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The IPNS single crystal diffractometer (SCD) has been operating since 1981 primarily as a general purpose instrument for small molecule crystallography and for surveying reciprocal space. The instrument has recently undergone a major upgrade involving replacement of most of the hardware and software. In particular, two new position-sensitive scintillator area detectors based on the Anger camera concept were designed, fabricated and installed on the SCD. Each of these detectors have active areas of 15×15 cm 2 with a spatial resolution of better than 2 mm. A new data acquisition system permits the use of larger data files with a larger wavelength bandwidth at higher time-of-flight resolution. Software developed with tools in the ISAW workbench provides graphical manipulation of three-dimensional arrays of data in histogram coordinates and in reciprocal space, and user-friendly wizards for routine data analysis. As described in this paper, the upgrade of the SCD provides significant improvements in resolution, speed of data collection, and ease of use.

Ionization cross sections differential in the angle and energy of the detected electrons were measured for electron impact on hydrogen gas at 200, 500, 1000, and 1500 eV. Measurements were made from 15\ifmmode^\circ\else\textdegree\fi{}--150\ifmmode^\circ\else\textdegree\fi{} and from a few eV to T-I where T is the primary electron energy and I the ionization potential of hydrogen. The results are compared to earlier measurements and to available calculations.

The effectiveness of the transplantation team in diffusing stress during the pretransplantation period is increasingly important the longer the patient remains on the transplant waiting list. This study describes the stressors and coping strategies of heart transplant candidates during the waiting period. Thirty-nine candidates on the active list for heart transplantation from four mid-East Coast transplantation centers participated. With a possible stress score of 0 to 243, the mean score for this sample was a low 72.84 (standard deviation = 37.47). The three most common stressors were (1) requiring a heart transplant, (2) having terminal heart disease, and (3) worrying family members. The three most common coping strategies were (1) thinking positively, (2) using humor, and (3) trying to keep life as normal as possible. The finding of low stress levels was surprising but may reflect the presence of hope or the patient's desire to spare family members worry--a concern commonly cited by patients. Another explanation is that patients desiring to be perceived as ideal transplant recipients may have underreported their stress. This suggests that the transplantation team should support positive coping strategies when possible and that both patient and family coping should be closely monitored throughout the waiting period.

The molecular components of complement are a major part of the armoury of the mammalian immune system, being required for the lysis of antibody-targeted cells. Several of the complement proteins are known to be encoded by genes within the major histocompatibility complex (MHC). Molecular analysis of these genes is providing new information on the basis of complement action and the possible roles of this system in autoimmune disease.

Four cosmid clones, each with an average insert size of 40 kilobase pairs and containing the factor B gene, were isolated from a human genomic DNA library. The clones were identified by hybridization with a 515-base-pair cDNA probe isolated by using a unique 17-base synthetic oligonucleotide probe from a human liver cDNA library. The cosmid clones were characterized by restriction endonuclease digestion and Southern blotting, and a partial restriction map of the DNA represented in the cosmids was constructed. The Bb portion of the factor B gene is about 4 kb in length. DNA sequence analysis has resulted in the determination of 3.3 kb of sequence at the 3' end of the gene. This region codes for amino acids 87-505 of Bb and includes the whole of the serine proteinase domain of the protein. The three active site residues of histidine, aspartic acid, and serine found at positions 267, 317, and 440 of the Bb sequence, respectively, lie on separate exons. Other functional regions within the serine proteinase domain are separated also by intervening sequences.

Radioimmune assays were developed to assay the binding of complement components C1q, C1s and C4 to antibody aggregates and to cell-bound antibody. The binding of the components was compared with the haemolytic activity and with the capacity to form the C3 convertase activity in the presence of excess C2. The destruction of whole complement and of C4 activity is similar per 1,000 molecules of antibody in aggregates and cell-bound antibody, as is the binding of C1g and C1s, the latter being in a 1:2 molar ratio. The binding of C4 is about 12 times greater, per 1,000 molecules of antibody, on cells than in aggregates. However, the effective C4 molecules, as judged by the formation of C3 convertase activity, are much more similar on cells and aggregates. An assembly mechanism of the early components of complement on antibody-coated cells, which is compatible with these results, is suggested.

The fourth component of complement, C4, was isolated from human serum in good yield, and in confirmation of previous reports was shown to be formed from three peptide chains, alpha, beta and gamma, with apparent mol.wts. 90 000, 80 000 and 30 000 respectively. Preparative methods are described for the isolation of the three peptide chains and their amino acid analyses reported. Component C4 contains 7.0% carbohydrate, alpha-chain 8.6% and the beta-chain 5.6%. The N-terminal amino acid sequences are given for 12 residues of the alpha-chain, eight of the beta-chain and 19 of the gamma-chain.

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4-A1, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4-A and C4-B proteins, respectively, apart from the switch in their antigenic properties.

