Protease-deficient Bacillus subtilis host strains for production of Staphylococcal protein A.

We constructed strains of Bacillus subtilis which produced very low levels of extracellular proteases. These strains carried insertion or deletion mutations in the subtilisin structural gene (apr) which were constructed in vitro by using the cloned gene. The methods used to construct the mutations involved the use of a plasmid vector which allowed the selection of chromosomal integrates and their subsequent excision by homologous recombination to effect replacement of the chromosomal apr gene by a derivative carrying an inactivating insert with a selectable marker (a cat gene conferring chloramphenicol resistance). The strains produced no subtilisin, no detectable extracellular metalloprotease activity, and residual extracellular serine protease levels as low as 0.5% of that of the standard strain from which they were derived. The strains proved to be superior host strains for the production of staphylococcal protein A, accumulating higher levels of intact protein than do previously available B. subtilis strains.     

In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human alpha 2-macroglobulin

Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.     

Mathematical analysis of cell-target encounter rates in two dimensions. The effect of chemotaxis.

The process by which cells encounter their targets is the first step of a number of cell functions involved in the immune response, such as cell-mediated cytotoxicity and phagocytic ingestion of foreign material. In many instances, this encounter may be rate-limiting, and therefore it is important to understand what factors influence the encounter rate. One key aspect of cell-target encounter is the motility behavior of the cell in the vicinity of a target. This movement may be entirely random, or there may be a directed, or chemotactic, component to it. In this paper we focus on the effects of cell motility properties, and particularly the chemotactic directional bias, on the rate of cell-target encounter. Specifically, we derive an expression for the mean encounter time of cells that meet targets in two dimensions as a function of the cells' directional orientation bias. We show that a modest degree of bias can reduce the mean encounter time by orders of magnitude, while nearly perfect directional bias offers little additional benefit. We illustrate the application of these results to a particular example system: alveolar macrophages removing inhaled particles and bacteria from the lung surface.     

Effects of hypoxia on lung mechanics in the newborn cat

The present study was designed to investigate the effects of hypoxia on lung mechanics in the newborn cat and to determine if vagal efferent innervation to the airways is involved in the response. We studied 11 animals, aged 2-7 days, anesthetized with a mixture of chloralose-urethane administered intraperitoneally. A tracheal cannula was inserted just below the larynx and following paralysis (pancuronium bromide), mechanical ventilation was initiated. A pneumothorax was created by a midline thoracotomy and an end-expiratory load was applied to maintain functional residual capacity. Animals were placed in a flow plethysmograph from which measurements of transpulmonary pressure, flow, and volume, mean inspiratory resistance, and dynamic compliance of the lung were calculated. The experimental protocol consisted of a series of 8-min trials, each preceded by a controlled volume history. The hypoxia challenge was composed of 1 min of ventilation with 40% O2, followed by 5 min exposure to 10% O2 and 2 min of recovery. In the majority of animals (7 out of 11), hypoxia had no effect on lung mechanics compared with control trials. Four animals responded to hypoxia with an increase in resistance and a decrease in compliance. Resistance remained elevated throughout the hypoxia with an average maximal increase of 47.2 +/- 22.2% (SD). Dynamic compliance was significantly decreased at the 2nd, 3rd, and 4th min only of hypoxia. Bilateral vagotomy abolished the response in the four animals and hypoxia had no effect on mechanics postvagotomy. Our data suggest that in most cases changes in lung mechanics do not play a causal role in the biphasic ventilatory response to hypoxia seen in the newborn.     

Cardiovascular effects of training for a marathon run in unfit middle aged men.

The effects of a 30 week exercise programme on serum lipid values, blood pressure, and cardiac function were assessed in a group of sedentary men aged 35-50 training for their first marathon. Mean serum cholesterol concentration (n = 33) fell by 12% from 6.54 (SE 0.18) to 5.76 (0.15) mmol/l (mean fall 0.78 mmol/l; 95% confidence interval 0.52 to 1.04 mmol/l), serum triglyceride concentration (n = 33) by 22% from 1.56 (0.17) to 1.21 (0.09) mmol/l (mean fall 0.34 mmol/l; 95% confidence interval 0.12 to 0.56 mmol/l), and mean blood pressure (n = 27) by 10% from 102 (2) to 92 (2) mm Hg (mean fall 10 mm Hg; 95% confidence interval 7 to 13 mm Hg). These changes were not explained by changes in body composition. Peak exercise left ventricular end diastolic volume (n = 16) increased with training; as a result of this and an increased exercise left ventricular ejection fraction peak exercise cardiac output increased from 19.9 (1.2) to 23.1 (3.0) l/min (mean rise 3.2 l/min; 95% confidence interval 1.5 to 5.0 l/min). Maximum oxygen consumption increased from 33.9 (1.6) to 39.0 (1.3) ml/kg/min (mean rise 5.0 ml/kg/min; 95% confidence interval 1.8 to 8.2 ml/kg/min). This study showed favourable effects on coronary risk factors and cardiac function and supports the place of regular exercise in coronary prevention programmes.     

