Genomic diversity of Astragalus cicer microsymbionts revealed by AFLP fingerprinting

DNA polymorphism among 36 Astragalus cicer nodule isolates and 9 reference mesorhizobia was evaluated by a simplified PstI based AFLP procedure with three selective primers: Pst-A, Pst-G, and Pst-GC. The DNA profiles were found to be highly specific for nearly each strain, although DNA bands characteristic for most A. cicer microsymbionts were also noted. The overall topologies of dendrograms, generated by AFLP patterns in PCR reaction with three primers, were very similar to one another and to that constructed by phenotyping. Also the strain compositions in the particular clusters on pheno- and genomograms were in good agreement. The obtained results indicate that AFLP technique can be a useful tool for typing of A. cicer rhizobia as well as for studying their diversity.     

Alpha-conotoxin residues that interact at close range with gamma-tyrosine-111 and mutant delta-tyrosine-113 on the Torpedo nicotinic acetylcholine receptor

The alpha-conotoxins MI and GI display stronger affinities for the alphagamma agonist site on the Torpedo californica electrocyte nicotinic acetylcholine receptor (ACHR) than for the alphadelta agonist site, while alpha-conotoxin SI binds with the same affinity to both sites. Prior studies reported that the arginine at position 9 on GI and the tyrosine at position 111 on the receptor gamma subunit were responsible for the stronger alphagamma affinities of GI and MI, respectively. This study was undertaken to determine if the alpha-conotoxin midchain cationic residues interact with Torpedo gammaY111. The findings show that lysine 10 on MI is responsible for the alphagamma selectivity of MI and confirm the previously reported importance of R9 on GI and on the SI analogue, SIP9R. The results also show that gammaY111 contributes substantially to the selective alphagamma high affinity of all three peptides. Double-mutant cycle analyses reveal that, in the alphagamma site, K10 on MI and R9 on SIP9R interact with the aromatic ring of gammaY111 to stabilize the high-affinity complex, while in contrast, R9 on GI does not. The substitution of Y for R at position 113 on the delta subunit converts the alphadelta site into a high-affinity site for MI, GI, and SIP9R through the interacting of deltaY113 with K10 on MI and with R9 on both GI and SIP9R. The overall data show that the residues in the two sites with which MI interacts, other than at gamma111/delta113, are either the same or similar enough to exert equivalent effects on MI, indicating that MI binds in the same orientation at the alphagamma and alphadelta sites. Similar findings show that SIP9R probably also binds in the same orientation at the wild-type alphagamma and alphadelta sites. The finding that R9 on GI interacts closely with deltaR113Y but not with gammaY111 means that GI binds in different orientations at the alphagamma and alphadelta sites. This report also discusses the molecular basis of the difference in the MI high-affinity sites on Torpedo and embryonic mouse muscle ACHRs.     

Model of creation and evolution of stable electropores for DNA delivery

Electroporation, in which electric pulses create transient pores in the cell membrane, is becoming an important technique for gene therapy. To enable entry of supercoiled DNA into cells, the pores should have sufficiently large radii (>10 nm), remain open long enough for the DNA chain to enter the cell (milliseconds), and should not cause membrane rupture. This study presents a model that can predict such macropores. The distinctive features of this model are the coupling of individual pores through membrane tension and the electrical force on the pores, which is applicable to pores of any size. The model is used to explore the process of pore creation and evolution and to determine the number and size of pores as a function of the pulse magnitude and duration. Next, our electroporation model is combined with a heuristic model of DNA uptake and used to predict the dependence of DNA uptake on pulsing parameters. Finally, the model is used to examine the mechanism of a two-pulse protocol, which was proposed specifically for gene delivery. The comparison between experimental results and the model suggests that this model is well-suited for the investigation of electroporation-mediated DNA delivery.     

A constituent of green tea, epigallocatechin-3-gallate, activates endothelial nitric oxide synthase by a phosphatidylinositol-3-OH-kinase-, cAMP-dependent protein kinase-, and Akt-dependent pathway and leads to endothelial-dependent vasorelaxation

Epidemiological studies suggest that tea catechins may reduce the risk of cardiovascular disease, but the mechanisms of benefit have not been determined. The objective of the present study was to investigate the effects of epigallocatechin-3-gallate (EGCG), the major constituent of green tea, on vasorelaxation and on eNOS expression and activity in endothelial cells. EGCG (1-50 microm) induced dose-dependent vasodilation in rat aortic rings. Vasodilation was abolished by pretreatment with Ng-nitro L-arginine methyl ester. In bovine aortic endothelial cells, EGCG increased endothelial nitric oxide (eNOS) activity dose-dependently after 15 min. Treatment with EGCG induced a sustained activation of Akt, ERK1/2, and eNOS Ser1179 phosphorylation. Inhibition of extracellular signal-regulated kinase (ERK)1/2 had no influence on eNOS activity or Ser1179 phosphorylation. Simultaneous treatment of cells with selective inhibitors for cAMP-dependent protein kinase (PKA) and Akt completely prevented the increase in eNOS activity by EGCG after 15 min, indicating that both kinases act in concert. Specific phosphatidylinositol-3-OH-kinase inhibitors yielded identical results. Akt inhibition prevented eNOS Ser1179 phosphorylation, whereas inhibition of PKA did not influence Akt and eNOS Ser1179 phosphorylation. Pretreatment of endothelial cells with EGCG for 4 h markedly enhanced the increase in eNOS activity stimulated by Ca-ionomycin, suggesting that Akt accounts for prolonged eNOS activation. Treatment of cells for 72 h with EGCG did not change eNOS protein levels. Our results indicate that EGCG-induced endothelium-dependent vasodilation is primarily based on rapid activation of eNOS by a phosphatidylinositol 3-kinase-, PKA-, and Akt-dependent increase in eNOS activity, independently of an altered eNOS protein content.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Glycocalyx restricts adenoviral vector access to apical receptors expressed on respiratory epithelium in vitro and in vivo: role for tethered mucins as barriers to lumenal infection

