Restriction analysis of lambda EMBL3 background recombinants: occurrence of lambda phages carrying a head to tail oriented left arm DNA sequence

Summary Eight representative recombinant background clones of λEMBL3 were analysed usingKpnI,BamHI,SalI,EcoRI andHindIII digestion. We found that λEMBL3 carries its own left arm in theBamHI cloning site. In this way, recombinant molecules were found to be generated which can grow onEscherichia coli strain NM539. In all cases analysed, the left arm DNA was inserted in a head to tail orientation. Seven clones carried a restoredBamHI site at thecos site-BamHI site connection. In the region where the inserted left arm and the right arm were ligated,BamHI cloning produces a large palindromic sequence consisting of two polylinkers. ThisBamHI site was incompletely cleaved in all cases analysed. We assume that a part of the λ DNA molecule in this region shows a cruciform structure prohibiting recognition or cleavage of this site by restriction endonucleaseBamHI.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.     

Characterization of the respiratory activity of mitochondria isolated from an insect cell line CP-1268 Laspeyresia pomonella

Mitochondria have been isolated from the codling moth Laspeyresia pomonella, CP-1268 cell line. The mitochondrial fraction was isolated from pooled 4 d, exponential growth phase, cultures. The mitochondria were determined to be intact based on the demonstration of respiratory control, the effects of 2,4 dinitrophenol and oligomycin on respiration, the inability to oxidize NADH, and the inability of cytochrome c to enhance respiration. The isolated mitochondria were able to oxidize succinate, pyruvate, malate, alpha-ketoglutarate, and alpha-glycerophosphate efficiently. Of the substrates tested, the CP-1268 mitochondria oxidized succinate most efficiently. The respiratory control ratios ranged from a high of 4.6 for pyruvate to a low of 1.7 with alpha-glycerophosphate. These findings confirm that the mitochondria were tightly coupled. The data also confirm the presence of three sites of oxidative phosphorylation because NAD-linked substrates had ADP-to-O ratios approaching 3 and flavoprotein linked substrates had values approaching 2.     

Core-cross-linked polymeric micelles as paclitaxel carriers

Cross-linkable di- and triblock copolymers of poly(epsilon-caprolactone) (PCL) and monomethoxyl poly(ethylene glycol) (MPEG) were synthesized. These amphiphilic copolymers self-assembled into nanoscale micelles capable of encapsulating hydrophobic paclitaxel in their hydrophobic cores in aqueous solutions. To further enhance their thermodynamic stability, the micelles were cross-linked by radical polymerization of the double bonds introduced into the PCL blocks. Reaction conditions were found to significantly affect both the cross-linking efficiency and the micelle size. The encapsulation of paclitaxel into the micelles was confirmed by the proton nuclear magnetic resonance (1H NMR) spectroscopy. Encouragingly, paclitaxel-loading efficiency of micelles was enhanced significantly upon micelle core-cross-linking. Both the micelle size and the drug loading efficiency increased markedly with increasing the PCL block lengths, no matter if the micelles were core-cross-linked or not. However, paclitaxel-loading did not obviously affect the micelle size or size distribution. The cross-linked micelles exhibited a significantly enhanced thermodynamic stability against dilution with aqueous solvents. The efficient cellular uptake of paclitaxel loaded in the nanomicelles was demonstrated by confocal laser scanning microscopy (CLSM) imaging. This new biodegradable nanoscale carrier system merits further investigations for parenteral drug delivery.     

