Variants of the group-specific component system as demonstrated by immunofixation electrophoresis. Report of a new variant, Gc Boston (Ge B).

Immunofixation electrophoresis is a relatively simple and reliable method for the genetic phenotyping of the group-specific component (Gc) of serum. This method permits direct comparison of electrophoretic mobilities and band concentrations, with no interference by other proteins. The variants Gc Ab and Gc Y appear identical by this technique; the Eskimo variant appears to be similar to Gc D but not to Gc Ab as previously reported. Gc Norway, also designated Gc 1C, is electrophoretically cathodal to the slower band of Gc 1 and therefore appears to be a distinct variant. A new variant, Gc Boston, is single banded with mobility between the two bands of Gc 1.     

There are two C4 genetic loci and a null allele in the chimpanzee

Genetic polymorphism in C4 in the chimpanzee was studied by agarose gel electrophoresis of desialated plasma and development of patterns by immunofixation with antiserum to human C4 and by a C4-sensitive hemolytic overlay. In general, immunofixation patterns showed multiple partially overlapping bands of which only the most cathodal had strong hemolytic activity. In analogy to human C4, the latter were designated C4B, whereas those detected by immunofixation which had little hemolytic activity were designated C4A. Chimp C4A and C4B reacted with human and mouse (monoclonal) anti-C4B and human anti-Ch1 but neither reacted with monoclonal anti-C4A or human anti-Ch2, Ch3, Rg1, or Rg2. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the alpha chain of C4B showed a slightly lower apparent relative mass than that of C4A at around M _r 93 000. There were three C4A variants and two C4B variants inherited in families as autosomal codominant traits, as C4A-C4B cosegregating pairs with no detectable crossing-over. These pairs were inherited with chimpanzee leukocyte antigen types C2 and BF variants without detectable crossing-over. Half-null C4 haplotypes with C4B ^*Q0 were observed in family studies. Nine BF, C2, C4A, C4B allelic haplotypic combinations (complotypes) were identified among presumably unrelated chimpanzees.Abbreviations used in this paper: ChLA chimpanzee leukocyte antigen - HLA human leukocyte antigen - EDTA ethylenediaminetetraacetate     

Genetic polymorphism of human plasminogen.

Using isoelectric focusing (IEF) in polyacrylamide gel of neuraminidase-treated serum or plasma samples and immunofixation or caseinolytic overlay after urokinase activation of gels, a common genetic polymorphism in human plasminogen has been delineated. Two alleles PLGN*A and PLGN*B, were observed with gene frequencies in whites of .69 and .30; in Orientals of .96 and .03; and in blacks of .80 and .18. Several rare alleles were also found. The distribution of phenotypes fits the Hardy-Weinberg equilibrium. Inheritance is autosomal codominant and fits the expectations of Mendelian inheritance. There is fetal synthesis, but no transplacental passage of plasminogen in either direction.     

The third component of complement: covalent attachment of a radioactive sugar to the labile binding site of C3 via the alternative pathway

A complement- (C) fixing particle consisting of agarose beads to which 5-thioglucose was attached by a --S--S-- bond (agarose-thioglucose) was employed to investigate the mechanism of attachment of C3 to surfaces. When whole serum containing [125I] C3 was incubated with agarose-thioglucose, labeled C3b was taken up in a form that was not removed by 2 M NaCl but was released by 10 mM dithiothreitol. Deposition of DTT-releasable C3b was dependent upon the alternative pathway of C activation. Gel electrophoresis of DTT-releasable C3b from similar experiments performed with unlabeled serum and agarose-[3H]thioglucose showed that the liberated C3b contained a molecule of radioactive thioglucose attached to the alpha'-chain by a covalent bond that was stable to mercaptoethanol. We propose that the thioglucose-alpha' chain bond was formed during the course of C activation by a reaction between the "labile binding site" of newly released C3b and the (then) particle-bound sugar. This formulation implies that the reaction by which C3b attaches to 5-thioglucose in this system is the reaction responsible for opsonization by C3b, and that the C3b-linked sugar represents a marker for the labile binding site. Incubation of the particle-bound C3b in serum resulted in the cleavage of the covalently linked alpha'-chain to several smaller polypeptides, the major cleavage product having a m.w. of 70,000.     

Evidence for differing modes of interaction of acriflavine with ultraviolet-induced lesions in an Hcr + bacterial strain

Summary In buffer suspensions of UV-irradiated Escherichia coli B/r WP2 Hcr⁺ (auxotrophic for tryptophan) acriflavine binds to DNA, but this treatment has little effect on killing and results in the appearance of fewer prototrophs on tryptophan-supplemented minimal agar. If plates contain a broth supplement, however, the buffer-acriflavine treatment greatly increases the yield of UV-induced prototrophs; but this increase does not depend on complete binding of acriflavine to the DNA as a whole, since it is observed with contact times too short for this to occur (as short as 20 seconds). The incorporation of acriflavine in both kinds of plating medium increases the yields of prototrophs. The maximum yield is observed when irradiated bacteria are exposed to acriflavine in buffer before they are plated on medium containing both acriflavine and a broth supplement. Thus post-irradiation effects of acriflavine cannot be accounted for in terms of a single mechanism of action. Our results support the suggestion that phenomena classed together as mutation frequency decline may not represent a single specific repair system.     

