Inhibitory Effect of Some Gaseous Hydrocarbons on the Cell-lysis of Micrococcus lysodeikticus by Egg-white Lysozyme

The cell-lysis of Micrococcus lysodeikticus by egg-white lysozyme was inhibited by some nonpolar hydrocarbons such as n-butane, isobutane or propane. The lysozyme-induced cell-lysis was, however, no longer inhibited when the same hydrocarbons were added after the cell-lysis had already begun. Bubbling air through the cell suspensions, in which the lysozyme-induced cell-lysis had been inhibited in advance by the hydrocarbons, restored the event of cell-lysis to some extents. The Lineweaver-Burk plots indicated the kinetics of competitive inhibition of the lysozyme-induced cell-lysis by these hydrocarbons. From these results, the gaseous hydrocarbons were assumed to exert little influence on the enzymatic activity of egg-white lysozyme itself, that is the hydrolysis of β-1,4-glucosaminide linkage of the mucopeptide chain in the cell wall, but inhibit competitively the binding of lysozyme to the cells.     

Heterologous protein secretion from Aspergillus niger in phosphate-buffered batch culture

Summary Aspergillus niger was grown in batch culture containing various initial concentrations of sodium phosphate buffer (pH 6.5). A wild-type strain of A. niger and a transformed strain producing hen egg-white lysozyme were studied. The maximum cell yield was attained in medium not supplemented with phosphate. In those cultures acidification of the medium resulted in a minimum of pH 2.0 before reverting to near neutrality. Increasing the initial levels of phosphate buffer reduced the fall in pH but lowered cell yields. Secreted levels of lysozyme were maximal in the 50–100 mm range of added phosphate buffer although mycelial yields were reduced by one third of mycelial yields in medium unsupplemented with phosphate buffer. Offprint requests to: D. B. Archer     

The T cell repertoire to a multideterminant antigen. Clonal heterogeneity of the T cell response, variation between syngeneic individuals, and in vitro selection of T cell specificities.

The specificity of lysozyme-induced T cell proliferative responses by individual B10.A mice was compared by using a panel of three peptides. A surprising degree of variation in the focus of the responses was observed among individual animals, both in the newly isolated lymph node cell populations and in long term T cell lines. The responses to each determinant after hen egg-white lysozyme immunization were not equal and in examining the mice as a group some determinants tended to be dominant. However, despite each animal favoring a restricted number of determinants, the responding T cell populations were still highly heterogeneous. The data suggest that many determinants are involved in the response to the whole Ag. The role of one or more dominant determinants can be overestimated because the degree of heterogeneity in long term T cell lines appears to be less than in freshly obtained lymph node cells, indicating that a process of in vitro selection occurs. We observed that the T cells responsive to one peptide, 46-61, appeared to have a selective advantage in vitro culture.     

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μ_max = 0.19 h⁻¹, K _s = 2.9 μg/l, K _i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE. Received: 16 July 1998 / Received revision: 11 November 1998 / Accepted 31 November 1998     

Chemosensory responses of Tetrahymena thermophila to CB2, a 24-amino-acid fragment of lysozyme

While lysozyme is a depolarizing chemorepellent in Tetrahymena, the entire lysozyme molecule is not necessary to activate the lysozyme receptor. Reduced lysozyme was cut into three fragments by cyanogen bromide cleavage and the fragments (CB₁, CB₂ and CB₃) were separated by HPLC. Behavioral bioassays showed that the carboxy-terminal 24-amino-acid fragment, which we call CB₂, is 100 times more active than intact lysozyme as a chemorepellent. CB₂ appears to activate the same receptor as lysozyme because behavioral cross-adaptation is seen between these two compounds and an antibody generated to the purified lysozyme receptor blocks responses to both lysozyme and CB₂. This is further supported by the observation that neomycin, which is a competitive inhibitor of lysozyme binding, also inhibits CB₂ responses. This inhibition may be due to the fact that neomycin is highly positively charged (+5 at pH 7.0) and CB₂ has a net charge of +4 at pH 7.0. Intracellular electrophysiological recordings documented that CB₂ elicits a transient, depolarizing receptor potential that is similar to the lysozyme-induced depolarizations except they are much smaller. CB₂ is a more potent and specific ligand for use in studies of the lysozyme receptor of Tetrahymena. Accepted: 21 February 1999     

