Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549

Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K⁺ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K⁺) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K⁺ channels are activated following reoxygenation but not during the hypoxia phase. The K⁺ currents decayed with time following reoxygenation. The decay characteristics of the K⁺ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K⁺ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K⁺ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K⁺ currents. © 1993 Wiley-Liss, Inc.     

Characterization of FP22, a large streptomycete bacteriophage with DNA insensitive to cleavage by many restriction enzymes

Bacteriophage FP22 has a very broad host range within streptomycetes and appeared to form lysogens of Streptomyces ambofaciens ATCC 15154. FP22 shared strong cross-immunity and antibody cross-reactivity with bacteriophage P23, but not with seven other streptomycete bacteriophages. FP22 particles had a head diameter of 71 nm and a tail length of 307 nm. The FP22 genome was 131 kb, which is the largest bacteriophage genome reported for streptomycetes. The G + C content of the genome was 46 mol% and restriction mapping indicated that FP22 DNA had discrete ends. NaCl- and pyrophosphate-resistant deletion mutants were readily isolated and the extent of the deletions defined at least 23 kb of dispensable DNA in two regions of the genome. The DNA was not cleaved by most restriction endonucleases (or isoschizomers) which have been identified in the streptomycetes, including the tetranucleotide cutter MboI (GATC).     

Genomic organization of the rat aromatic L-amino acid decarboxylase (AADC) locus: Partial analysis reveals divergence from the Drosophila dopa decarboxylase (DDC) gene structure

Aromatic L-amino acid decarboxylase (AADC) is responsible for the conversion of L-3,4-dihydroxyphenylalanine (L-DOPA) and L-5-hydroxytryptophan to dopamine and serotonin, respectively, which are important neurotransmitters. We characterized genomic clones derived from the rat AADC locus by Southern blot and nucleotide sequencing analyses to explore the exonal organization of the gene. Our results suggest that the rat AADC gene is relatively large, containing at least 12 exons and spanning at least 40 kb in the rat genome. In this study, nine exons corresponding to 71% of the published cDNA sequence were identified, the smallest of which was as short as 20 base pairs (bp). In the Drosophila dopa decarboxylase (DDC) gene, the sequences homologous to these nine exons are all present in the fourth exon. This implies that either multiple intron sequences have been added to the vertebrate AADC gene or alternatively, deleted from the invertebrate gene after the divergence of vertebrates and invertebrates during evolution.     

Assay of DNA-RNA hybrids by S1 nuclease digestion and adsorption to DEAE-cellulose filters.

A fast and accurate assay procedure for DNA-RNA hybrids is described in which exhaustive digestion of unhybridized DNA with S1 nuclease is followed by binding of hybrids to filter discs of DEAE-cellulose. The digested DNA can be efficiently washed from the filters so that background levels of 0.1-0.2% of input tracer DNA can be achieved, in contrast to the much higher (approximately 1-5%) backgrounds obtained using TCA precipitation procedures. Short duplexes, as small as 36 nucleotides in length, which are inefficiently bound to hydroxyapatite, are quantitatively bound to the DEAE-cellulose filters.     

Does carnitine have a role in fat absorption?

The effect of D-carnitine and tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyltransferase, on intestinal absorption of palmitic acid was determined. The proximal intestinal segment was ligated in adult male rats and filled with 0.5 microCi of 14C-palmitic acid alone or with either D-carnitine or TDGA. Thirty minutes later the radioactivity was determined in the intestinal lumen, intestinal wall and plasma. The absorption of palmitic acid was decreased in the presence of D-carnitine (10 mg/ml) as evidenced by significantly lower levels of radioactivity in the gut wall and the plasma and by significantly greater residual radioactivity in the lumenal contents. L-carnitine had no effect on plasma radioactivity but if D- and L-carnitine were given together the effect of D-carnitine was still in evidence. TDGA also inhibited intestinal absorption of palmitic acid.     

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors

Summary The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the -methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly₇₄ of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.