Adsorption of monovalent cations to bilayer membranes containing negative phospholipids.

The electrophoretic mobilities of multilamellar phosphatidylserine vesicles were measured in solutions containing monovalent cations, and the xi potentials, the electrostatic potentials at the hydrodynamic plane of shear, were calculated from the Helmholtz--Smoluchowski equation. In the presence of 0.1 M lithium, sodium, ammonium, potassium, rubidium, cesium, tetraethylammonium, and tetramethylammonium chloride, the xi potentials were -60, -62, -72, -73, -77, -80, -82, and -91 mV, respectively. Similar results were obtained with phosphatidylglycerol vesicles; different results were obtained with cardiolipin, phosphatidylinositol, and phosphatidic acid vesicles. The phosphatidylserine results are interpreted in terms of the Stern equation, a combination of the Gouy equation from the theory of the diffuse double layer, the Boltzmann relation, and the Langmuir adsorption isotherm. Evidence is presented that suggests the hydrodynamic plane of shear is 2 A from the surface of the membrane in solutions containing the alkali metal cations. With this assumption, the intrinsic association constants of the above monovalent cations with phosphatidylserine are 0.8, 0.6, 0.17, 0.15, 0.08, 0.05, 0.03, and 0 M-1, respectively. The validity of this approach was tested in two ways. First, the xi potentials of vesicles formed from mixtures of phosphatidylserine and a zwitterionic lipid, phosphatidylcholine, were measured in solutions containing different concentrations of sodium. All the data could be described by the Stern equation if the "relaxation" of the ionic atmosphere, which is predicted by classic electrostatic and hydrodynamic theory to occur at low salt concentrations and high potentials, was circumvented by using only large (diameter greater than 13 micrometers) vesicles for these measurements. Second, the fluorescent probe 2-(p-toluidinyl)naphthalene-6-sulfonate was used to estimate the potential at the surface of phosphatidylserine and phosphatidylglycerol vesicles sonicated in 0.1 M NaCl. Reasonable agreement with the predicted values of the surface potential was obtained.     

Synergistic tumor regressive activity observed following treatment of line-10 hepatocellular carcinomas with deproteinized BCG cell walls and mutant Salmonella typhimurium glycolipid

Summary Synergistic tumor-regressive activity was observed when the water-soluble portion of a phenol-water extract from mutant Salmonella typhimurium whole cells was combined with deproteinized cell walls from Mycobacterium bovis strain BCG. As little as 50 g deproteinized cell walls combined with 50 g water-soluble extract from the mutant salmonella produced 89–100% cures of line-10 dermal tumors in treated strain 2 guinea-pigs. However, none of the animals was cured following treatment with 50 g of deproteinized cell walls alone. Only 17% of treated animals were cured following treatment with 50 g of the water-soluble extract from the mutant salmonella. The deproteinized cell walls and water-soluble extract were suspended in oil-in-water emulsions and injected directly into 10 mm tumors. The deproteinized cell walls were prepared by treating BCG cell walls with proteolytic enzymes and denaturing agents (KCl, urea, Triton X-100, and guanidine hydrochloride). Urea or a combination of denaturing agents reduced the protein content of protease-treated cell walls from approximately 2% (w/w) protein to 0.7% protein. The antigenicity of the effectively deproteinized cell walls, as measured by skin testing in presensitized guinea-pigs, was reduced approximately ten-fold compared with untreated cell walls. Injection to mice of 500 g deproteinized cell walls in combination with 500 g water-soluble extract from the mutant salmonella produced transient, clinical signs of toxicity (malaise, conjunctivitis, diarrhea, and rough hair coats) lasting approximately 5 days. However, no deaths were observed. The synergistic antitumor effect of combining deproteinized BCG cell walls with the water-soluble extract from mutant salmonella may be useful for treatment of certain cases of spontaneous neoplastic disease.     

Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

Prevention of elevated cholesterolemia in monkeys.

Alfalfa root saponins prevented the expected increase in plasma cholesterol associated with the Ingestion of a semipurified high-butter, high-cholesterol diet in monkeys. Experiments in rats indicate that alfalfa root saponins decrease cholesterol intestinal absorption.     

Specificity of interactions of hapten side chains with the combining site of the myeloma protein MOPC 315.

The pKa values of the three histidine residues in the Fv fragment (variable region of the heavy and light chains) of the mouse myeloma protein MOPC 315, measured by high resolution n.m.r. (nuclear magnetic resonance), are 5.9, 6.9 and 8.2. The perturbation of the pKa of one of the histidines (pKa 6.9) on the addition of hapten and the narrow linewidth of its proton resonances suggests that it is at the edge of the combining site. References to the model of the Fv fragment [Padlan, Davies, Pecht, Givol & Wright (1976) Cold Spring Harbor Symp. Quant. Biol. 41, in the press] allows assignment of the three histidine residues, histidine-102H, histidine-97L and histidine-44L. The determination of the pKa of the phosphorus group, by 31P n.m.r., of a homologous series of Dnp- and Tnp- (di- and tri-nitrophenyl) haptens has located a positively charged residue. Molecular-model studies on the conformations of these haptens show that the residue is at the edge of the site. The model suggests that the positively charged residue is either arginine-95L or lysine-52H.     

