Study of the antigenic relationship between animal and human immunoglobulins using antisera to human immunoglobulins]

Cross-reactivity of monospecific antisera to human immunoglobulins with animal sera of 10 species was studied by immunoelectrophoresis and radial immunodiffusion. Antisera to IgG were shown to reveal IgG of all the species studied, antisera to IgM and especially to IgA cross reacted less extensively. The greatest number of cross reactions were given by the antisera obtained as a result of hyperimmunization. Hyperimmune monospecific antisera to human IgG, IgA, and IgM can be used for the identification of animal immunoglobulins during their isolation from the sera and for their quantitation by radial immunodiffusion.     

Structural comparisons between mouse and human prealbumin

In an attempt to construct model systems for familial amyloidotic polyneuropathy, prealbumin cDNA was cloned from a mouse liver cDNA library, using previously cloned human prealbumin cDNA as a hybridization probe. The primary structure of mouse prealbumin deduced from the cDNA sequence shows that it consists of 147 amino acids, including a whole prealbumin sequence (127 amino acids) and a putative signal sequence (20 amino acids). These numbers are in complete agreement with those determined for the human prealbumin. Among the 127 amino acid residues of the mature human prealbumin, 25 are replaced by different amino acids in the mouse prealbumin. Interestingly, 24 out of the 25 substituted amino acids are located at the outer surface of the protein, and the regions corresponding to the core and central channel of the protein are almost completely conserved. The cloned cDNA provided essential information for manipulating amyloidosis in mice.     

Structural studies of monoclonal human cryoprecipitable immunoglobulins

The light chain type, immunoglobulin class and when possible, heavy chain subclass of eleven monoclonal human cryoglobulins were correlated with the variable region subgroup of their light chains. The variable region subgroups were assigned by determining the primary amino acid sequence for the first 15 amino-terminal residues of these light chains. $\text{5}\text{5}$ IgM cryoglobulins which react with human IgG had light chains of the variable region-III kappa chain subgroup (vK-III). $\text{4}\text{4}$ IgG and $\text{2}\text{2}$ IgM cryoglobulins with undefined antibody specificity had both lambda and kappa light chains none of which were vK-III. The data support the concept that there is marked restriction of the IgM anti-IgG antibody response to the IgG auto-antigen.     

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.     

Reaction conditions affecting the relationship between thiobarbituric acid reactivity and lipid peroxides in human plasma.

The thiobarbituric acid (TBA) reactivity of human plasma was studied to evaluate its adequacy in quantifying lipid peroxidation as an index of systemic oxidative stress. Two spectrophotometric TBA tests based on the use of either phosphoric acid (pH 2.0, method A) or trichloroacetic plus hydrochloric acid (pH 0.9, method B) were employed with and without sodium sulfate (SS) to inhibit sialic acid (SA) reactivity with TBA. To correct for background absorption, the absorbance values at 572 nm were subtracted from those at 532 nm, which represent the absorption maximum of the TBA:MDA adduct. Method B gave values of TBA-reactive substances (TBARS) 2-fold higher than those detected with method A. SS lowered TBARS by about 50% with both methods, indicating a significant involvement of SA in plasma TBA reactivity. Standard SA, at a physiologically relevant concentration of 1.5 mM, reacted with TBA, creating interference problems, which were substantially eliminated by SS plus correction for background absorbance. When method B was carried out in the lipid and protein fraction of plasma, SS inhibited by 65% TBARS formation only in the latter. Protein TBARS may be largely ascribed to SA-containing glycoproteins and, to a minor extent, protein-bound MDA. Indeed, EDTA did not affect protein TBARS assessed in the presence of SS. TBA reactivity of whole plasma and of its lipid fraction was instead inhibited by EDTA, suggesting that lipoperoxides (and possibly monofunctional lipoperoxidation aldehydes) are involved as MDA precursors in the TBA test. Pretreatment of plasma with KI, a specific reductant of hydroperoxides, decreased TBARS by about 27%. Moreover, aspirin administration to humans to inhibit prostaglandin endoperoxide generation reduced plasma TBARS by 40%. In conclusion, reaction conditions affect the relationship between TBA reactivity and lipid peroxidation in human plasma. After correction for the interfering effects of SA in the TBA test, 40% of plasma TBARS appears related to in vivo generated prostaglandin endoperoxides and only about 60% to lipoperoxidation products. Thus, the TBA test is not totally specific to oxidant-driven lipid peroxidation in human plasma.     