The effect of streptozotocin-induced diabetes on phenylalanine hydroxylase expression in rat liver.

The impact of experimentally induced diabetes on the expression of rat liver phenylalanine hydroxylase has been investigated. A significant elevation in maximal enzymic activity was observed in diabetes. This was associated with significant increases in the amount of enzyme, the phenylalanine hydroxylase-specific translational activity of hepatic RNA and the abundance of phenylalanine hydroxylase-specific mRNA. These changes in phenylalanine hydroxylase expression were not observed when diabetes was controlled by daily injections of insulin. These results are discussed in relation to the hormonal control of phenylalanine hydroxylase gene expression.     

Transmembrane signaling: tumor promoter distribution

Diacylglycerol plays a critical role in transmembrane signaling by activating protein kinase C (PKC). The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) mimics that action, and in the human erythrocyte, TPA-activated PKC phosphorylates membrane proteins. Although molecular aspects of this process have been investigated, details of the interaction of TPA with plasma membranes remain elusive. Because TPA is hydrophobic, it has been assumed that it associates with the lipid bilayer. However, there is no direct evidence for its transbilayer distribution. Because knowledge of its location would limit molecular models proposed to explain its mode of action, we have used membrane-splitting techniques, based on freeze-fracture of planar cell monolayers, to quantify transmembrane partitioning of [3H]TPA. Under conditions where PKC-mediated phosphorylation was stimulated by [3H]TPA and where more than 90% of the [3H]TPA was associated with the human red cell plasma membrane, two-thirds of the TPA partitioned with the cytoplasmic leaflet after bilayer splitting. This represents the first direct topographic localization of TPA in a biological membrane and supports the hypothesis that the mechanism of TPA activation requires its association with the cytoplasmic leaflet of the bilayer.     

Nucleotide and deduced amino acid sequence of the hydrophobic surfactant protein SP-C from rat: expression in alveolar type II cells and homology with SP-C from other species

Pulmonary surfactant lowers surface tension in the lung. Its deficiency leads to the severe physiologic abnormalities seen in the respiratory distress syndrome. The hydrophobic surfactant proteins, SP-B and SP-C, appear to be especially important in the surface-spreading characteristics of pulmonary surfactant. We report the nucleotide sequence of cDNA clones for rat SP-C and compare the deduced amino acid sequence for SP-C from several species. A highly conserved domain exists within the confines of mature human SP-C. An Eisenberg plot of this region predicts a membrane-associated helix. We also demonstrate by Northern analysis the tissue-specific expression of SP-C. A comparison of signal strength between total lung RNA and RNA derived from isolated type II cells supports the idea that most SP-C messenger RNA in total lung can be accounted for by that present in alveolar type II cells.     

Detection of surface charge-related properties in model membrane systems by aqueous two-phase partition

Unilamellar vesicles composed of phosphatidylcholine (PC) and either phosphatidic acid (PA) or phosphatidylglycerol (PG) partition to the upper poly(ethylene glycol) (PEG)-rich phase of a charge-sensitive 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 7, consistent with the vesicles bearing a net negative charge. When prepared in the presence of a pH gradient (interior acidic), PC/PA vesicles exhibit an increased partition to the top PEG-rich phase, consistent with a redistribution of the PA from the inner to the outer monolayer of the vesicle bilayer. Conversely, when prepared in the presence of a pH gradient (interior basic), PC/PG vesicles exhibit a decreased top-phase partition, consistent with a redistribution of the PG from the outer to the inner monolayer of the vesicle bilayer. Unilamellar vesicles composed of PC and stearylamine partition to the lower dextran-rich phase of a 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 8.5, consistent with the vesicles bearing a net positive charge. When prepared in the presence of a pH gradient (interior acidic), conditions under which the stearylamine is trapped on the inner monolayer of the bilayer, the vesicles now partition predominantly to the interface in a manner similar to vesicles composed of PC alone. These results demonstrate that partitioning in aqueous two-phase polymer systems is a sensitive method for monitoring the asymmetry of charged lipids in model membrane systems and also suggests that partitioning in charge-sensitive systems depends only on the physical nature of the exterior surface of the membrane.