Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

High fidelity TNA synthesis by Therminator polymerase

Therminator DNA polymerase is an efficient DNA-dependent TNA polymerase capable of polymerizing TNA oligomers of at least 80 nt in length. In order for Therminator to be useful for the in vitro selection of functional TNA sequences, its TNA synthesis fidelity must be high enough to preserve successful sequences. We used sequencing to examine the fidelity of Therminator-catalyzed TNA synthesis at different temperatures, incubation times, tNTP ratios and primer/template combinations. TNA synthesis by Therminator exhibits high fidelity under optimal conditions; the observed fidelity is sufficient to allow in vitro selection with TNA libraries of at least 200 nt in length.     

Overexpression of the Na+/H+ exchanger and ischemia-reperfusion injury in the myocardium

In the myocardium, the Na(+)/H(+) exchanger isoform-1 (NHE1) activity is detrimental during ischemia-reperfusion (I/R) injury, causing increased intracellular Na(+) (Na(i)(+)) accumulation that results in subsequent Ca(2+) overload. We tested the hypothesis that increased expression of NHE1 would accentuate myocardial I/R injury. Transgenic mice were created that increased the Na(+)/H(+) exchanger activity specifically in the myocardium. Intact hearts from transgenic mice at 10-15 wk of age showed no change in heart performance, resting intracellular pH (pH(i)) or phosphocreatine/ATP levels. Transgenic and wild-type (WT) hearts were subjected to 20 min of ischemia followed by 40 min of reperfusion. Surprisingly, the percent recovery of rate-pressure product (%RPP) after I/R improved in NHE1-overexpressing hearts (64 +/- 5% vs. 41 +/- 5% in WT; P < 0.05). In addition, NMR spectroscopy revealed that NHE1 overexpressor hearts contained higher ATP during early reperfusion (levels P < 0.05), and there was no difference in Na(+) accumulation during I/R between transgenic and WT hearts. HOE642 (cariporide), an NHE1 inhibitor, equivalently protected both WT and NHE1-overexpressing hearts. When hearts were perfused with bicarbonate-free HEPES buffer to eliminate the contribution of HCO(3)(-) transporters to pH(i) regulation, there was no difference in contractile recovery after reperfusion between controls and transgenics, but NHE1-overexpressing hearts showed a greater decrease in ATP during ischemia. These results indicate that the basal activity of NHE1 is not rate limiting in causing damage during I/R, therefore, increasing the level of NHE1 does not enhance injury and can have some small protective effects.     

Determination of albuterol in plasma after aerosol inhalation by gas chromatography-mass spectrometry with selected-ion monitoring

Albuterol is a β₂-adrenergic agonist commonly used as a bronchdilator for the treatment of patients with asthma. We have developed an assay to determine plasma levels as low as 50 pg/ml of albuterol by gas chromatography-mass spectrometry (GC-MS). This assay utilizes isotopically labeled albuterol ([¹³C]albuterol) as an internal standard. In this assay albuterol and the internal standard are recovered from 1 ml of plasma using solid-phase extraction. The samples are then derivatized to trimethylsilyl ethers using N,O-bis(trimethylsilyl)trifluoro-acetamide with 1% trimethylchlorosilane. The samples are then analyzed by GC-MS with selected-ion monitoring (SIM) for the ions m/z 369.15 and 370.15. The method has been validated for a concentration range of 50–10000 pg/ml in plasma.     

PCR-based gene targeting of the inducible nitric oxide synthase (NOS2) locus in murine ES cells,a new and more cost-effective approach

Gene targeting by double homologous recombination in murine embryonic stem (ES) cells is a powerful tool used to study the cellular consequences of specific genetic mutations. A typical targeting construct consists of a neomycin phosphotransferase (neo) gene flanked by genomic DNA fragments that are homologous to sequences in the target chromosomal locus. Homologous DNA fragments are typically cloned from a murine genomic DNA library. Here we describe an alternative approach whereby the inducible nitric oxide synthase (NOS2) gene locus is partially mapped and homologous DNA sequences obtained using a long-range PCR method. A 7 kb NOS2 amplicon is used to construct a targeting vector where theneo gene is flanked by PCR-derived homologous DNA sequences. The vector also includes a thymidine kinase (tk) negative-selectable marker gene. Following transfection into ES cells, the PCR-based targeting vector undergoes efficient homologous recombination into the NOS2 locus. Thus, PCR-based gene targeting can be a valuable alternative to the conventional cloning approach. It expedites the acquisition of homologous genomic DNA sequences and simplifies the construction of targeting plasmids by making use of defined cloning sites. This approach should result in substantial time and cost savings for appropriate homologous recombination projects.     

Chemical synthesis of S-ribosyl-L-homocysteine and activity assay as a LuxS substrate

Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria. It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2). We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling. Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay. The chemical entities of products from the LuxS reaction were also established. Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.     

Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.     

Honey bee (Hymenoptera: Apidae) foraging in response to preconditioning with onion flower scent compounds

Onion (Allium cepa L.) seed production has long been plagued with yield problems because of lack of pollination by the honey bee, Apis mellifera L. To attempt to attract more pollinators to the onion seed production field, honey bees were conditioned to associate onion floral odor components with a reward. Isolated nucleus hives of honey bees were fed 30% sucrose solutions scented with a 0.2% solution of onion floral odor compounds. After feeding on these solutions for 6 wk, bees were not found to prefer onion flowers to two competing food sources, carrot and alfalfa flowers, at the 5% significance level. However, there was an overall trend indicating a change in honey bee behavior, with fewer "trained" bees visiting alfalfa and carrot and more visiting onion. Thus, it may be possible to alter honey bee behavior with preconditioning but probably not to a degree that would be economically significant.     

Role of claudin interactions in airway tight junctional permeability

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.