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Because staphylococcal protein A binds all the known subclasses of mouse IgG except IgG1, ethanol-fixed staphylococci were used as an adsorbent to prepare IgG1 fractions of anti-BALB/c alloantibody-containing globulins and normal globulins of the same strains. The loss of more than 99% of the IgG2 as a result of this adsorption was demonstrated by immunodiffusion. The IgG1 fractions of C3H and CBA anti-BALB/c globulins were tested for their effect on growth of the BALB/c plasmacytomas MOPC-315 and MOPC-460 in C3H and CBA mice by incubation with the tumor cells before transplantation and by injection periodically thereafter into the hosts. With alloantibody-containing globulins that showed slight enhancement of growth of these tumors, or none, the IgG1 preparations caused considerable enhancement of tumor growth. Control preparations of normal C3H or CBA globulins, or IgG1 fractions similarly prepared from the normal globulins, showed no enhancing effect on the growth of these tumors.

An exonuclease with a strong preference for single-stranded deoxyribonucleic acid over double-stranded deoxyribonucleic acid has been purified 500-fold from Xanthomonas oryzae. This enzyme liberates 5'-mononucleotides in a reaction which requires Mg2+.

A clear-plaque mutation (c2) of bacteriophage M13 has been shown to affect the regulation of viral DNA synthesis. This mutation increases the amount of the duplex replicative form DNA per cell while decreasing the synthesis of viral single strands. The relative synthesis of the M13 gene 5 protein is approximately half that observed in wild-type infections, suggesting that the effect of the c2 mutation on the regulation of viral DNA synthesis is a result of reduced expression of gene 5.

Bacteriophage phi X 174 gene D product, a protein required for single-stranded DNA synthesis by the phage, has been purified to near homogeneity. The protein is very abundant; approximately 10(5) monomers are present per infected cell when lysis is delayed. The protein has a monomer molecular weight of 15,200 and is normally a tetramer; however, it can form very large aggregates at high concentrations. Amino acid analysis shows an excess of arginine over lysine and a relatively high number of nonpolar residues. The protein carries a net negative charge at neutral pH. The first eight amino acids of the protein sequence have been determined; they are Ser-Gln-Val-Thr-Glu-Gln-Arg-Val. The carboxy-terminal residue is methionine. The protein has not yet been shown to bind directly to any single-stranded DNA; it does not adsorb to denatured calf thymus DNA-cellulose.

An exonuclease has been partially purified from XP-12-infected Xanthomonas oryzae which is not found in uninfected X. oryzae. Although both the phage-induced exonuclease and the major host exonucleolytic DNase released 5'-mononucleotides, these enzymes differed in their chromatographic behavior, pH optimum, salt inhibition, and heat sensitivity. These two exonucleases preferred different substrates. Nicked native DNA was the best substrate for the phage-induced enzyme, whereas denatured DNA was the best substrate for the host enzyme. Also, the host enzyme had a significant preference for denatured or nicked, normal cytosine-containing DNA (e.g., X. oryzae or T7 DNA) over similarly denatured or nicked 5-methylcytosine-rich DNA (namely, XP-12DNA), whereas the phage-induced enzyme hydrolyzed both types of DNA equally well.

XorI and XorII, two sequence-specific endonucleases, have been partially purified from Xanthomas oryzae. XorI and XorII were shown to be isoschizomers of PstI and PuvI, respectively. X. oryzae is a particularly good source of this PvuI isoschizomer because of the high yield of XorII, its simple purification scheme, and its relative stability. Furthermore, XorII was shown to cleave at different positions in its recognition sequence than do at least two of its known isochizomers; XorII cleaves between the C and the G at the 3'-end of its palindromic recognition sequence, 5'-CGATC G-3'. There is a single XorII site in each of the plasmid-cloning vehicles pBR313 and pBR322. Two unusual aspects of XorII digestion are discussed, namely, the kinetics of digestion of pBR313 and pBR322 and the resistance of human DNA to XorII.