Protein kinase activity of immunoglobulins in human blood plasma

Protein kinase activity of the immunoglobulins (Ig) fractions from blood plasma of clinically healthy humans has been studied. IgA, IgG and IgM preparations have been obtained using column chromatography on sorbents with rabbit antibody to H-chains of human Ig. The level of 32P incorporation in casein in the presence of [gamma-32P]ATP was used to determine protein kinase activity of the Ig-fractions. The protein kinase activity of the preparation of IgA (but not IgG or IgM) was defined. The high-purified preparation of IgA for studing the protein kinase activity has been obtained. Three stages of purifications were used--the separation of plasma proteins by polyethylenglycol 6000, gel-filtration on the column with Toyopearl HW-60 Fine and affinity chromatography on the column containing rabbit antibody to H-chains of human IgA. It was revealed that the fraction of IgA possesses the casein phosphorylation activity. Heparin and trifluoperazine completely and partially inhibited protein kinase activity of IgA while spermidine did not render essential influence. On the basis of the obtained results the conclusion is made that the blood of clinical by healthy humans contains IgA possessing the protein kinase activity.     

Structural relationship between tRNA2 Lys and tRNA4 Lys from mouse lymphoma cells

Summary Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNA₂ ^Lys and tRNA₄ ^Lys, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNA₄ ^Lys has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t⁶A^*, replacing t⁶A. This unidentified nucleotide is not a hypomodified form of t⁶A. Thus, tRNA4ys is not a simple precursor of tRNA₂ ^Lys. Both tRNAs have an unidentified nucleotide, U^**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNA₂ ^Lys and tRNA₄ ^Lys were obtained. The distinctions between tRNA₂ ^Lys and tRNA₄ ^Lys may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5,5-P₁,P₄-tetraphosphate, a putative signal in DNA replication.     

Sulfone-aromatic ligands for thiophilic adsorption chromatography: purification of human and mouse immunoglobulins.

New thiophilic matrices and new procedures were used for the purification of immunoglobulins both from human serum and from hybridoma cell cultures containing fetal calf serum. A range of aromatic and heteroaromatic ligands containing hydroxyl or amino groups have been coupled to divinyl sulfone-activated agarose. The resulting affinity matrices have the general formula M-O-CH2-CH2-SO2-CH2-CH2-X-Y, where M is the agarose matrix, X is oxygen or nitrogen, and Y is an aromatic or heteroaromatic compound. Contrary to earlier expectations these matrices showed pronounced thiophilic binding patterns when tested for the selective binding of immunoglobulins from human serum. The binding is influenced by the structure of the aromatic part of the ligand, the ligand concentration, and the concentration and type of lyotropic salt. 2-Hydroxypyridine coupled to divinyl sulfone-activated agarose was used to purify murine monoclonal antibodies (IgG1 and IgM) from hybridoma cell cultures containing fetal calf serum. Compared to previous methods, significantly increased binding capacity (300-1500%) was obtained by using 1.0-1.2 M ammonium sulfate. Purity of the monoclonal antibody may be optimized for each individual clone by washing the column with either a low concentration of ammonium sulfate or polyethylene glycol before elution.     

