Oral—parenteral immunization leads to the appearance of IgG auto-anti-idiotypic cells in mucosal tissues

The appearance of plasma cells, specific for antigen or autologous idiotypic antibody, in rabbits immunized systemically, orally, or orally-systemically was determined using immunofluorescence techniques. Oral or parenteral immunizations alone did not lead to the appearance of anti-idiotypic cells in mucosal tissues, although IgA antigen-binding cells were observed in these sites after the oral administration of antigen. However, when rabbits were primed with orally administered antigen, followed by a regimen of concurrent oral—parenteral immunizations, IgG anti-idiotypic plasma cells were demonstrated in mucosal lymphoid tissues.     

Bacterial mutagenesis and host cell DNA damage by chemical carcinogens in the Salmonella/hepatocyte system

Coincubation of isolated and intact rat hepatocytes and Salmonella typhimurium, (Salmonella/hepatocyte system) strain TA 98 was employed to determine both bacterial mutagenicity and DNA damage in the hepatocytes as measured by alkaline elution, following treatment with 2-acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Both the mutagenicity and the rate of DNA elution were dose-dependent for all three compounds. N-OH-AAF was 5 times more mutagenic and caused 80–100 times more DNA damage in the hepatocytes than AAF and AF when compared on a molar basis. The Salmonella/hepatocyte system may provide a more comprehensive evaluation of the potential genotoxic effect of chemicals than the currently used microbial mutagenesis sytems.     

Rabbit T cells with and without Fc receptors

Earlier, indirect evidence for rabbit subpopulations differing in Fc receptors and in response to mitogen has been directly tested. T cells were purified from spleen suspension by removal of adherent cells, followed by removal of Ig-bearing cells on petri dishes coated with antibody, directed against the light chain allotype of Ig receptors. The purified cells were further fractionated by formation of EA rosettes and separation on Ficoll-Hypaque. T cells which lacked Fc receptors had a larger response when stimulated with Con A or PHA than did T cells which possessed Fc receptors. Both subpopulations responded more when irradiated nonadherent B cells were added to the mixture, but the extent of help was the same for both cell populations. T cells which contained both Fc receptor-bearing cells and cells which lacked the receptor had a response which was intermediate between that of the two separated subpopulations.     

Dimeric intermediates of recombination in phage lambda

Biparental lambda phage DNA dimers formed by the Rec recombination system of E. coli were isolated in the absence of DNA replication and phage maturation. The RecA but not the RecB gene is required for dimer formation. Dimers are primarily circular but can also be branched circular or linear. In circular dimers the crossover points are distributed uniformly along the chromosome, even in the presence of the RecB-dependent Chi recombinational hotspots. Thus in the absence of DNA synthesis and maturation, the Rec system can act reciprocally both in the presence and absence of the RecB gene; this lack of RecB participation accounts for the observed lack of Chi activity.     

Protein interactions in the calf eye lens: interactions between β-crystallins are repulsive whereas in γ-crystallins they are attractive

Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.     

Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Codominant and recessive 9-beta-D-arabinofuranosyladenine-resistant mutations of baby hamster cells

9-beta-D-Arabinosyladenine (araA)-resistant mutants of baby hamster kidney (BHK) cells can be classified into 3 classes. In order to gain a better understanding of the mechanism(s) of resistance and the biochemical basis of cytotoxicity of various purine nucleosides, cell hybrids of the mutant and wild-type cells were made and analyzed. The class I araA-resistant, adenosine-kinase-deficient (AK-) allele was shown to be recessive to the wild-type araA-sensitive (AK+) gene. The class II mutant allele, which encodes an altered ribonucleoside diphosphate reductase, was shown to be codominant. The class III mutants show multiple phenotypes, araAr/dAdor/adenosine sensitive (Ados) and alteration in AK activity. The araA- and dAdo-resistant alleles of araS10d, ara-16c, and ara-19a in class III mutant/wild-type hybrid cells are all recessive to the wild-type allele, consistent with a common mechanism of resistance. In contrast the Ados allele of ara-S10d is dominant while those of ara-16c and ara-19a are recessive. The difference may be a reflection of two distinct mechanisms of enhanced Ado sensitivity or, alternatively, it suggests that the sensitivity of the hybrids to Ado is highly dependent on the level of AK activity.     

Kinetics of the reoxidation of succinate dehydrogenase.

The reoxidation phase of the catalytic cycle of succinate dehydrogenase was studied in Complex II preparations' by the rapid freeze-electron paramagnetic resonance (epr) technique. With the synthetic water-soluble Q₁ analog, 2,3-dimethoxy-5-methyl-6-pentyl-1, 4-benzoquinone (DPB), as the oxidant, the observed reoxidation of the epr-detectable components, previously reduced with dithionite or succinate, came to completion within a few milliseconds, well within the turnover time of the enzyme. Only ~80% of Fe-S center 1 and the HiPIP (the high-potential cluster) Fe-S center reacted rapidly with DPB, however; similarly incomplete reactions were observed previously in our studies of the reduction of the enzyme by succinate. The subsequent addition of ferricyanide, which appears to act as a chemical oxidant in these experiments, caused immediate reoxidation of the Fe-S centers and of the free radical. Ferricyanide and phenazine methosulfate (PMS) reoxidized all epr-detectable components in Complex II as well as in reconstitutively active, soluble preparations in' <6 ms, even at 0°C. Thus, reoxidation of the purified enzyme by PMS cannot be rate-limiting. Carboxamides and thenoyltrifluoroacetone inhibit strongly the reoxidation of the Fe-S center 1 and the HiPIP center by DPB, but not their reduction by succinate. These and other data suggest that these inhibitors block electron transport from the dehydrogenase to the Q pool on the O₂-side of the HiPIP center, but there is no evidence that they combine directly with the iron. A recent report that Wurster's blue reacts with soluble succinate dehydrogenase much more rapidly than does PMS could not be confirmed. The two oxidants react at equal rates with the purified soluble enzyme before and after it has been reincorporated into membranes.     

Author index for volume 31

Kilham rat virus (KRV) was isolated from the lymphocyte cytopathic 13762 summary adenocarcinoma tumor line described in part I of this report, as well as three other in vivo passaged rat tumors maintained in the same animal room. It could not, however, be isolated from the noncytopathic (CS8NT)D tissue culture line. Tests done with CsCl₂-purified KRV preparations showed that the virus could replicate in rat lymphocytes and could profoundly depress lymphocyte viability and lymphoproliferative responses. Heterologous anti-KRV antiserum could reverse the inhibitory effects of the purified virus preparation and the inhibitory effects of ultrasonically disrupted KRV-infected tumor cells, but could only partially reverse the inhibitory properties of X-irradiated whole 13762 tumor cells. The results suggest that KRV could account for some, if not all, of the inhibitory properties of the 13762 tumor line.