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery

A biodegradable gene transfer vector has been synthesized by linking several low molecular weight (MW) polyethylenimine (PEI, 1200 Da) blocks using an oligo(L-lactic acid-co-succinic acid) (OLSA, 1000 Da). The resulting copolymer P(EI-co-LSA) (8 kDa) is soluble in water and degrades via base-catalyzed hydrolytic cleavage of amide bonds. With regard to its application as a gene transfer agent, the polymer showed an interesting pH dependency of degradation. At pH 5, when DNases are highly active, the degradation proceeds at a slower rate than at a physiological pH of 7.4. PEI and P(EI-co-LSA) spontaneously formed complexes with plasmid DNA. Whereas the complexes formed with PEI were not stable and aggregated, forming particles of up to 1 microm hydrodynamic diameter, P(EI-co-LSA) formed complexes, which were about 150 nm in size and of narrow size distribution. The latter complexes were stable, due to their high surface charge (zeta-potential + 18 mV). Similar to low MW PEI, the copolymer exhibited a low toxicity profile. At the same time, the copolymer showed a significant enhancement of transfection activity in comparison to the low MW PEI. This makes P(EI-co-LSA) a promising candidate for long-term gene therapy where biocompatibility and biodegradability become increasingly important.     

Point mutation in kit receptor tyrosine kinase reveals essential roles for kit signaling in spermatogenesis and oogenesis without affecting other kit responses

The Kit receptor tyrosine kinase functions in hemato- poiesis, melanogenesis and gametogenesis. Kit receptor-mediated cellular responses include proliferation, survival, adhesion, secretion and differentiation. In mast cells, Kit-mediated recruitment and activation of phosphatidylinositol 3'-kinase (PI 3-kinase) produces phosphatidylinositol 3'-phosphates, plays a critical role in mediating cell adhesion and secretion and has contributory roles in mediating cell survival and proliferation. To investigate the consequences in vivo of blocking Kit-mediated PI 3-kinase activation we have mutated the binding site for the p85 subunit of PI 3-kinase in the Kit gene, using a knock-in strategy. Mutant mice have no pigment deficiency or impairment of steady-state hematopoiesis. However, gametogenesis is affected in several ways and tissue mast cell numbers are affected differentially. While primordial germ cells during embryonic development are not affected, Kit(Y719F)/Kit(Y719F) males are sterile due to a block at the premeiotic stages in spermatogenesis. Furthermore, adult males develop Leydig cell hyperplasia. The Leydig cell hyperplasia implies a role for Kit in Leydig cell differentiation and/or steroidogenesis. In mutant females follicle development is impaired at the cuboidal stages resulting in reduced fertility. Also, adult mutant females develop ovarian cysts and ovarian tubular hyperplasia. Therefore, a block in Kit receptor-mediated PI 3-kinase signaling may be compensated for in hematopoiesis, melanogenesis and primordial germ cell development, but is critical in spermatogenesis and oogenesis.     

Kit signaling through PI 3-kinase and Src kinase pathways: an essential role for Rac1 and JNK activation in mast cell proliferation.

The receptor tyrosine kinase Kit plays critical roles in hematopoiesis, gametogenesis and melanogenesis. In mast cells, Kit receptor activation mediates several cellular responses including cell proliferation and suppression of apoptosis induced by growth factor deprivation and gamma-irradiation. Kit receptor functions are mediated by kinase activation, receptor autophosphorylation and association with various signaling molecules. We have investigated the role of phosphatidylinositol 3'-kinase (PI 3-kinase) and Src kinases in Kit-mediated cell proliferation and suppression of apoptosis induced both by factor deprivation and irradiation in bone marrow-derived mast cells (BMMC). Analysis of Kit-/- BMMC expressing mutant Kit receptors and the use of pharmacological inhibitors revealed that both signaling pathways contribute to these Kit-mediated responses and that elimination of both pathways abolishes them. We demonstrate that the PI 3-kinase and Src kinase signaling pathways converge to activate Rac1 and JNK. Analysis of BMMC expressing wild-type and dominant-negative mutant forms of Rac1 and JNK revealed that the Rac1/JNK pathway is critical for Kit ligand (KL)-induced proliferation of mast cells but not for suppression of apoptosis. In addition, KL was shown to inhibit sustained activation of JNK induced by gamma-irradiation and concomitant irradiation-induced apoptosis.