The choroid plexus is a major site of CSF production. When primary cultures of bovine choroid plexus epithelial cells were exposed to 1 micrograms/ml cholera toxin, a 50-fold increase of intracellular cyclic AMP was found 1 h later. Exposure of cells to 10(-5) M isoproterenol, 10(-4) M prostaglandin E1, 10(-5) M histamine, and 10(-5) M serotonin caused increases of intracellular cyclic concentrations of 100-, 50-, 20-, and 4-fold, respectively. From 5 to 15 min were required for these maximal responses to occur. Many other molecules including prolactin, vasopressin, and corticotropin did not alter cellular cyclic AMP levels. The accumulation of cyclic AMP could be inhibited by specific antagonists: propranolol inhibited the isoproterenol-mediated stimulation while diphenhydramine and metiamide inhibited the histamine response. In addition, diphenhydramine inhibited serotonin-dependent cyclic AMP accumulation. Combinations of isoproterenol, prostaglandin E1, histamine, and serotonin elicited additive responses as measured by cyclic AMP accumulation with one exception, i.e., serotonin inhibited the histamine response. Our findings suggest that distinct receptor sites on choroid plexus epithelia exist for isoproterenol, prostaglandin E1, and histamine. Efflux of cyclic AMP into the extracellular medium was found to be a function of the intracellular cyclic AMP levels over a wide range of concentrations. Our studies provide direct evidence for hormonal regulation of cyclic AMP metabolism in epithelial cells of the choroid plexus.

A major site of cerebrospinal fluid production in vertebrates is the choroid plexus. The epithelial cells of the choroid plexus accumulate intracellular cyclic AMP in response to several effectors, including histamine. Since histamine is known to regulate fluid secretion in the stomach via H2 histamine receptors, we asked whether H2 receptors might also be present on epithelial cells of bovine choroid plexus. Using agonists and antagonists of histamine, we show that an agonist and antagonist pair specific for the H2 subtype were clearly more effective than an H1 agonist and antagonist pair in mimicking or inhibiting histamine stimulation of cellular cyclic AMP. Analysis by Schild plot allowed assignment of an apparent dissociation constant to the H2 antagonist metiamide which was 34-fold lower than that of its H1 counterpart, diphenhydramine. These results indicate that epithelial cells of the choroid plexus possess H2 histamine receptors.

The immunologic response of neonatal and of older rabbits to the tissue (transplantation) antigens of pooled rabbit leucocytes was studied, the test system being the suppression of formation of agglutinin to Shigella paradysenteriae by transferred lymph node cells which had been incubated with Shigella antigen. In the active induction of the suppressive effect on the transferred cells it was found that neonatal rabbits reacted as vigorously as 1 kg rabbits to the prior injection of a given number of rabbit leucocytes pooled from prospective donors of lymph node cells. The suppressive effect was dose-related, and within the range of number of leucocytes used was similar for both age groups of recipients. An attempt to detect a difference in the response during the first few days after leucocyte injection, before the full suppressive effect is reached, failed to show any difference between rabbits of the two age groups. Since it had been found possible to transfer the suppressive effect passively with sera obtained from older rabbits injected with rabbit leucocytes, attempts were made to do so with sera obtained from neonatal rabbits injected with similar numbers of pooled adult rabbit leucocytes. No consistent suppression of transferred lymph node cells was observed with sera from neonatal rabbits, even with relatively large amounts of such serum. In sera of rabbits which had been injected with rabbit leucocytes at the age of 4 to 6 weeks, suppressive antibody could be detected. When anti-rabbit-leucocyte serum obtained in adult rabbits was injected into neonatal and 1 kg recipients at a given volume per gram of animal weight the suppressive effect of the serum was of similar extent in the two groups of recipients. In the adoptive transfer of the lymph node cell suppressive effect, by cells of lymph nodes draining the sites of injection of pooled rabbit leucocytes, it was found that the popliteal lymph node cells of neonatal rabbits were as effective as those of 1 kg rabbits. Splenic cells of neonatal rabbits were also effective, when an adequate number of rabbit leucocytes had been injected intravenously. Thus, in conferring adoptive immunologic response, as in active immunization, the neonatal rabbits were as effective as the older rabbits in their response to homotransplantation antigens, in contrast to the considerable difference in concentration of the suppressive antibody in sera of neonatal and older rabbits injected with rabbit leucocytes.

