Mechanism of resistance to benzalkonium chloride by Pseudomonas aeruginosa.

The mechanisms of resistance of Pseudomonas aeruginosa to benzalkonium chloride (BC) were studied. The effluence of cell components was observed in susceptible P. aeruginosa by electron microscopy, but resistant P. aeruginosa seemed to be undamaged. No marked changes in cell surface potential between Escherichia coli NIHJC-2 and a spheroplast strain were found. The contents of phospholipids (PL) and fatty and neutral lipids (FNL) in the cell walls of resistant P. aeruginosa were higher than those in the cell walls of susceptible P. aeruginosa. The amounts of BC adsorbed to PL and FNL of cell walls of BC-resistant P. aeruginosa were lower than those for BC-susceptible P. aeruginosa. Fifteen species of cellular fatty acids were identified by capillary gas chromatography and gas chromatography-mass spectrometry. The ability of BC to permeate the cell wall was reduced because of the increase in cellular fatty acids. These results suggested that the resistance of P. aeruginosa to BC is mainly a result of increased in the contents of PL and FNL. In resistant P. aeruginosa, the decrease in the amount of BC adsorbed is likely to be the result of increases in the contents of PL and FNL.     

Evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin L in lysosomes

Our recent studies have shown that cathepsin L is first synthesized as an enzymatically inactive proform in endoplasmic reticulum and is successively converted into an active form during intracellular transport and we postulated that aspartic proteinases would be responsible for the intracellular propeptide-processing step of procathepsin L accompanied by the activation of enzyme (Y. Nishimura, T. Kawabata, and K. Kato (1988) Arch. Biochem. Biophys. 261, 64-71). To better understand this proposed mechanism, we investigated the effect of pepstatin, a potent inhibitor of aspartic proteinases, on the intracellular processing kinetics of cathepsin L analyzed by pulse-chase experiments in vivo with [35S]methionine in the primary cultures of rat hepatocytes. In the pepstatin-treated cells, the proteolytic conversion of cellular procathepsin L of 39 kDa to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 5-h chase periods. Further, the subcellular fractionation experiments demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the light density microsomal fraction after 2 h of chase. These results suggest that pepstatin treatment caused a significant inhibitory effect on the intracellular processing and also on the intracellular movement of procathepsin L from the endoplasmic reticulum to the lysosomes. These findings provide the first evidence showing that aspartic proteinase may play an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo. Therefore, we suggest that cathepsin D, a major lysosomal aspartic proteinase, is more likely to be involved in this proposed model in the lysosomes.     

Brefeldin A inhibits the targeting of cathepsin D and cathepsin H to lysosomes in rat hepatocytes

Effect of brefeldin A on the transport of lysosomal acid hydrolases (cathepsins D and H) was investigated in primary cultured rat hepatocytes. Both cathepsins were synthesized as proenzymes and progressively converted to mature enzymes in the control cells. However, BFA strongly inhibited the appearance of the mature enzymes in the cells in a dose dependent manner, suggesting that transport of newly synthesized lysosomal enzymes from the endoplasmic reticulum to lysosomes is blocked by the drug. The inhibitory effect by brefeldin A was reversible. Upon recovery from brefeldin A-intoxication, procathepsin D was effectively targeted into lysosomes, whereas a substantial amount of procathepsin H was found to be missorted, resulting in its secretion into the culture medium.     

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.     

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible involvement of actomyosin ADP complex in regulation of Ca2+ sensitivity in alpha-toxin permeabilized smooth muscle

The effects of substrate condition and ADP beta S on the pCa2+-tension relationships were investigated, using alpha-toxin permeabilized rabbit mesenteric artery at 37 degrees C. The contraction induced by 10 microM Ca2+ solution after permeabilization was as large as that induced by 145 mM K+ PSS solution containing 10 microM NE in the intact tissue, indicating that the majority of the cells were permeabilized. The Ca2+ sensitivity was greatly affected by the substrate condition and increasing the ratio of ATP/CP induced a leftward shift of the pCa2+-tension curve. Addition of 100 microM ADP beta S had a similar effect. When the ATP/CP ratio was high, the 0.1 microM Ca2+ solution relaxed the tissue precontracted by 10 microM Ca2+ solution more slowly showing hysteresis. One mM vanadate, which is reported to relax muscle by forming actomyosin-ADP-Vi (AM-ADP-Vi), completely inhibited both contractions induced by 0.18 microM Ca2+ solution containing 2 mM MgADP and 0.3 microM Ca2+ solution containing 0.3 microM PDBu. These results indicated that the population of AM-ADP complex in the crossbridge had increased due to the accumulation of ADP inside the tissue or activation of PKC and that the inhibition of ADP release from AM-ADP complex may be playing a key role in increasing Ca2+ sensitivity of myofilaments.     

