The effect of pressure on the specific resistance of yeast filter cakes during dead-end filtration in the range 30–500kPa

Suspensions of Kluveromyces marxianus var. marxianus NRRLy2415 and active dry bakers' yeast were dead-end filtered in the range 30–500kPa. In all cases, the specific cake resistance, , could be related to pressure, P, through the expression = 0 (1 kcP) where 0 and kc are empirical constants. For K. marxianus, the values of kc were 1.67 × 10–5 Pa–1 and 2.39 × 10×5 Pa–1 for suspensions with mean cell aspect ratios of 2.98 and 7.33 respectively. Values of kcfor active dry yeast were 10.56 × 10–5 Pa–1 in the case of unwashed cells with a mean aspect ratio of 1.21 and 7.94 × 10–5 Pa–1 for washed cells with a mean aspect ratio of 1.20. © Rapid Science Ltd. 1998     

The effect of 17-N substituents on the activity of the opioid κ receptor in nalfurafine derivatives

We have previously reported the essential structure of the opioid κ receptor agonist nalfurafine hydrochloride (TRK-820) for binding to the κ receptor. In the course of this study, we focused on the effect of the substituent at 17-N in nalfurafine on the binding affinity for the κ receptor. The exchange of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups, which are strong electron withdrawing substituents, almost completely diminished the binding affinities for the μ and δ opioid receptors, but the binding affinity for the κ receptor was still maintained. As a result, nalfurafine derivatives with 17-fluoro-substituted alkyl groups showed higher selectivities for the κ receptor than did nalfurafine itself. With regard to the κ agonistic activities, the conversion of the 17-N substituent in nalfurafine from cyclopropylmethyl to fluoro-substituted alkyl groups led to the gradual decrease of the agonistic activities in the order corresponding to their binding affinities for the κ receptor. In contrast, the derivative with the bulky 17-isobutyl group showed lower affinity and agonistic activity for the κ receptor than the derivatives with the smaller functional groups. This research suggested that both the electronic property and the steric characteristics of the 17-N substituent would have a great influence on the binding property for the κ receptor.     

The effect of staphylococcal δ-haemolysin on the secretory activity of the pancreatic β-cell

The secretion of insulin from isolated rat islets of Langerhans was found to be stimulated by the surface-active staphylococcal exotoxin, -haemolysin. The response was dependent on the concentration of -haemolysin, was rapid in onset, and could be maintained for at least an hour in the presence of the agent. The rate of secretion rapidly declined on removal of -haemolysin and the islets remained responsive to glucose follow!ng toxin treatment.Further characterization of the interaction of this agent with the -cell plasma membrane may provide valuable information concerning the role played by this membrane in the regulation of insulin secretion.     

The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.     

The effect of chondroitin sulphate–protein on the formation of collagen fibrils in vitro

1. It was found that the precipitation of collagen fibrils at 37 degrees from mixtures of chondroitin sulphate-protein and tropocollagen at physiological ionic strength and pH takes place in two distinct phases. The first occurs immediately on mixing either at 4 degrees or at 37 degrees , and the second occurs only at 37 degrees and after a lag phase whose magnitude depends on the proportions of components. 2. When the second stage of precipitation was inhibited by mixing the reactants at 4 degrees , the initial precipitate was found to contain ;native-type' collagen fibrils and chondroitin sulphate-protein. 3. On the basis of kinetic experiments it was concluded that aggregates of chondroitin sulphate-protein and tropocollagen form instantaneously and that these act as sites for the second stage of precipitation of fibrils. 4. The gels that result after continued incubation at 37 degrees are fibrous in appearance if formed in the presence of the initial precipitate of chondroitin sulphate-protein and tropocollagen. 5. On the basis of these experiments in vitro the authors propose a sequence of events for collagen fibrogenesis in vivo.     

The effect of high pressure homogenization on the activity of a commercial β-galactosidase

High pressure homogenization (HPH) has been proposed as a promising method for changing the activity and stability of enzymes. Therefore, this research studied the activity of β-galactosidase before and after HPH. The enzyme solution at pH values of 6.4, 7.0, and 8.0 was processed at pressures of up to 150?MPa, and the effects of HPH were determined from the residual enzyme activity measured at 5, 30, and 45?°C immediately after homogenization and after 1?day of refrigerated storage. The results indicated that at neutral pH the enzyme remained active at 30?°C (optimum temperature) even after homogenization at pressures of up to 150?MPa. On the contrary, when the β-galactosidase was homogenized at pH 6.4 and 8.0, a gradual loss of activity was observed, reaching a minimum activity (around 30?%) after HPH at 150?MPa and pH 8.0. After storage, only β-galactosidase that underwent HPH at pH 7.0 retained similar activity to the native sample. Thus, HPH did not affect the activity and stability of β-galactosidase only when the process was carried out at neutral pH; for the other conditions, HPH resulted in partial inactivation of the enzyme. Considering the use of β-galactosidase to produce low lactose milk, it was concluded that HPH can be applied with no deleterious effects on enzyme activity.     