Shigella paradysenteriae organisms were incubated in a solution of trypsin and then removed from the suspension by Seitz filtration. Serologic tests with anti whole Shigella serum indicated that the filtrate contained antigenic material derived from the organisms. When lymph node cells from rabbits not previously injected with Shigella were incubated in vitro with such filtrates and then transferred to irradiated recipients, agglutinins to Shigella appeared in the sera of the latter. The transfer of heated suspensions of cells was not followed by the appearance of agglutinins in irradiated recipient rabbits. Non-irradiated recipients of lymph node cells incubated with the filtrate also developed agglutinin titers, to a lower level than irradiated animals. Agglutinins did not appear in the sera of the majority of non-irradiated recipients of heated cells. The addition of excess antiserum to either the filtrate or the cell suspension before the incubation of these two materials resulted in a marked reduction in the subsequent agglutinin titers of recipient animals. However, if antiserum was added at the conclusion of the usual 30 minute incubation of cells and filtrate, or even after 5 minutes of incubation, there was no reduction in the agglutinin titers of the recipients. Cytologic examination of aliquots of a number of the suspensions of cells prepared for transfer revealed that approximately 98 to 99 per cent of the cells belonged to the lymphocytic series.

Antibody-producing cells have been identified among cells obtained from efferent lymphatic vessels, the thoracic duct, and peripheral blood. These cells, which produced plaques of hemolysis and which were quite rare (20 to 50 per million), due in most instances to 19S antibody, were located and studied by electron microscopy. Of the antibody-producing cells found in these three sites there were several features common to all: small size (5 to 8 micro), generally spherical shape, approximately central position of the nucleus, and retention in the nucleus of the condensations of chromatin characteristic of the lymphocyte. The differences among the cells of these sources were largely in the relative amount and state of organization of the organelles of the cytoplasm. In cells of the efferent lymphatic vessel and the thoracic duct, the endoplasmic reticulum showed a range from relative scarcity to considerable numbers of well organized channels. Between these extremes were cells with a considerable amount of endoplasmic reticulum, the channels disorganized and sectioned at various angles. The cytoplasmic matrix of all of these contained a profusion of polyribosomes. Antibody-producing cells obtained from peripheral blood showed, around the roughly spherical nucleus, a ring of cytoplasm which was narrow, but wholly organized into parallel lamellae of endoplasmic reticulum, with many polyribosomes between these, and a large Golgi body. Some similarities and some differences of these cells, in comparison with antibody-producing cells obtained from lymph nodes, have been indicated.

Leucocytes were obtained from the blood of rabbits, incubated in vitro with soluble antigenic material derived from Shigella paradysenteriae, washed, and transferred to irradiated recipient animals. On the 4th day after cell transfer, agglutinins to Shigella appeared in the sera of the majority of such recipients. The distribution of maximum agglutinin titers was substantially lower in these animals than in recipients of lymph node cells similarly incubated, as reported in earlier studies. Recipients of iodoacetate-treated cells showed no agglutinins. Similar results were obtained with cells of peritoneal exudates obtained 9 days after the intraperitoneal injection of lanolin and light mineral oil. When cells of peritoneal exudates obtained 1 or 2 days after the injection of this irritant were incubated with the antigenic material and transferred to irradiated recipients, agglutinins did not appear in the sera of the latter. Similar results were obtained with cells from peritoneal exudates obtained a few days after the intraperitoneal injection of heavy mineral oil. The preponderant cell types in the suspensions from these two sources were the polymorphonuclear neutrophils and monocytes, respectively.