Inefficient adenoviral vector (AdV)-mediated gene transfer to the ciliated respiratory epithelium has hindered gene transfer strategies for the treatment of cystic fibrosis lung disease. In part, the inefficiency is due to an absence of the coxsackie B and adenovirus type 2 and 5 receptor (CAR) from the apical membranes of polarized epithelia. In this study, using an in vitro model of human ciliated airway epithelium, we show that providing a glycosylphosphatidylinositol (GPI)-linked AdV receptor (GPI-CAR) at the apical surface did not significantly improve AdV gene transfer efficiency because the lumenal surface glycocalyx limited the access of AdV to apical GPI-CAR. The highly glycosylated tethered mucins were considered to be significant glycocalyx components that restricted AdV access because proteolytic digestion and inhibitors of O-linked glycosylation enhanced AdV gene transfer. To determine whether these in vitro observations are relevant to the in vivo situation, we generated transgenic mice expressing GPI-CAR at the surface of the airway epithelium, crossbred these mice with mice that were genetically devoid of tethered mucin type 1 (Muc1), and tested the efficiency of gene transfer to murine airways expressing apical GPI-human CAR (GPI-hCAR) in the presence and absence of Muc1. We determined that AdV gene transfer to the murine airway epithelium was inefficient even in GPI-hCAR transgenic mice but that the gene transfer efficiency improved in the absence of Muc1. However, the inability to achieve a high gene transfer efficiency, even in mice with a deletion of Muc1, suggested that other glycocalyx components, possibly other tethered mucin types, also provide a significant barrier to AdV interacting with the airway lumenal surface.     

Comparison of checkerboard and time-kill methods for the analysis of two antibiotics combined

Activity interaction analysis of two antibiotics by two methods: checkerboard and "time-kill" was compared during this study. Combinations of procaine penicillin, polymyxin B and bacitracin with neomycin and procaine penicillin with dihydrostreptomycin were examined. Checkerboard method is the most widely used technique for antimicrobials interactions analyses. The "time-kill" method, performed by the broth macrodilution technique, provides a dynamic picture of antimicrobial action and interaction over time (based on serial colony counts). Differences of "time-kill" method and the checkerboard technique, allow single visual examination (after 16 to 24 hours of incubation). Additive and inhibition effects were observed in combinations of neomycin with beta-lactam antibiotic (procaine penicillin) and peptide antibiotics (bacitracin and polymyxin B) on clinical strain S. Enteritidis IL 35 "Time-kill" method also confirmed observations mentioned above. In combinations of procaine penicillin with dihydrostreptomycin on strains E. coli IL 531 and E. coli IL 256 synergy effects on checkerboard technique were noticed. Such observation was not confirmed by the "time-kill" method. The methodologies and definitions of synergism are variable and not standardized. This situation should be improved, because comparison of the results obtained by different methods becomes a very difficult task.     

Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.     

A bioactive collagen-β tricalcium phosphate scaffold for tissue engineering

Collagen-phosphate composites (COL/β-TCP) are novel materials that have the potential to be used as bone analogues. The aim of our study was to develop a porous bioactive material composed of type I collagen, the main bone protein and tricalcium phosphate, the mineral phase of natural bone, and investigate their in vitro biocompatibility in a human dermal fibroblast culture system. In order to obtain the bioactive materials, type I collagen was isolated from bovine tendon and characterized by physicochemical methods. β-TCP was obtained from calcium carbonate by thermal decomposition at 900 °C temperature. The powder was examined with X-ray diffraction. Two variants of COL/β-TCP scaffolds (P1 and P2) were prepared and examined by scanning electron microscopy. Our results revealed a microporous structure with small white aggregates of β-TCP, non-homogenous scattered in the collagen framework without any preferential orientation. The biocompatibility of the obtained scaffolds was tested by biochemical and histological methods on human fibroblast cultures. Both materials acted as good subtrates for human dermal fibroblast proliferation and migration.     

Immunotropic influence of 900 MHz microwave GSM signal on human blood immune cells activated in vitro

In an earlier study we reported that G(o) phase peripheral blood mononulclear cells (PBMC) exposed to low-level (SAR = 0.18 W/kg) pulse-modulated 1300 MHz microwaves and subsequently cultured, demonstrate changed immune activity (Dabrowski et al., 2003). We investigated whether cultured immune cells induced into the active phases of cell cycle (G(1), S) and then exposed to microwaves will also be sensitive to electromagnetic field. An anechoic chamber of our design containing a microplate with cultured cells and an antenna emitting microwaves (900 MHz simulated GSM signal, 27 V/m, SAR 0.024 W/kg) was placed inside the ASSAB incubator. The microcultures of PBMC exposed to microwaves demonstrated significantly higher response to mitogens and higher immunogenic activity of monocytes (LM index) than control cultures. LM index, described in detail elsewhere (Dabrowski et al., 2001), represents the monokine influence on lymphocyte mitogenic response. The results suggest that immune activity of responding lymphocytes and monocytes can be additionally intensified by 900 MHz microwaves.