The inhibitory effect of human interferon α on the generation of lymphokine-activated killer activity

Summary The generation of lymphokine-activated killer (LAK) activity and the proliferative response to human recombinant interleukin-2 (IL-2) were significantly reduced by the presence of human recombinant leukocyte interferon (IFN) in cultures of human peripheral blood mononuclear cells (PBMC). Mature natural killer (NK) cells can be depleted from PBMC with the toxic lysosomotropic agentl-leucine methyl ester. The generation of cytotoxic cells from lymphocytes depleted in leucine methyl ester was also inhibited by indicating that the IFN- effect is not limited to mature cytotoxic NK cells. Depletion of adherent cells from PBMC did not affect the suppression of LAK induction by IFN-. Surface marker analyses of Tac antigen and transferrin receptor (TfR) showed that the presence of IFN throughout the culture period significantly suppressed the typical increase in IL-2-induced Tac- and TfR-positive cells. In contrast, IFN treatment before and after IL-2 culture enhanced LAK cytotoxic activity. Therefore, combinations of these biological response modifiers for clinical use should take into account the dual effect of IFN on key features of the IL-2 response.This work was supported in part by USPHS grant CA34442. Y. T. is a UCLA visiting scientist from the Department of Surgery, School of Medicine, Tokai University, Kanagawa, Japan and recipient of a fellowship from the University of California Cancer Research Coordinating Committee. N. E. is a UCLA visiting scientist from the Department of Surgery, School of Medicine, Tohoku University, Sendai, Japan     

Structure–function studies on jaburetox,a recombinant insecticidal peptide derived from jack bean (Canavalia ensiformis) urease

Background

Ureases are metalloenzymes involved in defense mechanisms in plants. The insecticidal activity of Canavalia ensiformis (jack bean) ureases relies partially on an internal 10 kDa peptide generated by enzymatic hydrolysis of the protein within susceptible insects. A recombinant version of this peptide, jaburetox, exhibits insecticidal, antifungal and membrane-disruptive properties. Molecular modeling of jaburetox revealed a prominent β-hairpin motif consistent with either neurotoxicity or pore formation.

Methods

Aiming to identify structural motifs involved in its effects, mutated versions of jaburetox were built: 1) a peptide lacking the β-hairpin motif (residues 61–74), JbtxΔ-β; 2) a peptide corresponding the N-terminal half (residues 1–44), Jbtx N-ter, and 3) a peptide corresponding the C-terminal half (residues 45–93), Jbtx C-ter.

Results

1) JbtxΔ-β disrupts liposomes, and exhibited entomotoxic effects similar to the whole peptide, suggesting that the β-hairpin motif is not a determinant of these biological activities; 2) both Jbtx C-ter and Jbtx N-ter disrupted liposomes, the C-terminal peptide being the most active; and 3) while Jbtx N-ter persisted to be biologically active, Jbtx C-ter was less active when tested on different insect preparations. Molecular modeling and dynamics were applied to the urease-derived peptides to complement the structure–function analysis.

Major conclusions

The N-terminal portion of the Jbtx carries the most important entomotoxic domain which is fully active in the absence of the β-hairpin motif. Although the β-hairpin contributes to some extent, probably by interaction with insect membranes, it is not essential for the entomotoxic properties of Jbtx.

General significance

Jbtx represents a new type of insecticidal and membrane-active peptide.     

The effect of divalent cations on the catalytic activity of the human plasma 3′-exonuclease

The 3′-exonuclease from human plasma is a soluble form of nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) (EC 3.1.4.1/EC 3.6.1.9). Here, the possibility of divalent cation influence for the 3′-exonuclease activity was investigated using the phosphorothioate congener of oligonucleotide containing all phosphorothioate internucleotide linkages of the [R_P]-configuration ([R_P-PS]-d[T₁₂]) as the substrate for this enzyme. It was found that the 3′-exonuclease is a metalloenzyme, i.e. its phosphodiesterase activity was completely abolished at 0.8 mM concentration EDTA and, in turn, it was restored in the presence of Mg²⁺ or Mn²⁺ ions. In addition, Mg²⁺ can be replaced effectively by Ca²⁺, Mn²⁺, or Co²⁺, but not by Ni²⁺ and Cd²⁺ during the hydrolysis of the phosphorothioate substrate in human plasma. In addition, the mechanism is postulated, by which a single internucleotide phosphorothioate bond of the S_P-configuration at the 3′-end of unmodified phosphodiesters (PO-oligos), or their phosporothioate analogs (PS-oligos) protects these compounds against degradation in blood.     