Efferent lymph of the popliteal lymph nodes of rabbits was collected 4 days after a single footpad injection of SRBC. Thin-layer agar plating was done to isolate plaque-forming cells of the lymph for electron microscope examination, and the numbers of plaque-forming cells (PFC) in cells from the lymph and lymph nodes were determined. Of 71 PFC of lymph isolated and examined, 93% were lymphocytes, most of them with signs of substantial levels of physiologic activity. The cytoplasm showed an abundance of free ribosomes and many finger-like projections. The endoplasmic reticulum (ER) was barely detectable in most of the active lymphocytic PFC, and in some, a few short narrow channels of ER could be seen. Approximately one-fifth of the lymphocytic PFC presented an appearance of senescence, with signs of degeneration: rounded cells, with amorphous nuclear chromatin, and very few microvilli. The remaining 7% of the PFC of the lymph showed an unusual combination of features: small round cells with a narrow ring of cytoplasm which, however, contained well-organized channels of ER. Such cells had been found only among PFC of peripheral blood of the rabbit. The number of PFC per million cells was higher in the lymph than in the suspensions of lymph node cells. In both the contralateral lymph node and its efferent lymph, the number of PFC was less than 1% that of the injected side.

The transfer to rabbits of homologous lymph node cells which have been incubated in vitro with Shigella-trypsin filtrate leads to the appearance of agglutinins to Shigella in the sera of the recipients. In the present study it has been found that the prior injection of the prospective recipients with blood leucocytes from the donor animals prevented the appearance of anti-Shigella agglutinins. The following observations have been made in this system: 1. The degree of the pre-injection effect was found to be a function of the number of leucocytes injected and of the interval between such pre-injection and the transfer of the antigen-incubated lymph node cells. 2. The pre-injection of leucocytes at appropriate intervals could also cause the failure of antibody to appear in sera of recipients of lymph node cells when these were obtained from donor rabbits injected with Shigella, 1, 2, or 3 days prior to cell transfer. 3. Agglutinins failed to appear in cell-transfer experiments after the pre-injection not only of blood leucocytes, but also of lymph node cells, peritoneal exudate cells, or thymus cells of rabbits. This effect was not brought about by pre-injection of erythrocytes of rabbits or leucocytes of chicken, cow, or horse. The pre-injection of leucocytes of human blood had an effect of partial suppression. 4. When the leucocytes for pre-injection were pooled from groups of rabbits, either the prospective donors of the lymph node cells or other rabbits, essentially complete suppression of agglutinin titers occurred regularly. When the leucocytes for pre-injection were obtained from an individual rabbit and the lymph node cells from another rabbit the suppression of the recipients' titers occurred sporadically. 5. When the recipient's own leucocytes were pre-injected the subsequent agglutinin titers were somewhat lower than those of the non-pre-injected controls. When the recipient's whole blood was re-injected as the source of leucocytes the subsequent agglutinin titers were as high as those of the non-pre-injected controls. 6. The pre-injection effect was not obtained if the leucocytes had been heated, frozen and thawed, suspended in distilled water, lyophilized, or treated with sodium iodoacetate. However, sonic oscillation or x-irradiation of the leucocytes had no effect on their capacity to bring about the pre-injection effect.

In earlier studies it was shown that if rabbit lymph node cells were incubated with Shigella-trypsin filtrate and transferred to other, recipient, rabbits, agglutinins to Shigella appeared subsequently in the sera of the recipients. However, if leucocytes from the donor rabbits were injected into the recipients at a suitable interval prior to lymph node cell transfer, agglutinins to Shigella did not appear after cell transfer. In the present study, a number of experiments were done which bear on the immunologic character of this suppressive effect on the transferred cells, the "pre-injection effect." X-irradiation of the recipients before the injection of donor leucocytes led to a decrease in the extent of the pre-injection effect, i.e. to the appearance of higher agglutinin liters, in the recipient animals. It was found possible to transfer the pre-injection effect by cells of rabbit lymph nodes draining sites of injection of rabbit leucocytes. No such effect was obtained with heated aliquots of these cell suspensions or with lymph node cells from sheep-erythrocyte-injected donors. It was also possible to transfer the pre-injection effect passively by serum. Suppression of transferred lymph node cells was observed regularly after injection of serum pooled from groups of rabbits which had been injected with leucocytes pooled from the blood of 60 to 70 rabbits. The active material in anti-leucocyte serum could be precipitated in the globulin fraction of the serum and could be removed by absorption with rabbit lymph node cells. In the course of experiments on the passive transfer of the pre-injection effect by antisera of individual rabbits to leucocytes of individual donor rabbits, evidence was obtained of the existence in the sera of some normal rabbits of antibodies to some of the rabbit leucocyte antigens.