The lysozyme of Nephthys hombergii (annelid)

Summary The enzymatic properties ofNephthys hombergii lysozyme were compared with those of hen egg-white lysozyme. The observed results allowed to conclude that if similarities between both enzymes could be established (variation of the initial velocity of lysis as a function of the ionic strength, influence of urea and histamine), it was nevertheless possible to differentiate them clearly by their affinity constants forM. luteus cells, by the inhibitory power of N-acetylglucosamine and by their action on chitopentaose, chitotetraose and colloidal chitin.94th communication on lysozymes. This paper is based in part on a thesis submitted by J.-P. P. in partial fulfillment of the requirements for the degree of Docteur ès Sciences, Paris (AO 6057).     

Regulation of protein synthesis in hen's oviducts. II. Extracellular ovalbumin as an inhibitor of ovalbumin synthesis in the oviducts.

Washed, preincubated minced hen's oviducts, which contained low levels of extracellular and intracellular proteins, synthesized egg-white proteins actively. The addition of ovalbumin to the incubation medium resulted in inhibition of the synthesis of egg-white proteins by the washed, preincubated oviduct cells, while the addition of bovine serum albumin seemed to stimulate protein synthesis and hen's egg-white lysozyme had no effect. The inhibitory or stimulatory effect on protein synthesis was proportional to the amount of protein added to the medium. The inhibitory effect of added ovalbumin was shown not to be due to the incorporation of ovalbumin into the oviduct cells from the incubation medium. Egg-white proteins added to the medium also inhibited protein synthesis inside the cells and the extent of the inhibition appeared to correspond to the amount of ovalbumin present in egg-white.     

General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

Mutant of Bacillus subtilis Demonstrating the Requirement of Lysis for Growth

A temperature-sensitive mutant of Bacillus subtilis which has impaired ability to autolyze was isolated. The growth rate of mutant cells at high temperature can be increased by the addition of egg-white lysozyme or a B. subtilis autolysin. Experiments with this mutant show that lysis is essential for cell growth.     

Host plant constituents as oviposition stimulants for a generalist herbivore: European corn borer

Oviposition response of the polyphagous European corn borer,Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae), to chemical constituents in host plants was investigated in the laboratory using two-choice bioassays. Foliar extracts of corn (Zea mays L.), pepper (Capsicum annuum L.) and potato (Solanum tuberosum L.) were prepared using the solvents pentane, acetone and methanol. In all three host plants, chemicals soluble in pentane stimulated oviposition. In potato, chemicals extractable in acetone also elicited a positive oviposition response. When presented with a choice between pentane extracts of corn and pepper, females preferred corn. No preferences were exhibited between pentane extracts of corn and potato or pepper and potato. Pentane extracts of corn husks, tassels, silk, and corn leaves from plants at early whorl and tassel (pre-pollen shed) stages of development also stimulated oviposition. Similar extracts from plants at 2-leaf and blister (when kernels resemble blisters) stages were not stimulatory. This indicates that plant phenology affects chemically mediated oviposition response in European corn borer. The potential use of plant chemicals for management ofO. nubilalis in the field is suggested.     

Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.     

On the inhibition of hen egg-white lysozyme activity by aminoglycosidic antibiotics.

Lytic activity of hen egg-white lysozyme towards bacterial cells of Micrococcus lysodeikticus was pH-dependent inhibited by several aminoglycosidic antibiotics, the structure of which is related to the saccharidic substrates of the enzyme.Inhibition extent suggests the role of the positive charges of this type of antibiotics on the mechanism of lysozyme activity inhibition.     

Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

Human airway lysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-1 at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteristic of the peptide backbone (e.g., beta-sheet; C alpha-C, C alpha-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% alpha-helix, 25% and 20% beta-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.     