The effect of degradation on κ-carrageenan/locust bean gum/konjac glucomannan gels at acidic pH

The feasibility of textural and rheological modification of gels containing κ-carrageenan (KC) and locust bean gum (LBG) by addition of konjac glucomannan (KGM) was investigated. Special attention was paid to the effect of polysaccharide degradation during heating at acidic pH. The general effect of polysaccharide degradation was to decrease the Young's modulus, while the fracture strain in extension was scarcely affected unless the degradation was very severe.     

The effect of some ions on the activity of β-galactosidase and the inhibitory effect of tris (hydroxymethyl) aminomethane

The stimulating effect of phosphate and the inhibitory effect of tris-HCl on the activity of β-galactosidase inEscherichia coli was studied. The phosphate anion antagonizes the inhibitory effect of chloride. Since a similar effect is displayed by sulphate and arsenate no specific “stimulating” effect of phosphate can take place. The tris cation has also an inhibitory effect which is antagonized by univalent cations (K⁺). The resulting β-galactosidase activity reflects the antagonisms between cations and anions present in the reaction medium.     

The effects of pH and citrate on the activity of nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria

1. The effect of pH on the co-operative activation of the NAD-specific isocitrate dehydrogenase from pea mitochondria by isocitrate is shown. 2. The interlinked effects of pH on the affinity of the NAD-specific isocitrate dehydrogenase for isocitrate and the dependence of the pH optimum on the substrate concentration are presented. 3. A consideration of the conditions of pH and substrate concentration under which citrate activates the NAD-specific isocitrate dehydrogenase demonstrates similarities between the binding of isocitrate and citrate. 4. A comparison of the effects of citrate and pH on the gross structure of the enzyme is investigated by density-gradient centrifugation. 5. The kinetic interpretations of these results are briefly considered. 6. The metabolic significance of these studies is discussed.     

Structure–activity relationships in aminosterol antibiotics: The effect of stereochemistry at the 7-OH group

Squalamine and three aminosterol analogs have been shown to inhibit bacterial cell growth and induce lysis of large unilamellar phospholipid vesicles. The analogs differ in the identity of the polyamine attached at C3 of the sterol, and the stereochemistry of a hydroxyl substituent at C7. Analogs with a tetraammonium spermine polyamine are somewhat more active than analogs with a shorter trisammonium spermidine polyamine, and analogs with an axial (α) hydroxyl substituent at C7 are more active than analogs with the corresponding equatorial (β) hydroxyl group. There is some variability noted; the 7β-OH spermine analog is the most active compound against Escherichia coli, but the least effective against Pseudomonas aeruginosa. Lytic activity correlates well with antimicrobial activity of the compounds, but the lytic activity varies with the phospholipid composition of the vesicles.     

The Effect of pH on the Electrical Capacitance of Phosphatidylcholine–Phosphatidylserine System in Bilayer Lipid Membrane

This paper reports measurements on the pH dependence of the electrical capacitance of lipid membranes formed by 1:1 phosphatidylcholine-phosphatidylserine mixtures. A theoretical model was developed to describe this dependence, in which the contributions of functional groups (as the active centers of adsorption of the hydrogen and hydroxide ions) to the overall membrane capacitance were assumed to be additive. The proposed model was verified experimentally using electrochemical impedance spectroscopy. The theoretical predictions agreed with the experimental results over the measured pH range. A minimum corresponding to the isoelectric point appeared in both the theoretical equation and the experimental data.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

The effect of pH on the characteristics of the binding of nicotinamide–adenine dinucleotide by nicotinamide–adenine dinucleotide specific isocitrate dehydrogenase from pea mitochondria

1. The effect of pH on the V(max.) and concentration of NAD(+) at half-maximum velocity at a constant isocitrate concentration was examined, and the results were related to the requirements for binding of H(+) ions to the enzyme. 2. The effect of varying the NAD(+) concentration on the pH optimum with constant isocitrate concentration was studied. 3. A comparison has been made between the effect of isocitrate concentration on the characteristics of binding of NAD(+) and the effect of NAD(+) concentration on the characteristics of isocitrate binding at three different pH values. 4. The mechanistic and metabolic significance of these studies is considered.     

The effect of energization on the apparent Michaelis–Menten constant for oxyge in nmitochondrial respiration

Lineweaver-Burk plots of 1/v against 1/[O(2)] for rat liver mitochondrial respiration with succinate or ascorbate+NNN'N'-tetramethyl-p-phenylenediamine as substrates are non-linear. In state 3u (uncoupled by trifluoromethoxycarbonyl cyanide phenylhydrazone) such plots tend to be concave upward, whereas in state 4 (energized) the plots were concave downward. The apparent K(m) for oxygen is larger in state 4 than in state 3u, despite the higher turnover in the latter system. It is postulated that at least one reversible reaction occurs between cytochrome c and cytochrome c oxidase, whose rate is increased on energization (reversed electron transfer); a model including such a reaction is proposed which accounts semiquantitatively for the observations.