The heavy chain of a pathological human immunoglobulin IgG and also the Fd fragment have been isolated. No free alpha-amino group was present on either and the N-terminal sequence of both has been identified as pyrrolid-2-one-5-carbonylvalylthreonine. Splitting at the four methionine residues of the heavy chain with cyanogen bromide gave five fractions. The fraction from the C-terminal end of the chain was isolated in high yield and the amino acid sequence was: His-Glu-Ala-Leu-His-Asp(NH(2))-His-Tyr-Thr-Glu(NH(2))-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly These results give strong support to the view that the heavy chain of immunoglobulin is a single peptide chain.

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.

CLASSICAL PATHWAY OF COMPLEMENT ACTIVATION ... . . . . . . ...... . Structure 0/ Components . CI (Clq. Clr. and CIs) . Component C2 . Component C4 ....... . . Components C3 ant!.. C5 . Control proteins (CI inhibitor and C4b binding protein) concerned with regulation of cT and C4b . C Obl�:n�;�!�':��b.2�:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: C5 convertase C4b.2a.3b . Covalent Bond Formation by Complement Compon ents .. Origin of the postulated reactive acyl group formed on activation of C3. C4. and a,-macroglobulin .

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.

The complex series of interactions among complement components which leads to the lysis of cellular antigens may be initiated in several ways. The most well-defined at present are: 1. The classical pathway initiated by the antibody—antigen reaction which leads to activation of the first component Cl, which in turn activates C4 and C2, which together react with C3, leading to changes in the subsequent components, giving rise to a C8–C9 complex which is responsible for cell lysis. 2. The alternate pathway in which activation of C3 may be initiated by different mechanisms, only one of which is believed to arise from antibody-antigen aggregation. Components Cl, C4, and C2 are not involved, but events subsequent to the activation of C3 appear to be the same in both pathways. A recent summary of present knowledge has been given by Lachmann (1973).

The structure of immunoglobulin raises several obvious questions about the structural genes coding for their peptide chains.

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.

An alternative method of isolation from human plasma is described for C3b inactivator, C3bINA, the proteinase that in conjunction with either beta 1H or C4b-binding protein will hydrolyse respectively C3b or C4b, the activation products of the third, C3 and fourth, C4, components of complement. The purification is by chromatography of plasma on columns of QAE-Sephadex, wheat-germ agglutinin-Sepharose, hydroxyapatite and Sephacryl S-200. The yield of C3bINA (6 mg from 500ml of plasma) is severalfold higher than in previously described methods. The sensitivity of the assay for C3bINA has been increased by including optimal amounts of beta 1H, and it was observed that beta 1H was essential for hydrolysis by C3bINA of C3b, whether the C3b was in solution or bound to a cell surface. Native C3 is not hydrolysed by C3bINA + beta 1H, but the haemolytically inactive form that appears on prolonged storage at 4 degrees C or on freezing and thawing is hydrolysed and gives fragments of the alpha-chain of 75000 and 43000 apparent mol.wt. As the alpha'-chain of C3b, which has lost an N-terminal peptide C3a, gives fragments of 67000 and 43000 apparent mol.wt. when incubated with C3bINA + beta 1H, this suggests that the larger fragment is N-terminal and the smaller one C-terminal. The pH optimum of C3bINA with soluble substrates is 6.0, but no clear classification of the type of proteinase to which this enzyme belongs has been obtained.

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.

A remarkable difference has been observed between the reactivity of the two forms of human complement component C4. C4B binds twice as effectively as C4A to antibody-coated red cells, but the reverse occurs with protein-antigen complexes. C4B reacts much more effectively with hydroxyl groups than C4A and this is reversed for reaction with amino groups in spite of the very small difference in amino acid sequence between the two forms of C4. No other differences in stability, activation or inactivation were observed. These findings emphasise the biological advantage of the duplication of the C4 gene in its reaction with a wide range of antigenic structures. The correlation of the presence of different forms of C4 with susceptibility to autoimmune diseases may be explicable by these big differences in binding reactivity.

The heavy chain of rabbit immunoglobulin G exists in three major allotypic patterns, Aa1–Aa3. A comparison of the amino acid compositions of the heavy chains isolated from immunoglobulin IgG homozygous for each allotypic determinant revealed the presence of an additional methionine residue per chain in the Aa3 allotype relative to the Aa1 and Aa2 allotypes. The position of the additional methionine residue was determined by cyanogen bromide cleavage and by tryptic digestion of the γ-chains; it coincided with the inter-Fd–Fc area of the chain. Isolation and characterization of the corresponding tryptic peptides of 31 amino acid residues from each of the allotypes showed the presence of a methionine-for-threonine replacement in the Aa3 allotype, but only in about 70–80% of the molecules. No other allotypic variations were seen in this tryptic peptide. Allotypically related variations in composition were also detected in the N-terminal cyanogen bromide-cleavage peptide.

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.