Possible involvement of bone cells in a new cementum formation

Cells from the gingival lamina propria, bone-derived granular tissues and periodontal ligament (PDL) were isolated after periodontal surgery and subsequently cultured in vitro. The resulting cells were defined as gingival cells, bone cells and PDL cells, respectively. Under a phase contrast microscope, the cultured cells exhibited a spindle and/or a polyhedral shape. On the basis of their appearance under an electron microscope, spindle-shaped cells and polyhedral-shaped cells were identified as fibroblasts and osteoblasts, respectively. Bone cells, a homogeneous population of osteoblasts, had a more rapid growth ability than PDL cells, which were a heterogeneous population of fibroblasts and osteoblasts. Of particular interest was that only bone cells produced bone matrix in the multilayers in vitro. These results support the hypothesis that the phenotype expressed by cells from the alveolar bone establishes a new concept for progenitor cells in the formation of cementum.     

High cell density suspension culture of mammalian anchorage independent cells: Oxygen transfer by gas sparging and defoaming with a hydrophobic net

Gas sparging directly into the culture-broth is not done in cell culture, except when the gas flow rate is very small, because much foaming occurs.During screening of defoaming methods, foam was observed to be broken up effectively when it made contact with a net fabricated from hydrophobic materials. Providing a highly efficient oxygen supply to suspension culture was tried using the new defoaming method. In a 5 1 reactor equipped with the foam-eliminating net fabricated with polysiloxane, oxygen was transferred at 21 mmole/l·h equivalent to an about forty-fold higher rate than in conventional surface aeration. This was equivalent to a consumption rate of 1×10⁸ cells/ml, even at a low oxygen gas flow rate of 0.1 cm/s corresponding to a fourth of the gas flow rate when foam leaked through the net.Perfusion culture of rat ascites hepatoma cell JTC-1 was successfully carried out in the 51 scale culture system with the net and a hydrophobic membrane for cell filtration. The viable cell concentration reached 2.7×10⁷ cells/ml after twenty-seven days, in spite of the nutrient-deficient condition of the lower medium exchange rate, that is, a working volume a day, and viability was maintained at more than 90%. In a 1.21 scale culture of mouse-mouse hybridoma cell STK-1, viable cell concentration reached 4×10⁷ cells/ml. These results showed that oxygen transfer by gas sparging with defoaming was useful for high density suspension culture. A foam-breaking mechanism was proposed.Abbreviations Eagle's MEM Eagle's minimal essential medium - Dulbecco's modified Eagle MEM Dulbecco's modified Eagle minimal essential medium     

Synthesis of a gene for the protein kinase domain of the epidermal growth factor receptor and its expression in Escherichia coli

A gene encoding the protein kinase domain of the epidermal growth factor receptor has been chemically synthesised, cloned and expressed in Escherichia coli. The 942-base-pair gene was constructed by enzymatic ligation of 56 oligonucleotides and cloned into an expression vector downstream of the E. coli trp promoter. Production of active gene product was confirmed by means of a protein kinase assay, demonstrating that the enzymatic activity of the protein kinase domain of the epidermal growth factor receptor is retained after expression in E. coli.     

Sequence-dependant polymorphism of DNA duplex in solutions

Raman spectra of six synthetic polydeoxyribonucleotide duplexes with different base sequences have been examined in aqueous solutions with different salt or nucleotide concentrations. Detailed conformational differences have been indicated between B and Z forms of poly[d(G-C)] X poly[d(G-C)], between B forms of poly[d(G-C)] X poly[d(G-C)] and poly[d(G-m5C)] X poly[d(G-m5C)], between A and B forms of poly(dG) X poly(dC), between B and "CsF" forms of poly[d(A-T)] X poly[d(A-T)], between B forms of poly[d(A-U)] X poly[d(A-U)] and poly[d(A-T)] X poly[d(A-T)], and between low- and high-salt (CsF) forms of poly(dA) X poly(dT). The Raman spectrum of calf-thymus DNA in aqueous solution was also observed and was compared with the Raman spectra of its fibers in A, B, and C forms.