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Cardiac output was determined in nine mongrel dogs 2 and 3 hours after induction of anesthesia with urethane, 750 mg/kg, and chloralose, 75 mg/kg. Cardiac output at 3 hours declined significantly from 2-hour postanesthetic values. The only other significant hemodynamic change was an increased pulmonary resistance. When chloralose and urethane anesthesia are employed in animal experimentation, sequential hemodynamic changes should be appreciated to avoid serious experimental error.

Fragment C-1, the N-terminal half of the heavy chain of rabbit immunoglobulin G, was prepared by cyanogen bromide cleavage from the heavy chain of immunoglobulin G obtained both from the pooled serum of normal rabbits and from specific anti-dinitrophenyl antibody. Tryptic digestion of fragment C-1 after the lysine residues had been allowed to react with S-ethyl trifluorothioacetate led to the isolation of six peptides from inert immunoglobulin G and specific antibody that appear to account for most of this section of the heavy chain. This approach should make possible comparative sequence studies of the Fd section of the heavy chain from different allotypes and from specific antibodies.

The partial amino acid sequence of two large peptides is described. These were prepared from the N-terminal half of the heavy chain of immunoglobulin G from pooled normal rabbit serum by tryptic digestion after the ∈-amino groups of the lysine residues had been blocked with S-ethyl trifluorothioacetate. These peptides are believed to account for about 145 residues of fragment C-1, the N-terminal section of rabbit immunoglobulin G heavy chain prepared by cyanogen bromide cleavage. The evidence from the present paper and the preceding paper (Cebra, Givol & Porter, 1968) suggests that it may be possible to deduce a predominant amino acid sequence for most, if not all, of this section of the molecule.

The complement system has two pathways of activity, both dependent on the sequential conversion of proteolytic zymogens to active proteases leading to a common lytic complex and both with control proteins which inhibit or inactivate different steps in the cascade. Three of the components--C2, factor B and C4--are coded by closely linked genes in the MHC of man and mouse and have been placed relative to each other. The genes are polymorphic, particularly C4, with variable numbers of loci as well as many mutant forms. Some alleles of C4 show strikingly different reactivities in their haemolytic activity and this may be relevant to the association of susceptibility to autoimmune diseases with particular haplotypes in this section of HLA.

Genes in the major histocompatibility complex code for three major groups of glycoproteins, now referred to as classes I, II, and III. Susceptibility to some autoimmune diseases and to systemic lupus erythematosus is associated with the presence of particular haplotypes of genes in these three classes. An attempt has been made to correlate these finding on the basis of the observation that different polymorphic forms of complement component C4 show varying efficiencies of complement activation. It is suggested that susceptibility to these diseases will be related to the varying efficiency of complement cell lysis and of immune aggregate dissolution by complement. This in turn will depend on the strength of interaction of the different polymorphic forms of C4 with other proteins (some also polymorphic) in the scheme of activation and inactivation of complement. Such an arrangement would lead to preferential association of certain alleles of C2, C4 and factor B and possibly also of class I and II antigens as potential targets of complement reaction.

Rapid progress has been made in establishing linkages and in chromosome allocation of the genes of some 9 complement components. In the MHC, C2, Factor B, and two C4 or C4 related genes have been placed in some detail in both man and mouse. The gene coding for the cytochrome P-450 21-hydroxylase has been shown to be duplicated and immediately 3' to the two C4 genes, though it appears to be functionally and structurally unrelated to the complement components. Thus six genes have been mapped to this region where particular haplotypes are associated with increased susceptibility to a number of diseases, some of which are autoimmune in character. The complete gene structure of Factor B has been solved in man and rapid progress is being made with the C2 and C4 genes. The structural basis of the polymorphisms of these genes is being established. In C4, the polymorphism is exceptionally complex with varying numbers of loci and probably more than 50 allotypes occurring in man. A structural basis has also been found for the big differences in the biological activity of some of the C4 allotypes in man. Apart from the genes in the MHC, linkage has been found between the genes coding for C4bp, CR1, and Factor H. Remarkably there are sequence homologies between these proteins and C2 and Factor B, probably related to the ability to bind to one or other of the structurally similar proteins C3b and C4b. The complete cDNA sequences of C3 and C4 in mouse and man have given much information on the many posttranslational modifications of these proteins. A partial structure has been obtained for the C3 gene and the homology shown between C3, C4, C5, alpha 2-macroglobulin, and pregnancy zone protein. Although the amount of detailed information in the molecular genetics of complement components is accumulating rapidly, there appears to be a reasonable prospect that linkages and homologies will classify the data into a comprehensible form.

Polymorphism of complement components, recognized by differences in either their antigenic specificity or their electrophoretic mobility, together with studies of inherited deficiencies, has enabled many of their structural genes to be mapped. In humans, three genes (for C2, C4, and factor B) have been placed between HLA-D and HLA-B on chromosome 6 and in mice, C4 between H2-I and H2-D, chromosome 17. Structural studies show that these components have exceptional features. C2 and factor B which contain the proteolytic active site of the C3 and C5 convertases are of the classical and alternative pathway respectively and are similar in structure and function. Both are novel types of serine proteases. C4 (as C3) contains an intrachain thioester bond essential for hemolytic activity. Molecular genetic investigations are determining the relative positions of these genes, and their precise structure, and should clarify their relation to the inherited diseases which are associated with defects in this section of the human genome.

THE method developed by Mishell and Dutton1 for the in vitro stimulation of an apparently primary response to red cell antigens offers advantages over in vivo experiments in analysing the cellular basis of the immune response. The possible uses of the in vitro system would be much increased were it possible to get reproducible responses to a well defined hapten.

Hapten-antibody complexes prepared at equivalence with the bivalent hapten bis-DNP-octamethylene-diamine and purified rabbit anti-DNP antibody were fractionated by Sepharose gel-filtration and the fractions examined by electron microscopy. Individual fractions were tested for whole-complement fixation and C1 fixation. Dimer forms did not show this type of biological activity, while fractions containing tetramers and larger polymers exhibited both C and C1 fixation, which could be inhibited by prior exposure of the complexes to the univalent hapten epsilon-DNP-caproic acid. The dose-response result indicated that the C-fixation observed was not due to interpolymeric cooperative effects. It was concluded that in the generation of biological activity by soluble antigen-antibody complexes made with complement-fixing antibody, quaternary structural changes following specific combination with antigen may be as important as any tertiary structural alterations that occur in the individual immunoglobulin molecule.