William J. Mandy

The rabbit CD1 and the evolutionary conservation of the CD1 gene family.

A comparison of the genes encoding the CD1 leucocyte differentiation antigens in man and mouse shows important differences which prompted us to analyze theCD1 genes of the rabbit. We have found that the rabbit genome contains multipleCD1 loci. Upon cloning and sequencing, one of these loci was found to encode the known rabbit CD1-like antigen (R-Ta) and to be closely related to the humanCD1b gene, which is absent in the mouse, while a second rabbit gene is closely related to both the humanR3 and the mouseCD1 genes. The data reinforce the notion of the existence of two classes ofCD1 genes, one of which is conserved in all species, while the other, albeit also evolutionarily old, has been deleted in mice as well as in other rodents.

Rabbit immunoglobulin allotype A12: a new agglutinating specificity.

A new allotype, A12, present on rabbit IgG is described. This allotype is detected by inhibition-of-agglutination techniques similar to those employed for the previously described allotype A11. The specificity is on the H chain in the hinge region of IgG. It can be associated with any of the H chain group a allotypes. A11 and A12 are transmitted by codominant autosomal genes.

Characterization of allotype A11 in rabbits: A specificity detected by agglutination☆

Abstract Inhibition of agglutination techniques were used to characterize a new allotypic determinant, A11, of rabbit immunoglobulin. Agglutinator antiserum specific for the A11 determinant was used to agglutinate rabbit type F erythrocytes sensitized by coating with anti-F antibody bearing the A11 determinant. Agglutinator was prepared by injecting rabbits lacking the A11 determinant with IgG of the same group a and group b allotypes but possessing the A11 determinant. Several sensitizors for detecting the A11 specificity were prepared by immunizing rabbits possessing the A11 determinant with type F rabbit erythrocytes. The determinant was found to be associated with the IgG class of immunoglobulins by filtration and by DEAE chromatography. Studies with enzymatic fragments and subunits of IgG show that the A11 determinant is carried on the heavy ( H ) chain. Although the determinant was detectable on the (Fab′) 2 fragments obtained by pepsin hydrolysis of IgG, the requirement for the integrity of the inter- H chain disulfide bond precluded its detection on isolated H or light ( L ) chains. The results further suggest that certain structures on both H chains of a given IgG molecule require proper orientation for interaction with the anti-A11 serum.

Rabbit IgC heavy chains: Evolutionary divergence of genes coding for constant region

Abstract This investigation elaborates further on the question of shared immunoglobin allotypes among the present day lagomorphs. The serum and IgG preparations of cottontial rabbits and hares were tested against various alloantisera bearing specificities for the groups a , b , c , d and e allotypic markers. Indirect radioimmune precipitin techniques were used to identify certain group a and group b markers on hare (Fab′) 2 fragments. Hemagglutination-inhibition tests estabilished the presence of the e 15 marker on cotton tail and hare IgG; the group d ( d 11 and d 14) and e 14 markers were undetected. The results are consistent with a hypothesis that the ancestral lagomorph C H gene coded for e 15 allotypic specificities.

The action of cyanogen bromide on rabbit IgG molecules of allotypes A11 and A12.

Abstract Cyanogen bromide acts on A12 rabbit IgG in HCl to yield 5S fragments and on A11 rabbit IgG to yield 3.5S fragments plus some 5S fragments. Separation of the 3.5S and 5S cleavage products by ultracentrifugation or by gel filtration with Sephadex® G200 may thus be used to confirm serological typing of rabbit IgG for A11 and A12 and to estimate the relative amounts of each of these allotypes in IgG samples. The group a (H chain) allotypic specificities of the fragments may be determined by their ability to block the precipitation of radioiodinated IgG by homologous antiallotype serum. Thus these procedures may be used to study the association of A11 and A12 allotypic specificities with those of group a.

The substrate specificity of L-asparaginase from Alcaligenes eutrophus

The tumor suppressive effects of L-asparaginase have been well documented [ I] . All clinical studies reported to date have utilized the enzyme from Escherichia coli. However, continual therapy with the enzyme from this source has commonly resulted in problems of toxicity, including immunological sensitivity to the foreign protein [2], indicating the need for alternative sources of L-asparaginase. Towards that end we have been examining the serological and chemical properties of a variety of bacterial L-asparaginases. The present paper deals with some of the kinetic properties of L-asparaginase from Alcaligenes eutrophus (formely Hydrogenomonas eutropha) [3] . A comparison of the substrate specificities for L-asparaginase from A. eutrophus and E. coZi showed striking differences.

Constant region IgG allotypes in hares: Group e allelic polymorphism

Abstract Alloantisera prepared against domestic rabbit IgG allotypes were used in hemagglutination-inhibition and direct radioimmune binding tests to detect the Cγ region allotypes in hares from several geographical locations. The d11, d12 and e14 allotypes were absent in all the hare samples tested. The hemagglutination-inhibition and the radioimmune binding tests gave differing results in the classification of hares with respect to the e15 allotype. The apparent enigma was resolved by taking into consideration the concept of multiple antigenic determinants comprising the e15 allotypic system. The results are presented and discussed in terms of possible allelic genetic variants of the Cγ gene in hares.

Constant region IgG allotypes in cottontail rabbits: group E allelic polymorphism.

Abstract Alloantisera prepared against domestic rabbit IgG allotypes were used in hemagglutination-inhibition and direct radioimmune binding tests to detect the Cγ region allotypes in cottontail rabbits. The absence of the group d allotypes (d11 and d12) in cottontail rabbits was confirmed. However, allelic variants of the group e markers (e14 and e15) were found in some of the cottontail rabbits examined. For example, all cottontails exhibited the e15 marker and 6 out of 10 animals exhibited the e14 marker as well. Several experiments were conducted to show that the e14 and e15 antigens of domestic rabbit and cottontail IgG are identical. Other results are consistent with the hypothesis that the e14 and e15 antigens in cottontail rabbits are products of allelic genes.

Lagomorph IgG hinge region: Allotype associated amino acid sequence variations☆

Abstract Amino acid composition and amino terminal sequence analyses of peptic and tryptic peptides of the Cγ hinge region for domestic rabbit, cottontail rabbit and hare IgG are compared. The failure to detect the group d (d11 and d12) allotypes in cottontail rabbits and hares can be attributed to additional variability in amino acid sequences that correspond to the variability between d11 and d12 noted in domestic rabbits. The results are consistent with the hypothesis that the Cγ genes of present day lagomorphs arose from a common ancestral gene through a series of point mutations.

Chemical modification of a rabbit immunoglobulin allotypic specificity

Abstract The group d allotypes ( d 11 and d 12) of rabbit IgG can be distinguished serologically by specific antisera and chemically by a methionine/threonine substitution. The methionine residue which correlates with d 11 was modified by chloramine T and iodoacetic acid to methionine sulfoxide and carboxymethyl-methionine, respectively. The chemical modification of the d 11 protein was monitored by the loss in d 11 antigenic specificity and by resistance to CNBr attack.