Comparative study on the interaction of cell-penetrating polycationic polymers with lipid membranes

Cell-penetrating peptides are arginine- and lysine-rich cationic peptides that can readily enter cells not only by themselves but also carrying other macromolecular cargos. In fact, we have reported that polycationic polymer such as poly-l-lysine (PLL) and poly-l-arginine (PLA) translocate through negatively charged phospholipid liposome membranes. In this work, we made a comparative study of the interaction of PLL or PLA with lipid membranes consisting of negatively charged phospholipids to understand the role of basic amino acid residue (i.e. arginine and lysine) in the membrane-penetrating activity of polypeptides. PLA and PLL translocated into giant unilamellar vesicle composed of soybean phospholipids. ζ-potential and turbidity measurements demonstrated the electrostatic binding of PLL and PLA to large unilamellar vesicle (LUV). Fluorescence studies using membrane probes revealed that the binding of PLA and PLL to LUV affects the hydration and packing of the membrane interface region, in which the membrane insertion of PLA appeared to be greater than PLL. Differential scanning calorimetry showed that the enthalpy of the gel to liquid-crystalline phase transition for dipalmitoyl phosphatidylglycerol vesicle was greatly reduced by binding of PLL and PLA, in which the reduction is much larger in PLA than in PLL. Circular dichroism measurements in 2,2,2-trifluoroethanol/water mixture or in the presence of LUV indicated that the propensity of PLA to form α-helical structure is greater than PLL. Consistently, attenuated total reflection-Fourier transform infrared spectroscopy revealed that there is greater α-helical structure in PLA bound to LUV compared to PLL, which has much less ordered structure. Furthermore, isothermal titration calorimetry measurements demonstrated that the contribution of enthalpy to the energetics of binding to LUV is two-fold larger in PLA than in PLL. These results suggest that the stronger interaction of arginine residue with negatively charged phospholipid membranes compared to lysine residue appears to facilitate the conformational change in cationic polypeptide and its insertion into lipid membrane interior.     

Primary pulmonary leiomyosarcoma. Report of a case diagnosed by fine needle aspiration cytology

BACKGROUND: Primary pulmonary leiomyosarcoma is a rare but important entity. We report a case diagnosed by fine needle aspiration cytology. CASE: A 73-year-old male presented with an asymptomatic, right, pulmonary, subpleural nodule detected by computed tomography during follow-up for chronic obstructive pulmonary disease. Fine needle aspiration cytology showed cellular smears with numerous single or loosely cohesive groups of spindle-shaped to round cells. The tumor cell nuclei were blunt ended (cigar shaped), with fine to fine-granular chromatin, prominent nucleoli and an irregular nuclear rim. The tumor cells were positive for desmin and negative for cytokeratin and S-100 protein by immunocytochemistry. Right upper lobectomy with lymph node dissection was performed. Pathologic diagnosis after microscopic, immunohistochemical and electron microscopic studies was leiomyosarcoma. CONCLUSION: To our knowledge, this is the first reported case of primary pulmonary leiomyosarcoma arising in the subpleural region diagnosed by fine needle aspiration cytology. Immunocytochemistry was useful in establishing the diagnosis in this case.     

Engineering neoglycoproteins with multiple O-glycans using repetitive pentapeptide glycosylation units

Controlled protein remodeling with O-linked glycans has been limited by our incomplete understanding of the process of glycosylation. Here we describe a secretable fibroblast growth factor (FGF) with multiple mucin-type O-glycans produced by introducing a minimum pentapeptide glycosylation unit in a decarepeat format at its N- or C-terminus. Expressed in Chinese hamster ovary cells, chemical and biochemical analyses of the resultant proteins (Nm10-FGF and Cm10-FGF, respectively) demonstrated that all O-glycosylation units were glycosylated and the dominant structure was sialylated Gal[1–3]GalNAc. This indicates that minimum O-glycosylation unit in multirepeat format serves as a remarkably efficient acceptor in CHO cells. The Nm10-FGF and Cm10-FGF proteins maintained the mitogenic activity to vascular endothelial cells. In addition, intact Cm10-FGF and its desialylated form interacted with several lectins in the same way as mucin-type glycoproteins. The intact Cm10-FGF with multiple sialylated O-glycans exhibited a longer lifetime in circulating blood, whereas the Cm10-FGF with desialylated O-glycans exhibited a shorter lifetime than the deglycosylated form of Cm10-FGF. Our approach would thus appear to be highly effective for engineering neoglycoproteins, the characteristics of which are determined by their multiple mucin-type O-glycans.     

Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4

Rhodococcus erythropolis strain PR4 has been isolated as an alkane-degrading bacterium. The strain harbours one linear plasmid, pREL1 (271 577 bp) and two circular plasmids, pREC1 (104 014 bp) and pREC2 (3637 bp), all with some sequence similarities to other Rhodococcus plasmids. For pREL1, pREC1 and pREC2, 298, 102 and 3 open reading frames, respectively, were predicted. Linear plasmid pREL1 has several regions homologous to plasmid pBD2 found in R. erythropolis BD2. Sequence analysis of pREL1 and pBD2 identified common metal-resistance genes on both, but pREL1 also encodes alkane-degradation genes not found on pBD2, with enzyme constituents some of which are quite different from those of other organisms. The alkane hydroxylase consisted of a cytochrome P450 monooxygenase, a 2Fe-2S ferredoxin, and a ferredoxin reductase. The ferredoxin reductase amino acid sequence resembles the AlkT (rubredoxin reductase) sequence. A zinc-containing alcohol dehydrogenase further oxydizes alkanols, alkane oxidation products catalysed by alkane hydroxylase. Of the circular plasmids, the pREC1 sequence is partially similar to the sequence of pREAT701, the virulence plasmid found in Rhodococcus equi. pREC1 has no pREAT701 virulence genes and encodes genes for beta-oxidation of fatty acids. Thus, joint actions of enzymes encoded by pREL1 and pREC1 may enable efficient mineralization of alkanes.     

NMR based structure-activity relationship analysis of an antimicrobial peptide, thanatin, engineered by site-specific chemical modification: Activity improvement and spectrum alteration

Activity improvement of an antimicrobial peptide, thanatin, has been achieved up to 4-fold higher than natural original one by site-specific chemical modifications with tert-butyl group at two cysteine residues which form an intramoleular disulfide bridge. The chemically modified thanatin (C11tBu/C18tBu) exhibited improved antimicrobial activity toward Gram-positive bacteria, Micrococcus luteus, whereas lowered activity toward Gram-negative bacteria, Escherichia coli. This finding suggests that disulfide-bridge formation is not only indispensable for exhibition of antimicrobial activity of thanatin but also closely related to the activity specificity towards bacteria. NMR analysis indicates that thanatin acts against E.coli stereospecifically by taking advantage of its C-terminal β-hairpin structure, while the activity against M. luteus does not relate to structures and correlates very well to side-chain hydrophobicity.     

Renadirsen,a Novel 2′OMeRNA/ENA® Chimera Antisense Oligonucleotide,Induces Robust Exon 45 Skipping for Dystrophin In Vivo

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by out-of-frame or nonsense mutation in the dystrophin gene. It begins with a loss of ambulation between 9 and 14 years of age, followed by various other symptoms including cardiac dysfunction. Exon skipping of patients’ DMD pre-mRNA induced by antisense oligonucleotides (AOs) is expected to produce shorter but partly functional dystrophin proteins, such as those possessed by patients with the less severe Becker muscular dystrophy. We are working on developing modified nucleotides, such as 2′-O,4′-C-ethylene-bridged nucleic acids (ENAs), possessing high nuclease resistance and high affinity for complementary RNA strands. Here, we demonstrate the preclinical characteristics (exon-skipping activity in vivo, stability in blood, pharmacokinetics, and tissue distribution) of renadirsen, a novel AO modified with 2′-O-methyl RNA/ENA chimera phosphorothioate designed for dystrophin exon 45 skipping and currently under clinical trials. Notably, systemic delivery of renadirsen sodium promoted dystrophin exon skipping in cardiac muscle, skeletal muscle, and diaphragm, compared with AOs with the same sequence as renadirsen but conventionally modified by PMO and 2′OMePS. These findings suggest the promise of renadirsen sodium as a therapeutic agent that improves not only skeletal muscle symptoms but also other symptoms in DMD patients, such as cardiac dysfunction.     

The Long-Term Effects of Pitavastatin on Blood Lipids and Platelet Activation Markers in Stroke Patients: Impact of the Homocysteine Level

To examine the impact of the plasma homocysteine level on the anti-atherosclerotic effects of pitavastatin treatment, we retrospectively examined 59 patients who had a history of stroke and had been prescribed pitavastatin for the treatment of dyslipidemia at the Neurology department of Toho University Ohashi Medical Center Hospital. The patients were classified into two groups according to their homocysteine levels. Carotid artery plaque progression was determined before and after pitavastatin treatment. Plasma levels of high-sensitivity C-reactive protein, platelet molecular markers, and von Willebrand factor were measured. Pitavastatin treatment had beneficial effects on the lipid profiles of these patients and slowed atherosclerosis progression. These effects were observed in both the high and low homocysteine groups. Proactive lipid intervention using pitavastatin may inhibit the progression of atherosclerosis and contribute to secondary prevention of stroke in high-risk patients. We conclude that this statin could inhibit progression at any stage of disease and should therefore be proactively administered to these patient groups, regardless of disease severity.     

Cancer preventive potential of trichothecenes from Trichothecium roseum

Bioassay-guided separation of extracts from the culture broth and mycelium of the fungus Trichothecium roseum, aiming at the discovery for cancer preventive agents, resulted in the isolation of three new trichothecene sesquiterpenes, trichothecinols A-C (1-3) together with three known analogues, trichothecin (4), trichodermol (5) and trichothecolone (6). Compounds 1-6 exhibited remarkably potent inhibition against Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further compound 1 strongly inhibited TPA-induced tumor promotion on mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in two-stage carcinogenesis tests. These results suggest that compound 1 might be a valuable lead for further evaluation as a cancer preventive agent. In addition to their cancer preventive activity, compound 2 was found to show modest antifungal activity against Crypotcoccus albidus and Saccharomyces cerevisiae.