acid decarboxylase involved in flavor formation

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.