The Steady State and Time-resolved Fluorescence Studies on the Lysozyme-Ligand Interaction

The conformational change of hen egg-white lysozyme (EC 3.2.1.17) induced by the interaction with tri-N-acetyl-D-glucosamine were investigated by steady state and time-resolved fluorescence spectroscopy. To identify more clearly the conformation of hen egg-white lysozyme interacting with the ligand, the fluorescence decay kinetics of the lysozyme and its complex with the ligand were precisely measured at their full spectral regions. The spectral analysis based on the time-resolved studies showed that the binding of the ligand affected not only the Trp62 directly linked to the ligand but its influence was extended to the vicinity of Trp108 and further to the hydrophobic matrix box region. Near the binding site, the intramolecular distance between Trp108 and Glu35 was expanded or contracted depending on the pH of the buffer solution. On the other hand, the interaction of Trp28 and/or Trp111 with their surroundings was reduced by restriction of fluctuational motions at the hydrophobic matrix box region.     

Lysis of lyophilizedEscherichia coli cells with egg-white lysozyme without ethylenediaminetetraacetic acid

A simple method of lysis of lyophilized cells ofEscherichia coli is described, using egg-white lysozyme in the absence of ethylenediaminetetraacetic acid (EDTA).     

Segmental Motion of Subsite C of Hen Egg-White Lysozyme: Fluorescence Depolarization Studies of Kyn62-Lysozyme as an Active Analogue

The dynamical properties of subsite C of hen egg-white lysozyme were investigated using Kyn62-lysozyme as an active analogue. Time-resolved fluorescence depolarization studies showed that the segmental motion of kynurenine which was important in subsite C was described with two components of which the rotational correlation times were ?₁ = 150 ps and ?₂ = 1.4 ns, respectively. Although these two segmental motions retained 90% of motional freedom, the slower motion was completely restricted and the degree of freedom was lost to 40% during the interaction with oligomers of N-acetyl-D-glucosamine.     

Elevated breath pentane in heart failure reduced by free radical scavenger

Pentane, a product of lipid peroxidation, has been detected in situations involving ischemic injury. Such injury may be limited if lipid peroxidation can be controlled by antioxidants. The role of lipid peroxidation in chronic heart failure (CHF) was assessed by measuring breath pentane in patients with CHF vs. age matched controls. The effect of a free radical scavenger on pentane released during CHF was also measured. Pentane levels were correlated with the daily dose of captopril, a sulfhydril-containing drug used to treat CHF, which is an angiotensin converting enzyme inhibitor. To separate the scavening effects of captopril from the pharmacologic effects of converting enzyme inhibitors, a crossover study using a nonsulfhydril inhibitor was used. Patients with CHF excreted (p < 0.005) hich concentrations of pentane (5.7 ± 2.1 vs. control 3.6 ± 1.2 nmol/l). Patients treated with captopril also had significantly higher (p < 0.05) excretion of pentane than the control patients (4.7 ± 1.3 vs. 3.6 ± 1.2 nmol/l). The dose of captopril was inversely proportional to the concentration of pentane excreted (r = 0.55, p < 0.05). Pentane excretion during captopril therapy was significantly lower before (p < 0.01) and after (p < 0.02) nonsulfhydril inhibitor therapy. Conclusion: breath pentane is elevated in CHF and it can be reduced by a free radical scavenger. This reduction of pentane excretion is not a converting enzyme inhibitor class effect.     

Lysis efficiency of standard DNA extraction methods for Halococcus spp. in an organic rich environment

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.     

Electron microscopy of Staphylococcus aureus cell wall lysis

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Mu?oz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.     

Electrical conductivity of single crystals of lysozyme

The dc electrical conductivity of large single crystals of hen egg-white lysozyme has been measured. The samples were grown from aqueous solution and dried in air with silica gel. The temperature dependence of the conductivity obeyed the relation σ = σ₀ exp(? ΔE/kT), with ΔE = 1.2 eV. The ΔE value agreed with most of the previous results for various proteins in the form of lyophilized powder. On the other hand, log σ₀, being between 7 and 11, was much larger than the previously reported values and differed among the samples. An irreversible decrease in σ₀, without affecting ΔE, was observed on heating the samples above 85°C. It was shown that the set of results can be explained if the charge carriers responsible for the observed conduction are regarded as protons, originating from residual water molecules. Photoresponse of the samples to uv radiation below 305–315 nm was also observed. Reproducible and reliable results were obtained relatively easily in the present experiments, which is thought to be the main advantage of using single-crystalline samples.