The high polymorphism of classical Mhc molecules found in mammals is not simply the result of strong selection for pathogen resistance in the recent past, since there are virtually no examples of diseases caused by infectious pathogens for which resistance is determined by particular Mhc haplotypes, and in the best-studied case, a particular aspect of malaria in humans, the selection is remarkably weak. We discuss three possibilities to explain high polymorphism in mammals: accumulating, merging and boosting. The mammalian Mhc is complicated and redundant, so that every Mhc haplotype may give some level of resistance due to multiple classical Mhc genes as well as other disease resistance genes; this frustrates the attempts to demonstrate selection for disease resistance. We have looked at two vertebrate groups that may represent two extreme examples of selection for Mhc polymorphism. Birds, like mammals, have highly a polymorphic Mhc that determines strong allograft rejection. However, chickens have a much smaller, compact and simpler Mhc than mammals, as though the Mhc has been stripped down to the essentials during evolution. The selection on a single Mhc gene should be much stronger than on a large multigene family and, in fact, there are a number of viral diseases for which resistance and susceptibility are determined by particular chicken Mhc haplotypes. We have determined the peptide motifs for the chicken class I molecules from a number of haplotypes, which may explain some disease associations quite simply. On the other hand, salamanders have very low Mhc polymorphism and slow allograft rejection. We have isolated axolotl Mhc molecules and shown that they cosegregate with the locus that determines graft rejection in the axolotl, have only a few alleles and only weakly stimulate axolotl T lymphocytes in mixed lymphocyte culture. We believe that salamanders have classical Mhc molecules but most T cells do not recognize them, so that there is no strong selection for polymorphism.

A B lymphocyte that produces the immunoglobulin heavy (H) chain mu may switch to the production of another heavy chain class: gamma, epsilon, or alpha. Since the new heavy chain retains the original variable (V) region, antigenic specificity is maintained. The switch is accompanied by a large deletion of DNA at the heavy chain locus. To explain how this deletion is generated, three models have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, both by-products of looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch. One requirement for switching may be transcription of the constant (C) region to which the cell switches. The switch rearrangement is catalyzed by a switch recombinase, and the isolation of the components of this putative enzyme system is in progress. Although the switch deletion is an accepted fact, the discussion is enlivened by scenarios for switching without DNA rearrangement; such suggestions include processing at the RNA level and trans-splicing.

The diaminobenzidine (DAB) method was adapted for the microphotometric determination of cytochrome c oxidase (cyt ox) in the rat hippocampus. The qualitative and quantitative investigations at the light microscopic level showed that acetone and cytochrome c pretreatment of cryostat sections resulted in a significant increase of demonstrable cyt ox activities. The final incubation medium consisted of 7.5 mM DAB, 2% polyvinylalcohol (PVA) and 6% dimethyl sulfoxide in 0.1 M Hepes buffer; final pH 7.5. PVA was used to keep DAB and artificially oxidized DAB in solution. In the kinetic and endpoint measurements a linear response of the reaction with highest slope was observed only in the initial 5-6 min of reaction. Thereafter the slope decreased. Ultracytochemical demonstrations, which were performed as a topochemical control, showed reaction product only in mitochondria (cristae and intermembranous space). In contrast to vibratome sections all mitochondria reacted positively in cryostat sections of aldehyde-fixed hippocampi. The enhancement of reaction after acetone pretreatment of cryostat sections (light microscopic level) and after a freezing step in ultracytochemistry is discussed in connection with diffusion problems of DAB through mitochondrial membranes.

Unlike most salamanders, the Mexican axolotl (Ambystoma mexicanum) fails to produce enough thyroxin to undergo anatomical metamorphosis, although a "cryptic metamorphosis" involving a change from fetal to adult hemoglobins has been described. To understand to what extent the development of the axolotl hemopoietic system is linked to anatomical metamorphosis, we examined the appearance and thyroxin dependence of class II molecules on thymus, blood, and spleen cells, using both flow cytometry and biosynthetic labeling followed by immunoprecipitation. Class II molecules are present on B cells as early as 7 weeks after hatching, the first time analyzed. At this time, most thymocytes, all T cells, and all erythrocytes lack class II molecules, but first thymocytes at 17 weeks, then T cells at 22 weeks, and finally erythrocytes at 26-27 weeks virtually all bear class II molecules. Class II molecules and adult hemoglobin appear at roughly the same time in erythrocytes. These data are most easily explained by populations of class II-negative cells being replaced by populations of class II-positive cells, and they show that the hemopoietic system matures at a variety of times unrelated to the increase of thyroxin that drives anatomical metamorphosis. We found that administration of thyroxin during axolotl ontogeny does not accelerate or otherwise affect the acquisition of class II molecules, nor does administration of drugs that inhibit thyroxin (sodium perchlorate, thiourea, methimazole, and 1-methyl imidazole) retard or abolish this acquisition, suggesting that the programs for anatomical metamorphosis and some aspects of hemopoietic development are entirely separate.

We applied cytochemical procedures to demonstrate the presence of acid and alkaline phosphatase in the visceral yolk-sac endoderm of rats using frozen, aldehyde-fixed tissue with cerium as the capture agent. This procedure allowed more detailed topochemical localization than was possible using unfrozen tissue or with lead as the capture agent. Acid phosphatase was found to be present in lysosomes as well as in a small number of apical canaliculi, which are thought to be recycling structures of the cell membranes in endodermal cells. Reaction products of alkaline phosphatase were observed on the outer surface of apical, lateral, and basal cell membranes. In addition, some apical vacuoles contained alkaline phosphatase, and more apical canaliculi were positive for alkaline phosphatase than for acid phosphatase. However, most of the apical canaliculi were negative for both enzymes. It is suggested that acid and alkaline phosphatase are taken up by different numbers of apical canaliculi during the detachment of apical canaliculi from lysosomes and resorption vacuoles.