The influence of UV-B radiation on the physicochemical nature of tobacco (Nicotiana tabacum L.) leaf surfaces

Relationships between leaf wettability and surface physicochemicalcharacteristics were examined in two genotypes of tobacco (Nicotianatabacum L. cv. Samsun) grown under controlled conditions atthree different levels of biologically effective ultraviolet-B(UV-B_BE; 280–320 nm) radiation; 0 (control), 4.54 and5.66 kJ m^–2d^–1. Leaf wettability, assessed by measuringleaf-water droplet contact angles, was positively correlatedwith epicuticular wax chemical composition and trichome density,but not the amount of wax on the surface of leaves. Tobaccowax comprised a mixture of C₁₉–C₃₃ n-alkanes (59%) withhomologues containing an odd number of carbon atoms predominating,C₂₈–C₃₂ br-alkanes (38%), and a small quantity (3%) offree C_l6–C₁₈ fatty acids. Significant effects of UV-Bradiation upon wax production and chemical composition wererestricted to the adaxial surface of leaves. Enhanced UV-B radiationreduced the quantity of epicuticular wax in the more sensitivegenotype [GR32-3], assessed from effects on dry matter accumulation,partitioning and changes in leaf morphology, and resulted inmarked changes in wax composition and homologue distributionsin both genotypes. UV-B-induced increases in branching, andshifts toward the synthesis of shorter-chain homologues providedevidence for a fundamental effect of UV-B radiation on wax biosynthesis,with the observed effects consistent with a highly specificand direct effect of UV-B radiation on microsomal-based elongasesin the epidermis. UV-B radiation also reduced the density oftrichomes on the adaxial leaf surface, whilst increasing thenumber of trichomes on the abaxial leaf surface. Changes inwax composition and trichome density induced by UV-B radiationwere associated with increases in leaf surface wettability whichwere particularly pronounced on the adaxial surface. The subtle,though possibly far-reaching, physiological consequences ofsuch UV-B-induced changes in surface wettability are discussedin the light of other recent findings. Key words: Epicuticular wax chemistry, wax quantity, leaf wettability, trichome density, ultraviolet-B radiation     

Modelling of underwater light in freshwater lakes using survival and failure time analysis

1. This study presents a new approach to modelling subsurface irradiance using concepts from survival and failure time analysis. The model applies a modified Weibull distribution function to predict downwelling irradiance. Data sets from forty-seven Norwegian sites show extremely high coefficients of determination, up to 99.99%, when analysed by the Weibull model.
2. The uncritical use of a single k _d value to model underwater light conditions is likely to result in poor estimates of received irradiance. This error may amount to several hundred per cent. The practice of force-fitting linear least-squares regressions to log-transformed irradiance data inevitably leads to highly biased estimates of the true fraction of incident irradiance entering the water.
3. Wave effects causing fluctuations of subsurface irradiance are modelled with synthetic data and compared with field observations. Fluctuations of surface elevation by waves produce skewed frequency distributions of the underwater light field. The result of these effects, which are to reduce the accuracy of estimated model parameters, can be largely eliminated by carefully designing field procedures used for the acquisition of subsurface light data.     

Use of a pyrimidine nucleoside that functions as a bidentate hydrogen bond donor for the recognition of isolated or contiguous G-C base pairs by oligonucleotide-directed triplex formation.

Synthesis of the nucleoside building block of the 6-keto derivative of 2'-deoxy-5-methylcytidine (m5oxC) as an analog of an N3-protonated cytosine derivative is described. A series of 15mer oligonucleotides containing either four or six m5oxC residues has been prepared by chemical synthesis. Complexation of the 15 residue oligonucleotides with target 25mer duplexes results in DNA triplexes containing T-A-T and m5oxC-G-C base triplets. When the m5oxC-G-C base triplets are present in sequence positions that alternate with TAT base triplets, DNA triplexes are formed with Tm values that are pH independent in the range 6.4-8.5. A 25mer DNA duplex containing a series of five contiguous G-C base pairs cannot be effectively targeted with either m5C or M5oxC in the third strand. In the former case charge-charge repulsion effects likely lead to destabilization of the complex, while in the latter case ineffective base stacking may be to blame. However, if the m5C and M5oxC residues are present in the third strand in alternate sequence positions, then DNA triplexes can be formed with contiguous G-C targets even at pH 8.0.