Murine transfer factor. I. Description of the model and evidence for specificity

A model for studying transfer of delayed-type sensitivity to mice with cellfree materials is described. The results with a particulate antigen (Candida) and 4 soluble protein antigens (PPD, ferritin, cytochrome c, and horseradish peroxidase) suggest that the phenomenon is antigen specific. Identical preparations from the spleens of insensitive donors were not active. This murine model should facilitate characterization of the immunologic and chemical properties of transfer factor.     

Mass Production of the Mosquito Parasite Romanomermis culicivorax: Effect of Density

When numbers of Romanomermis culicivorax Ross and Smith were varied in containers with a constant surface area and depth of sand, densities of 12-24 nematodes per cm² yielded significantly more preparasites than higher densities. When container surface area and numbers of nematodes were constant and sand volume and depth were varied, yields did not differ significantly. When numbers of nematodes and sand volume were constant and surface area and sand depths were varied, yields were significantly higher for a density of 24 nematodes per cm². Yields of preparasites were tripled by simply setting up three cultures, each containing 5 g of nematodes, instead of a single culture containing 15 g.     

Magnetic resonance studies of the spatial arrangement of glucose-6-phosphate and chromium (III)-adenosine diphosphate at the catalytic site of hexokinase.

The interaction of CrADP, an exchange-inert paramagnetic analogue of Mg-ADP, with yeast hexokinase has been studied by measuring the effects of CrADP on the longitudinal nuclear relaxation rate (1/T1) of the protons of water and the protons and phosphorus atom of enzyme-bound glucose-6-P. The paramagnetic effect of CrADP on 1/T1 of water protons is enhanced upon complexation with the enzyme. Titrations measuring this paramagnetic effect at several enzyme concentrations in the presence of glucose-6-P yielded a characteristic enhancement factor for 1/T1 of water protons and the dissociation constant of CrADP from the ternary enzyme . ADPCr . glucose-6-P complex. The latter value (2 mM) is similar to that obtained from kinetic inhibition studies (Danenberg and Cleland [1975]. Biochemistry. 14:28). The presence of glucose-6-P increased the enhancement of the water relaxation rate by enzyme-bound CrADP, suggesting the formation of an enzyme . CrADP . glucose-6-P complex. The existence of such a complex was confirmed by the observation of a paramagnetic effect of enzyme-bound CrADP on the l/T1 of the 31P-nucleus and protons of enzyme-bound glucose-6-P. From the paramagnetic effects of enzyme-bound CrADP on the relaxation rates of the 31P-nucleus and the carbon-bound protons of glucose-6-P in the enzyme . ADPCr . glucose-6-P complex, using the correlation time of approximately 0.7 ns, determined from the magnetic field-dependence of 1/T1 of water protons over the range 24.3-360 MHz, a Cr3+ to phosphorus distance of 6.6 +/- 0.7 A and Cr3+ to alpha- and beta-anomeric proton distances of 8.9 and 9.7 A were calculated. These results imply the absence of a direct coordination of the phosphoryl group of glucose-6-P by the nucleotide-bound metal on hexokinase but indicate van der Waals contact between a phosphoryl oxygen of glucose-6-P and the hydration sphere of the nucleotide-bound metal. The distances are consistent with a model that assumes molecular contact between the phosphorus of glucose-6-P and a beta-phosphoryl oxygen of ADP suggesting an associative phosphoryl transfer. Because after phosphorylation of ADP, the metal ion is coordinated to the transferred phosphoryl group, the overall migration of the phosphoryl group during the phosphoryl transfer is approximately 3.6 A toward the nucleotide-bound metal. Little or no catalysis of phosphoryl transfer from glucose-6-P to alpha, beta-bidentate or beta-monodentate CrADP ( less than or equal to 0.05% of the rate found with MgADP) occurred in the presence of hexokinase, as monitored by glucose formation in a coupled assay system using glucose oxidase and peroxidase. The ability of beta, gamma-bidentate CrATP to act as a substrate (Danenberg and Cleland [1975].     

Specific protection from phenocopy induction by heat shock

Mild heat treatments applied to whole animals or cell cultures of Drosophila prior to lethal heat shocks result in increased survival and protection against phenocopy induction. The optimal condition for the preliminary mild heat treatment is that which induces the synthesis of heat-shock proteins but does not turn off the protein synthesis that is in progress. Recovery of protein synthesis but not RNA synthesis following a drastic heat shock is much enhanced by the pretreatments. The results suggest that the protection for survival and against phenocopy induction is due to storage of messenger RNA.     

Growth characteristics of atypical mycobacteria in water and their comparative resistance to disinfectants.

With the increasing significance of group IV atypical mycobacteria as etiological agents in a variety of infections, studies were conducted to determine their growth capabilities in water and their comparative resistance to disinfectants used to decontaminate hospital equipment. Isolates of Mycobaterium chelonei (TM strains) from peritoneal fluids of patients and peritoneal dialysis machines were able to multiply in commercial distilled water, with generation times at 25 degrees C ranging from 8 to 15 h. Levels of 10(5) to 10(6) cells per ml were attained, and these stationary-phase populations declined only slightly over a 1-year period. Results of studies to determine resistance to disinfectants showed the following. (i) TM strains of M. chelonei cultured in commercial distilled water showed survivors in 2% aqueous formaldehyde (HCHO) solutions up to 24 h; in 8% HCHO, only a 2-log reduction in viable counts was observed over a 2-h sampling period. Reference ATCC strains of M. chelonei and M. fortuitum were rapidly inactivated, with no survivors after 2 h of exposure to 2% HCHO or 15 min of exposure to 8% HCHO. (ii) In 2% alkaline glutaraldehyde, TM strains survived 60 min. whereas ATCC strains showed no survivors after 2 min of contact time. (iii) All M. chelonei and M. fortuitum strains survived 60 min of exposure to concentrations of 0.3 and 0.7 microgram of free chlorine per ml at pH 7.     

The effect of oxygen concentration on the steady-state kinetics of the solubilized cytochrome c oxidase.

1. The steady-state kinetics of ascorbate oxidation as a function of oxygen concentration was measured with a solubilized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) preparation. 2. Linear double reciprocal plots were obtained at various fixed concentrations of ascrobate, cytochrome c and cytochrome aa3. 3. The results are interpreted in terms of an oxidase model similar to that put forward by Minnaert in 1961 (Minnaert, K. (1961) Biochim. Biophys. Acta 50, 23-34). 4. The Km for oxygen at infinite cytochrome c concentration is 0.95 muM and the intramolecular rate constant for the transfer of electrons from cytochrome c to cytochome aa3 is 400 s(-1). According to the model, this implies that the second order rate constant for the reaction between oxygen and the oxidase is 9.5 X 10(7)M(-1)-s(-1).     

The electric dipole moment of rhodopsin solubilized in Triton X-100.

The electric dipole moment of solubilized rhodopsin was determined with dielectric dispersion measurements. Rhodopsin was extracted from disc membranes of cattle rod outer segments with the nonionic detergent Triton X-100. The dipole moment of rhodopsin at its isoionic point in the detergent micelle is 720 D (150 charge-A). This value is comparable to dipole moments of nonmembrane proteins, especially those which tend to aggregate or polymerize. Flash irradiation of the rhodopsin results in an increase in the dipole moment of about 25 D (5 charge-A). The light-induced increase in dipole moment appears to be composed of two parts--a faster component related to a change in the number of protons bound by rhodopsin and a slower component apparently independent of the change in proton binding.