Thermodynamics of antibody-antigen reactions. 2. The binding of bivalent synthetic random coil antigens to antibodies having different antigen precipitating properties

The objects of this study were the equine IgG and IgG(T) classes of antibodies with immunologic specificity for the dinitrophenyl group and bivalent antigens consisting of linear poly(ethylene glycol) polymers which terminated at both ends in dinitrophenyl groups. Complex formation between antibodies of both classes and one of several sharp fractions of antigen having number average molecular weights in the range 25 000 to 75 000 were studied by measuring the light scattered from solutions containing equimolar amounts (approximately 5 x 10(-6) mol/L) of one of the antibodies and one size fraction of antigen, and variable amounts of monovalent hapten. The data were analyzed in the context of a model that accounted for the formation of linear and cyclic complexes of all extents of aggregation. Two parameters in addition to the intrinsic antibody-dinitrophenyl group association constant were found to be necessary in the assumed equilibrium model to account for the behavior of the system. One of these accounted for the looses in configuration entropy that resulted when a random-coil polymer became bound at one end to a space-occupying antibody. The other was a ring closure factor for the formation of cyclic complexes. Ring closure factors for the formation of larger cyclic complexes (present in only small amounts under the conditions studied) were related to the ring closure factor for the formation of the smallest, which was found to increase as antigen size decreased, and for each antigen size to be consistently higher for IgG(T) antibody than for IgG antibody. Comparison of the theoretically estimated values of the two parameters within their measured values indicated that the average conformation of IgG antibodies in solution is open ("T" shaped) but the average inter-Fab are angle in IgG(T) antibodies is approximately 60 degrees or less.     

Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.     

Microbiological transformations of delta6a10a-tetrahydrocannabinol.

A screening program was conducted to find microorganisms that catalyze transformation reactions with cannabinoids. Three hundred fifty-eight cultures, consisting of 97 bacteria, 175 actinomycetes, and 86 molds, were incubated in media containing 0.5 mg of Delta(6a,10a)-tetrahydrocannabinol (Delta(6a,10a)-THC) per ml. After 120 h of cultivation, ethyl acetate extracts of the cultures were examined by thin-layer chromatography (TLC) for transformation products. About 18% of the cultures modified Delta(6a,10a)-THC. The ability to modify the substrate did not predominate among any particular group of microorganisms. After purification, the products from three cultures were analyzed by high-resolution mass spectrometry, 100-mHz proton magnetic resonance spectrometry, ultraviolet spectrometry, and infrared spectrometry. These spectral data indicated that a Mycobacterium sp. oxidized Delta(6a,10a)-THC to cannabinol and a diastereomeric pair of 6a-hydroxy-Delta(10,10a)-THC isomers; a Streptomyces sp. and a Bacillus sp. oxidized Delta(6a,10a)-THC to 7-keto-Delta(6a,10a)-THC and 4'-hydroxy-Delta(6a,10a)-THC, respectively. The occurrence of these products and the presence of others that have not yet been isolated or identified indicate that microbial transformation may be a useful tool for the preparation of new cannabinoids that have desirable pharmacological properties.     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

The microbiological basis of process control in methanogenic fermentation of soluble wastes

The essential requirement for anaerobic digestion of industrial wastes is that the process should operate reliably at high performance. In the digestion of dilute, soluble wastes it is necessary to retain the active biomass within the digester at short liquid retention times for the process to be economically feasible and this is reflected in digester design. Performance of digesters can only be assessed by interpretation of measurable parameters such as pH₂, E_h, pH, volatile fatty acid concentrations, temperature, gas production, biomass content and feed rate and composition. The effects of changes in these parameters on the microbiology of methanogenic digestion and the application of this knowledge in control of the process is discussed.     

Proteolytic degradation of heterologous proteins expressed inAspergillus niger

Summary Proteolytic degradation of heterologous proteins expressed in the filamentous fungusAspergillus niger reduces the yield of authentic target protein. The activities ofA. niger proteases are differentiated by their effects on two proteins expressed and secreted fromA. niger: hen egg-white lysozyme and porcine pancreatic phospholipase A₂.     

