The presence or absence of a polysaccharide capsule on the human pathogen Neisseria (N.) gonorrhoeae is still a topic of controversy. For this reason we compared the results obtained by light ...microscopy (dry India ink-Fuchsin stain) and electron microscopy (Alcian blue-lanthanum nitrate stain) of encapsulated strains of N. meningitidis and Streptococcus (S.) pneumoniae and of non-encapsulated strains of S. pneumoniae and Escherichia (E.) coli with those obtained using the same methods on strains of pilliated and non-pilliated N. gonorrhoeae. After staining with India ink-Fuchsin no capsules could be demonstrated on any of the N. gonorrhoeae strains studied. If present the capsules on these cells are too delicate to be identified by light microscopy. After treatment with Alcian blue-lanthanum nitrate sections of cells of N. meningitidis and S. pneumoniae generally showed the presence of a capsular layer. Sections of cells of the non-encapsulated strain of S. pneumoniae which possess C (common)-polysaccharide also showed surface associated capsule-like material. Similarly the surface of the cells of the E. coli strain showed material which appeared to be tufts of pili and/or M (mucoid)-antigen. In experiments where the N. gonorrhoeae cells were harvested as early as after six hours of growth a capsule-like material was demonstrated on cells of all strains studied.
BACKGROUNDPancreas transplantation is the only curative treatment option for patients with juvenile diabetes. Organ shortage and restrictive allocation criteria are the main reasons for increasing ...waitlists, leading to severe morbidity and mortality. We designed a study to increase the donor pool with extended donor criteria (EDC) organs (donor age, 50-60 years; body mass index, 30-34 kg/m).
METHODSUtilization of EDC organs required the implementation of a new allocation system within Eurotransplant. The study was a prospective, multicenter, 2-armed trial. The primary endpoint was pancreas function after 3 months. Rejection episodes, kidney function, and waitlist time were secondary endpoints. Patients receiving an EDC organ were study group patients; recipients of standard organs were control group patients. Follow-up was 1 year.
RESULTSSeventy-nine patients were included in 12 German centers, 18 received EDC organs and 61 received standard organs. Recipient demographics were similar. Mean EDC donor age was 51.4 ± 5 years versus 31.7 ± 12 in the control group. Insulin-free graft survival was 83.3% for EDC and 67.2% for standard organs (P = 0.245) after 3 months. One-year pancreas survival was 83.3% and 83.5% in the EDC versus standard group. One-year kidney allograft survival was approximately 94% in both groups. Rejection episodes and morbidity were similar.
CONCLUSIONSThe Extended Pancreas Donor Program (EXPAND) shows in a prospective trial that selected EDC organs of donors older than 50 years can be used with outcomes similar to standard-criteria organs, therefore showing potential to reduce organ shortage and waiting times. This study substantiates the full implementation of EDC organs in a pancreas allocation system.
Heterografted molecular bottle brushes, i.e., poly(glycidol-graft-ε-caprolactone-acetyl)-co-(glycidol-g raft-methyl methacrylate) P(G-graft-CLAC)-co-(G-graft-MMA) and ...poly(glycidol-graft-ε-caprolactone-acetyl)-co-(glycidol-g raft-n-butyl methacrylate) P(G-graft-CLAC)-co-(G-graft-BMA) were prepared in two steps starting with a linear polyglycidol. In the first step an approximately 50% homografted polymer poly(glycidol-graft-ε-caprolactone-acetyl)-co-glycidol P(G-graft-CLAC)-co-G was obtained via ring-opening polymerization of ε-caprolactone using polyglycidol as a multifunctional macroinitiator and Novozyme 435 (Lipase B from Candida antarctica (CALB) immobilized on a macroporous resin) as a catalyst. Selective acetylation of the hydroxy groups at the graft ends was achieved via enzymatic acetylation with vinyl acetate, and the hydroxy groups at the backbone were acylated with 2-bromo-2-methylpropionyl bromide. Finally poly(methyl methacrylate) or poly(n-butyl methacrylate) grafts were attached by atom transfer radical polymerization. The heterografted molecular bottle brushes show monomodal elution curves in gel permeation chromatographic analyses. Differential scanning calorimetry confirms the existence of phase separated domains for both polymers.
Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca2+ signaling in the cell cycle control and survival ...of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca2+ concentration (Ca2+i) was measured by fura-2 Ca2+ imaging. Nuclear activity of Ca2+/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G2/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G2/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca2+ entry as evident from fura-2 Ca2+ imaging. Similarly, IR stimulated a Ca2+-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca2+ entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G2 phase was delayed upon buffering Ca2+i with the Ca2+ chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca2+ entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.
Copolypeptides with propargyl side chains were functionalized with different thiols (1-thio- beta -d-glucose tetraacetate, 7-mercapto-4-methylcoumarin, and methyl-3-mercaptopropionate) by ...initiator/catalyst-free thiol-yne photochemistry. No more than 1.5 instead of ideally 2 equivalents of thiol could be added to the propargyl units even when the thiol was used in excess, attributable to steric constraints (close proximity of the reaction site to the copolypeptide backbone) or conjugation (extended aromatic system). Nevertheless, this procedure enables a facile and modular synthesis of highly functionalized homo- and hetero-functional copolypeptides.
An approach is introduced to characterize conformational ensembles of intrinsically unstructured peptides on the atomic level using two-dimensional solid-state NMR data and their combination with ...molecular dynamics simulations. For neurotensin, a peptide that binds with high affinity to a G-protein coupled receptor, this method permits the investigation of the changes in conformational preferences of a neurotransmitter transferred from a frozen aqueous solution via a lipid model phase to the receptor-bound form. The results speak against a conformational preorganization of the ligand in detergents in which the receptor has been shown to be functional. Further extensions to the study of protein folding are possible.
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