Endotoxemia increases relative perfusion to dorsal-caudal lung regions.

Changes in the spatial distribution of perfusion during acute lung injury and their impact on gas exchange are poorly understood. We tested whether endotoxemia caused topographical differences in perfusion and whether these differences caused meaningful changes in regional ventilation-to-perfusion ratios and gas exchange. Regional ventilation and perfusion were measured in anesthetized, mechanically ventilated pigs in the prone position before and during endotoxemia with the use of aerosolized and intravenous fluorescent microspheres. On average, relative perfusion halved in ventral and cranial lung regions, doubled in caudal lung regions, and increased 1.5-fold in dorsal lung regions during endotoxemia. In contrast, there were no topographical differences in perfusion before endotoxemia and no topographical differences in ventilation at any time point. Consequently, endotoxemia increased regional ventilation-to-perfusion ratios in the caudal-to-cranial and dorsal-to-ventral directions, resulting in end-capillary PO2 values that were significantly lower in dorsal-caudal than ventral-cranial regions. We conclude that there are topographical differences in the pulmonary vascular response to endotoxin that may have important consequences for gas exchange in acute lung injury.     

Oat mesophyll protoplasts: their response to various feeder cultures

Oat (Avena sativa L.) mesophyll protoplasts were recently demonstrated to be capable of dedifferentiation, repeated divisions, and colony formation. Since the development of oat mesophyll protoplasts is decisively influenced by the nature of the used feeder culture (species, variety and concentration), we conducted a systematic study of this parameter. Generally, graminaceous feeders promoted protoplast proliferation, while dicot species repressed protoplast divisions. The beneficial effect of those feeders that promote divisions was counterbalanced by a factor that causes necrosis. The correct balance between promotion of divisions or necrosis depended on the nature of the feeder and its plating density.     

Insertion of new sequences into the catalytic domain of an enzyme

Activities of enzymes can be modified by the replacement of active-site amino acids with residues that strengthen specific interactions with substrates or that alter the specificity. The scope for engineered enzymes would be broadened if additional, new sequences could be inserted into a catalytic domain. Properly designed, these sequences could encode new ligand binding sites, be intermediates in the construction of chimeric enzymes, or alter the internal flexibility and "breathing" modes of the active-site region. As a first step toward this objective, we inserted oligopeptides of up to 14 amino acids into various locations within an 82 amino acid region of the adenylate synthesis domain of Escherichia coli methionyl-tRNA synthetase. These sites include ones that are flanked by sequences that are conserved between the proteins from E. coli and the yeast Saccharomyces cerevisiae and those that are essential for activity and stability. We found that all of the insertional mutants are stable and some have catalytic parameters for adenylate synthesis that are comparable to those of the wild-type enzyme. Thus, such an approach may provide for a variety of novel applications.     

Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments

Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.     

Synthesis of two polypeptide subunits of an aminoacyl tRNA synthetase as a single polypeptide chain

E. coli aminoacyl tRNA synthetases are typically comprised of a single type of polypeptide chain. Glycine tRNA synthetase is an exception, and is comprised of two different subunits. Previous work showed that glyS encodes both subunits in a tandem arrangement of coding regions which are in the same reading frame. Nine nucleotides separate the TAA stop of the first coding segment (alpha-subunit) from the ATG start of the second one (beta-subunit). A plasmid containing glyS was put into four different ochre suppressor strains. In three of them, significant quantities of an alpha-beta fusion protein were synthesized in maxicells, in genetic backgrounds which retained cellular proteases. This shows that the fusion protein is stable in vivo and suggests that Gly-tRNA synthetase is operationally a single polypeptide which is the ancestor of the two subunits.     

A mutation in the small (alpha) subunit of glycyl-tRNA synthetase affects amino acid activation and subunit association parameters

A strategy was designed to isolate mutants of glycyl-tRNA synthetase that are altered at the amino acid binding site, including a class with altered amino acid specificity. For this purpose, the plasmid pBR322 was mutated so that the codon (AGC) of the active site Ser-68 in the beta-lactamase gene was changed to the glycine codon GGC to inactivate the encoded enzyme. Suppressors that increase the amount of beta-lactamase activity of the Gly-68 allele of beta-lactamase were isolated and some mapped to the gene encoding glycyl-tRNA synthetase (glyS). While in vitro misaminoacylation of tRNA(Gly) with serine was not detected for any of the mutants, glycyl-tRNA synthetase activity was altered. One severely affected glyS mutant (N302) was studied in more detail. For this mutant, a single Pro-61----Leu substitution in the alpha chain confers an elevation of the Km values for glycine (25-fold) and for ATP (45-fold) in the aminoacylation reaction, but only a minor perturbation of the Km for tRNA. There also was a severely reduced adenylate synthesis activity (greater than 100-fold). In addition, a nonlinear dependence between aminoacylation activity and enzyme concentration was observed which implies that the alpha chain Pro-61----Leu mutation has disrupted the functionally essential subunit interactions of the holoenzyme. The results of the preceding paper have shown that the alpha chain and parts of the beta chain are required for aminoacylation and adenylate synthesis activity. The results of this study suggest that the alpha chain specifically contributes to amino acid and to ATP binding in a way that is affected by proper subunit interactions.