Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from normal class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i. e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.     

Electro-insertion of xeno-glycophorin into the red blood cell membrane

The electroporation technique, with field strengths slightly below the critical value Ec for electroporation of red blood cells (RBC), enables the insertion of xeno-proteins into the RBC membrane without damaging the cells. The electro-insertion has been used to insert biotinylated human glycophorin into human RBC membrane and human glycophorin into murine RBC membrane. Binding anti-human glycophorin antibody (10F7) to the murine RBC bearing human glycophorin indicates extracellular orientation of inserted glycophorin. Insertion of about 10(5) glycophorin molecule per cell has been estimated by whole cell ELISA.     

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion

HLA class 11 molecules were isolated from mouse L cells transfected with a DR gene and an allele, 52a, of locus DR III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described DR III locus. The TR81 specificity was also present on the DR I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.     

Detection of complex alleles by direct analysis of DNA heteroduplexes

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.     

Iron(III)-adriamycin and Iron(III)-daunorubicin complexes: physicochemical characteristics, interaction with DNA, and antitumor activity

Fe(III) complexes of two anthracyclines, adriamycin and daunorubicin, have been studied. Using potentiometric and spectroscopic measurements, we have shown that adriamycin and daunorubicin form two well-defined species with Fe(III), which can be formulated as respectively Fe(HAd)3 and Fe(HDr)3. In these formulas, HAd and HDr stand for adriamycin and daunorubicin in which the 1,4-dihydroxy-anthraquinone moiety is half-deprotonated. Both complexes are six-membered chelates. The stability constant is beta = (2.5 +/- 0.5) X 10(28) for both complexes. Interaction with DNA has been studied showing that, despite strong coordination to Fe(III), anthracyclines are able to intercalate between DNA bases pairs, releasing the metal. These complexes display antitumor activity against P 388 leukemia that compares with that of the free drug. Fe(HAd)3, unlike adriamycin, does not catalyze the flow of electrons from NADH to molecular oxygen through NADH dehydrogenase. Moreover, it is shown that the triferric adriamycin compound so called "quelamycin" is in fact a mixture of Fe(HAd)3 and polymeric ferric hydroxide.     

Leukocyte LFA-1, OKM1, p150,95 deficiency syndrome: functional and biosynthetic studies of three kindreds

Three patients (2 female, 1 male) with recurrent infection, granulocytosis, impaired pus formation, and/or delayed umbilical cord separation were identified. Assessments of polymorphonuclear leukocytes (PMN)/monocyte function in each patient revealed profound abnormalities of adherence and adherence-dependent functions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of their PMN lysates demonstrated a deficient or absent protein(s) of 138 kilodaltons (gp 138). Na3HB4 labeling demonstrated the absence of a major cell surface glycoprotein complex in each patient. Among parental and sibling PMN suspensions, functional assessments revealed no consistent abnormalities, although variably diminished gp138 was identified by SDS-PAGE and Na3HB4 labeling. Analysis by fluorescence-activated cell sorting and monoclonal antibodies (MAb) to LFA-1 alpha, OKM1 alpha, and their common beta subunit demonstrated a severe or total deficiency of PMN/monocyte surface expression of each protein among all patients; intermediate values were observed for parental and affected sibling suspensions, findings consistent with an autosomal recessive mode of inheritance for this disorder. Cell surface labeling (125I) and immunoprecipitation with the same MAb demonstrated the absence of these glycoproteins in addition to a 150-kilodalton protein (p150,95). Identical abnormalities of surface expression of patient lymphocytes blast-transformed with phytohemagglutinin (PHA) or Epstein-Barr virus were demonstrated. Further, significantly diminished natural killer cell cytotoxicity was observed for each patient tested. PHA blast-transformed patient lymphocytes labeled with [35S]methionine demonstrated a total absence of the beta molecule but indicated the presence of an LFA-1 alpha precursor. These findings indicate that LFA-1 alpha synthesis and surface expression require beta association. It is concluded that impaired inflammatory function in this disorder is casually related to a heritable deficiency of critical "adhesive" leukocyte glycoproteins.     

Analysis of the coliphage T5 DNA ejection process with free and liposome-associated TonA protein.

Outer membrane protein TonA, the receptor for coliphage T5, has been partially purified and incorporated into the phospholipid bilayer of liposomes. Adsorption of the phage to its receptor in either a free or liposome-associated form is fast and sufficient to trigger the ejection of encapsidated DNA. In both in vitro systems the exit of DNA from the phage capsid is a very slow process. Ejected DNA can partially accumulate inside the liposome aqueous compartment, but the transfer from the phage head to the liposome internal space is never complete, perhaps because the liposome volume is too small. The presence of polyamines or divalent cations (magnesium) or both in the incubation medium diminished the extent of DNA ejection, possibly by stabilizing DNA inside the head. DNA movement was slowed as the temperature was decreased from 37 to 18 degrees C. Furthermore, incubation at 4 degrees C totally prevented this DNA movement, even if a large part of the DNA had already exited the capsid.     

Serological discrimination of DR4 haplotypes by radioimmunoassay

Three new alloantigenic specificities of human major histocompatibility complex class 11 molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/DW10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class 11 molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.     

Mechanism of Excretion of a Bacterial Proteinase: Factors Controlling Accumulation of the Extracellular Proteinase of a Sarcina Strain (Coccus P)

It has been known that the extracellular proteinase of Coccus P is found only in cultures grown in the presence of Ca²⁺. It is now shown that this cation is required neither for synthesis, excretion, or activation of a zymogen nor as a prosthetic factor necessary for enzymatic activity. The only function of Ca²⁺ is to stabilize the active structure of the enzyme molecule, presumably by substituting for absence of S-S bridges. In the absence of Ca²⁺, the excreted proteinase undergoes rapid autodigestion and, instead of the active protein, its hydrolytic products are accumulated in the culture fluid. In minimal medium and under conditions of enzyme stability [presence of Ca²⁺ and Ficoll (Pharmacia)], Coccus P accumulates the proteinase at a gradually reduced speed although the rate of cultural growth remains constant. It is shown that this decline in rate of accumulation is caused by the excreted proteinase itself, possibly acting on its own precursor emerging from the cell in a form susceptible to proteolytic attack and not amenable to Ca²⁺ protection. A proteinase precursor is actually demonstrable in a calciumless culture at the onset of the enzyme accumulation which follows Ca²⁺ addition. It is suggested that excreted proteins require an unfolded (or incompletely folded) structure to cross the cell envelope.     

Complement genes C1r and C1s feature an intronless serine protease domain closely related to haptoglobin

The exon-intron structure of the human complement C1s gene displays a striking similarity with that of the gene encoding haptoglobin, a peculiar transport protein distantly related to the serine proteases. While the protease regions of the serine zymogens are typically encoded by multiple exons, the protease domains of C1s and of its genetically linked and functionally interacting homolog C1r are encoded as intronless domains, not unlike a region of haptoglobin, which in fact is devoid of proteolytic activity. The close similarity of the C1s gene with haptoglobin includes the precise conservation of exon-intron junctions and it extends to upstream exons encoding the short repeats typical of several complement components, but found also in other functionally unrelated proteins. Additional evidence of the common ancestry of C1r, C1s and haptoglobin is the presence, within the protease domain, of a set of sequence markers that distinguish these three proteins from all known serine proteases. The finding of vertebrate serine protease genes with an uninterrupted protease-encoding exon supports the definition of a novel evolutionary branch of this gene family and rules out the hypothesis that regards this unusual exon as an irrelevant byproduct of the extravagant functional divergence of haptoglobin.