Constants of the Alper and Howard-Flanders oxygen equation for damage to bacterial membrane, deduced from observations on the radiation-induced penicillin-sensitive lesion

Energy deposited in the bacterial envelope of E. coli B/r induces lesions which are lethally attacked by penicillin in concentration insufficient to affect unirradiated bacteria. The critical lesions are probably in the membrane moiety. Bacteria were irradiated in the presence of 100 per cent oxygen, oxygen-free nitrogen and mixtures of 1.01, 0.59, 0.3, 0.1 and 0.06 per cent oxygen in nitrogen. Changes in sensitivity with pO2 conformed with the Alper and Howard-Flanders equation, for bacteria treated after irradiation by penicillin as well as for the untreated ones. The values of m were respectively 4.8 and 3.3; the values of K were identical, within experimental error, i.e. 4.4 mmHg. Sensitivity to induction of the penicillin-sensitive lesion was calculated from the difference in the reciprocals of D0 values proper to untreated and treated bacteria, for every gas used. The value of m could not be directly calculated because the effect of penicillin on anoxically irradiated bacteria was not detectable. For that reason, a transformation of the oxygen equation was used which allowed estimates to be made of both m and K, provided the results conformed with the equation. Within experimental error they did so conform. The calculated values of m and K for induction of the penicillin-sensitive lesion were respectively 8 and 5.9 mmHg, but it is shown that the oxygen enhancement ratio was probably underestimated and the K value overestimated. On the assumptions that these values of m and K are specific for radiation damage to bacterial membrane, and that radiation-induced killing is attributable to lethal lesions in the membrane as well as the DNA, the results demonstrate that any interaction of oxygen with sites of energy deposition in the DNA must play a very much smaller role in radiosensitization than does interaction with sites of energy deposition in the membrane.     

An unusual "morphologic" variant of BF S

In the course of family studies of haplotypes of the alleles of the sixth chromosome loci HLA-A, C, B, D/DR, BF, C2, C4A, C4B, and glyoxalase I, we encountered an unusual BF variant. Its mobility was similar to BF F but it appeared to have a lesser intensity after straining with Coomassie Blue, and it was demonstrated by crossed immunoelectrophoresis to be present in lower concentration. It was therefore designated BF FQL. This variant was found on the haplotype HLA-A1, B17, DR7, BF*FQL, C2*C, C4A*6, C4B*1, GLO2. All other haplotypes of this type so far identified carry the BF variant BF S. Following activation of serum samples with zymosan, BF was analyzed by both agarose electrophoresis and isoelectric focusing and immunofixation. On both treatments, serum with BF SFQL produced a Ba pattern identical to that of a sample which was BF S. The Bb pattern for F and S are similar but differ from those of the rare variants BF F1 and BF S1. The Bb pattern of BF FQL was, thus, as expected, the same as BF F or BF S. Hence, we conclude that the variant is a mutant from BF S with mobility similar to BF F. The mutation seemed also to have resulted in a lower concentration of product than normal.     

Adding two components of radiosensitization by oxygen

It has been shown, or inferred, in various contexts that radiosensitization of cells by oxygen is the sum of two (or more) components. If the component sensitivities conform with the Alper and Howard-Flanders equation their sum cannot also conform, but, in practice, even the most meticulous experimental techniques will fail to reveal lack of conformity unless one of the component K values is at least nine times the other. Thus, despite the many results that have demonstrated conformity with the equation, the existence of at least two components may well be a general phenomenon. The killing of cells by radiation is attributable to a summation of lesions in different structures; different K values for the contributing components are therefore to be expected, since neither oxygen nor its competitors are likely to be present in uniform concentration in all elements of the cell nucleus. Provided the components have intrinsic values of o.e.r. greater than one, their addition results in sensitivity that increases monotonically with PO2, approaching asymptotically to the overall o.e.r. which is a weighted average of the component o.e.r.s. In a curve plotted with PO2 on a linear scale a point of inflection can occur only if one component o.e.r. has a value less than one (i.e. oxygen is protective for that component), and then only if relationships between the other parameters satisfy certain conditions. In cases in which points of inflection in the sensitivity curve has been observed these are unlikely to be accounted for by the addition of two components. The analysis of the consequences of adding two components of oxygen sensitization could apply also to chemical sensitization of hypoxic cells.