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Rabbit antisera against highly purified L-asparaginase from Serratia marcescens and from Escherichia coli showed up to 60% inhibition of the catalytic amidohydrolysis of L-asparagine when combined with the homologous enzyme. This inhibition was diminished somewhat against the heterologous enzyme. Kinetic studies in the presence of these antisera showed an increased Kmapp for both homologous and heterologous enzymes using L-asparagine as substrate. In contrast, kinetic studies employing the poor substrate, L-glutamine, showed activation attributable to specific antibodies. This was seen in lower Kmapp values and up to twofold increases in the Vmax over the normal rabbit serum controls. The high degree of cross-inhibition (approximately 80%) and the low degree of cross-reactivity in the quantitative precipitin test (approximately 34%) suggest that these two enzymes possess structural similarities located mainly in the regions of the catalytic sites.

The factors which govern the interaction and the inhibition of horse liver alcohol dehydrogenase (H-L-ADH) by specific antibody have been examined in detail. Both guinea pig and rabbit antisera inhibited the enzymic activity of H-L-ADH to varying degrees, with residual activity remaining even in the presence of a large excess of antibody. The inhibitory capacity of the antisera increased with prolonged immunization. The extent of enzymic inhibition was dependent on the molecular composition and the size of the immune complex, the order of addition of the reactants and the concentration of the substrate and the coenzyme. Inhibition of H-L-ADH was instantaneous and was not influenced by the presence of guinea pig complement. A number of possible mechanisms which could account for the inhibition of H-L-ADH by antibody are discussed.

Varying concentrations of DDT in solution in different solvents were tested by painting copper screen wire of 18 meshes per inch. Tests were made at varying time intervals after painting, the tested wire being kept under differing conditions of exposure. The aims of the experiments were to demonstrate the following points; 1. The length of contact with the painted wire required to kill within selected subsequent arbitrary time period. 2. The original lethal effect of the newly painted wire and the period over which subsequent lethal effect would persist. 3. The effect of different DDT solvents on primary lethal effect, and the duration of lethal effect as modified by different solvents. 4. The effect of varying concentrations of DDT in different solvents on the primary lethal effect, and the duration of lethal action. 5. The influence of different types of “weathering” on the lethal effect of DDT in solution.

Serum samples from 1,258 normal adult blood donors were assayed for complement activity. The average complement titer for the entire sample was 38.14. There was no difference in complement activity with respect to sex, blood group or Rh***(D) type. The level of circulating complement appears to be higher in older humans and in heavier individuals. In this kind of experimentation, the need for a standard complement for assay of different batches of sheep erythrocytes is emphasized.

Marucci, Americo A. (Upstate Medical Center, Syracuse, N.Y.). Mechanism of action of staphylococcal alpha-hemolysin. II. Analysis of the kinetic curve and inhibition by specific antibody. J. Bacteriol. 86: 1189–1195. 1963.—At least two steps are necessary before the rabbit erythrocyte is lysed by staphylococcal alpha-hemolysin. The first step involves the reaction of alpha-hemolysin with the red cell. The second step, leading to the release of hemoglobin, is an intrinsic reaction of the damaged red cell and takes place without further participation of hemolysin. The speed of this intrinsic reaction is temperature-dependent. Erythrocytes taken from the same rabbit do not vary in their susceptibility to the alpha-hemolysin. From the results of the experiments described herein, a preliminary hypothesis on the mechanism of action of staphylococcal alpha-hemolysin is given.

Summary At 37°C EAC′1,4,2 becomes unreactive with C′2 according to first order kinetics with a half-life of 10.3 minutes. C′2 is taken up by EAC′1,4,2, as determined by activity measurements and by analyses with radio-iodinated C′3. Under the usual experimental conditions only a small proportion of C′3 is fixed by EAC′1,4,2, leaving a major part free in the fluid phase. From these findings it follows that the reaction between EAC′1,4,2 and C′3 comes to a standstill because of the decay of the cell substrate. The same is true in the reaction between Ea and C′. There is no lag in the reaction between EAC′1,4,2 and C′3, indicating that the resulting formation of a damaged cell is essentially a single event with respect to time. The velocity of reaction between EAC′1,4,2 and C′3 is directly proportional to the concentration of EAC′1,4,2 and varies as the second or third power of the concentration of C′3. This may mean that two or three “molecules” of C′3 must combine simultaneously, or nearly simultaneously, with EAC′1,4,2 for a productive reaction.

Mice which had been infected with scrapie 4 to 5 weeks earlier were inoculated with soluble and particulate antigens. There was no evidence that the scrapie agent had any immuno-suppressive effect.

Antiperoxidase has been prepared in the rabbit, cat, mouse, hamster and chicken. All of these antisera inhibit peroxidase activity.

SummaryA method for determining ion transportation through the mucosa of the urinary bladder is described. This method has been used on normal and artificially constructed bladders in dogs.Rapid and continuous absorption of iodide, sodium and phosphorus ions takes place through the mucous membrane lined bladders, while only minimal amounts of these ions are absorbed through the normal bladder and through bladders constructed by means of seromuscular grafts.