Deletion-mutant epidermal growth factor receptor in human gliomas: effects of type II mutation on receptor function.

Malignant human glioma D-298 MG amplifies a rearranged epidermal growth factor receptor (EGFR) gene (c-erbB proto-oncogene), resulting in an in-frame deletion of 83 amino acids in domain IV of the extracellular domain of the EGFR. EGF and transforming growth factor-a (TGF-a) bound to the mutant EGFR with high affinity and enhanced the intrinsic mutant EGFR kinase activity. The mutant EGFR was capable of transducing EGF-stimulated glioma cell proliferation and invasiveness in an in vitro three-dimensional spheroid model. The deletion-mutant EGFR in D-298 MG is capable of being activated by growth factor; this suggests that overexpression of this mutant EGFR protein rather than structural alteration may be the more significant biologic event.     

Dynamics and aggregation of the peptide ion channel alamethicin. Measurements using spin-labeled peptides.

Two spin-labeled derivatives of the ion conductive peptide alamethicin were synthesized and used to examine its binding and state of aggregation. One derivative was spin labeled at the C-terminus and the other, a leucine analogue, was spin labeled at the N-terminus. In methanol, both the C and N terminal labeled peptides were monomeric. In aqueous solution, the C-terminal derivative was monomeric at low concentrations, but aggregated at higher concentrations with a critical concentration of 23 microM. In the membrane, the C-terminal label was localized to the membrane-aqueous interface using 13C-NMR, and could assume more than one orientation. The membrane binding of the C-terminal derivative was examined using EPR, and it exhibited a cooperativity seen previously for native alamethicin. However, this cooperativity was not the result of an aggregation of the peptide in the membrane. When the spectra of either the C or N-terminal labeled peptide were examined over a wide range of membrane lipid to peptide ratios, no evidence for aggregation could be found and the peptides remained monomeric under all conditions examined. Because electrical measurements on this peptide provide strong evidence for an ion-conductive aggregate, the ion-conductive form of alamethicin likely represents a minor fraction of the total membrane bound peptide.     

Voltage-dependent conductance for alamethicin in phospholipid vesicles. A test for the mechanism of gating.

The ion currents induced by alamethicin were investigated in unilamellar vesicles using electron paramagnetic resonance probe techniques. The peptide induced currents were examined as a function of the membrane bound peptide concentration, and as a function of the transmembrane electrical potential. Because of the favorable partitioning of alamethicin to membranes and the large membrane area to aqueous volume in vesicle suspensions, these measurements could be carried out under conditions where all the alamethicin was membrane bound. Over the concentration range examined, the peptide induced conductances increased approximately with the fourth power of the membrane bound peptide concentration, indicating a channel molecularity of four. When the alamethicin induced currents were examined as a function of voltage, they exhibited a superlinear behavior similar to that seen in planar bilayers. Evidence for the voltage-dependent conduction of alamethicin was also observed in the time dependence of vesicle depolarization. These observations indicate that the voltage-dependent behavior of alamethicin can occur in the absence of a voltage-dependent phase partitioning. That is, a voltage-dependent conformational rearrangement for membrane bound alamethicin leads to a voltage-dependent activity.     

The distribution of neuropeptides in the ocular tissues of several mammals: a comparative study.

1. The distribution of several neuropeptides (vasoactive intestinal peptide, substance P, somatostatin and neurotensin) was assessed in ocular tissues from the cow, sheep, rabbit and rat. 2. Vasoactive intestinal peptide was most abundant in the choroid and sclera in all species except the rat. Substance P was most abundant in the retina of cow and rat and in the iris/ciliary body of sheep and rabbit. Somatostatin and neurotensin were most abundant in the retina of all species examined. 3. Regulatory peptides thus display distinct regional distributions within the ocular tissues of a single species of mammal and, in addition, exhibit interspecific variation.