A monoclonal mouse antiallotype antibody reacts with certain human and other vertebrate immunoglobulins: Genetic and phylogenetic findings

A new mouse monoclonal antibody, 18.1, recognizes an allotypic determinant on mouse IgG₁ of the a allotype. We found that this alloantibody reacts with immunoglobulins of evolutionary distant vertebrate species including man, and with only certain isotypes or allotypes in some of these species. Most mammalian sera are reactive, except those from lagomorphs, marsupials, and monotremes. Avian and reptilian sera are also positive, while the amphibian and fish sera tested were negative. In human sera, 18.1 detects an isotypic marker on IgG2 and an allotypic marker on IgG3. This reactivity parallels the distribution of the Gm non-g determinant. These findings are discussed in relation to the phylogeny of IgG and its subclasses.     

A monoclonal mouse antiallotype antibody reacts with certain human and other vertebrate immunoglobulins: genetic and phylogenetic findings

A new mouse monoclonal antibody, 18.1, recognizes an allotypic determinant on mouse IgG1 of the "a" allotype. We found that this alloantibody reacts with immunoglobulins of evolutionarily distant vertebrate species including man, and with only certain isotypes or allotypes in some of these species. Most mammalian sera are reactive, except those from lagomorphs, marsupials, and monostremes. Avian and reptilian sera are also positive, while the amphibian and fish sera tested were negative. In human sera, 18.1 detects the isotypic marker on IgG2 and an allotypic marker on IgG3. This reactivity parallels the distribution of the Gm non-g determinant. These findings are discussed in relation to the phylogeny of IgG and its subclasses.     

Coronary artery reactivity in human vessels: some questions and some answers

Spasm of a conduit coronary artery, converting it into a major resistance vessel impeding myocardial blood flow, may have severe short- or long-term effects on cardiac rhythm and systolic ejection of blood. It is now clear that human coronary arteries in vitro contract to acetylcholine but that relaxation is the only response observed in dog coronary vessels. Acetylcholine is as powerful a constrictor of human coronary arteries, in terms of tension induced, as 5-hydroxytryptamine (5-HT) or histamine and is a substantially more powerful constrictor than norepinephrine. Field stimulation of coronary artery strips caused a vasoconstriction that was partially antagonized by atropine (3.45 X 10(-6) M). An enhanced reactivity of the epicardial arteries of cardiac and older patients to several agonists was also observed and appears to provide a background against which a number of vasoactive agents might induce spasm. Coronary tissue from cardiac patients also contains stores of 5-HT and histamine, and the histamine levels are substantially increased above the values in vessels from noncardiac patients. Coronary artery spasm or contraction probably can be initiated by diverse intrinsic and extrinsic influences, including autonomic discharge from either the parasympathetic or sympathetic nervous system or from histamine or 5-HT, and probably no one agent or entity is causative in all cases.     

The relationship between mutagenicity and carcinogenicity of some nitrosamines.

The carcinogenicity and mutagenicity of 26 nitrosamines were compared to help validate the predictability of a short-term in vitro test. 80% of the compounds showed agreement between the two characteristics, while 20% did not. Of the latter group, 8% were false positives and 12% were false negatives.     

Surface immunoglobulins on mouse myeloma cells.

Plasma cells from 22 different transplantable mouse myelomas (PCT) were tested by 14 different class-specific and type-specific anti-immunoglobulin antiserums for cytotoxicity effects using a trypan blue-exclusion method. Seven IgF,κ tumors were all sensitive to anti-IgF and anti-κ antiserums. None of the other antiserums showed a cytotoxic effect. Five IgG,κ and four IgH,κ tumors were lysed by anti-κ serums, but not by anti-IgG or anti-IgH or the others. One of two IgA,κ tumors was lysed by anti-κ, but neither was lysed by anti-IgA or the other serums.One IgM,λ tumor was lysed by anti-IgM and anti-λ. Two λ Bence Jones tumors were not lysed by anti-λ, but both of these and the IgM,λ tumor were lysed by anti-κ. One κ Bence Jones tumor was lysed only by anti-κ serum.Only the IgF tumors and an IgM tumor were lysed by the appropriate anti-heavy chain serum, and $\text{21}\text{22}$ lines had κ surface determinants.