Two methods have been developed which permit use of the unlabeled antibody immunohistochemical technique for detection of human antibody, without the need for immunization of humans with peroxidase. Human antibody to herpes simplex virus (HSV) reacted with human cell cultures infected with HSV was the experimental system. In the first method an attempt was made to employ rabbit peroxidase-antiperoxidase (PAP) soluble complexes in connectin with human antibody. This was done by sequential addition to the HSV-infected cells of (a) human anti-HSV, (b) rabbit antihuman globulin, (c) guinea pig antirabbit globulin (the bridging reagent) and (d) rabbit PAP. Strong specific staining of HSV-infected cells was obtained; however, difficulties were encountered with nonspecific reactions on uninfected cells. In the second method PAP soluble complexes prepared with baboon antiperoxidase were bridged to the human anti-HSV antibody by rabbit antihuman globulin. Because of the phylogenetic relatedness of human and baboon globulins this resulted in firm binding which gave strong specific staining of HSV-infected cells without significant reaction in uninfected cells.

A quantitative micro-complement fixation test capable of detecting nanogram quantities of antigen is described. The test is simple to perform and is highly reproducible. Typical results from three antigen-antibody systems are given.

Immunochemical properties of l -asparaginases from Serratia marcescens and Escherichia coli were compared by means of quantitative precipitin reactions and quantitative microcomplement-fixation reactions employing homologous and heterologous antisera. The enzyme preparations used in these tests produced one arc of precipitate upon immunoelectrophoresis. According to our results with the precipitin and micro-complement fixation reactions, both enzymes are related immunochemically. On the basis of these tests, distinct structural differences exist between the two enzymes. From results with the micro-complement fixation test, we estimated that the two enzymes could differ by 10 to 15 amino acid residues.

Summary A modification of the peroxidase-labelled antibody technique was applied to study the distribution of virus antigens in chicken cells infected with two serotypes (A and B) of avian leukosis virus (ALV). Type-specific chicken antisera reacted only with cells infected with virus of the homologous envelope serotype. When unfixed cells were exposed to type-specific antivirus serum, only antigens located at the cell surface were stained, while cells exposed to type-specific antibodies after fixation revealed both surface and intracytoplasmic virus antigen. Cytoplasmic antigen was usually concentrated in discrete granules which often had a vesicular structure. Hamster antibodies against ALV group-specific (gs) antigen reacted with fixed infected cells regardless of envelope serotype, and the distribution of antigen was as shown with chicken type-specific antibodies. Intranuclear gs antigen may have been present in a few cells. Unfixed infected cells did not react with hamster gs antibody and confirmed the location of ALV gs antigen within the virus particle. Some chicken antisera with neutralizing antibodies against a single envelope serotype of ALV contained both type-specific and gs antibodies. These antisera reacted with fixed cells infected with virus of either the homologous or heterologous serotype and stained both surface and cytoplasmic antigens. With unfixed infected cells, these antisera combined only with the surface of cells infected with virus of the same serotype as the neutralizing antibodies in the serum. Thus, the reaction in heterologously infected fixed cells was with internal gs antigen. This confirms independently that chickens are not naturally tolerant to their homologous ‘C type’ gs antigens.

Soluble immune complexes of ferritin and chicken antiferritin have been prepared. This has been used with unlabeled chicken antibody to avian leukosis virus and rabbit antichicken globulin to demonstrate the presence of specific viral antigens in tissue culture by electron microscopy.

1. 1. Quantitative precipitin studies with crystalline urease and antiurease have been performed. The resulting data provide information concerning the combining proportions of the enzyme and antibody on a weight basis. 2. 2. Urease is inhibited by rabbit anti-urease. 3. 3. The extent of inhibition of the enzyme by specific antiserum is dependent upon the ratio of antibody to antigen employed. The greatest inhibition, about 80 %, was achieved with a reaction mixture containing a large excess of antibody. 4. 4. Under the conditions of this study, complement was not found to play a role in the inhibition of urease by anti-urease.

Marucci, Americo A. (Upstate Medical Center, Syracuse, N.Y.). Mechanism of action of staphylococcal alpha-hemolysin. I. Some factors influencing the measurement of alpha-hemolysin. J. Bacteriol. 86: 1182–1188. 1963.—A kinetic method for the accurate and reproducible measurement of the action of staphylococcal alpha-hemolysin on rabbit erythrocytes is described. The activity of the alpha-hemolysin depends upon the temperature used for measurement. At 37 C the hemolysin lyses cells faster, but it in turn is rather quickly inactivated. At 0 C there is no inactivation, but the rate of lysis is greatly decreased. There is no change in the activity with change in total reaction volume, providing that the concentrations of cells and hemolysin are kept constant. The fraction of rabbit red cells lysed by a given amount of hemolysin in a given time is constant and independent of the total number of cells in the reaction mixture.

http://archive.org/details/boilingheattrans109459449 U.S. Coast Guard (U.S.C.G.) author.