Partial characterization of the human serum transferrin epitope reactive with the monoclonal antibody TRC-2.

A murine monoclonal antibody (MAb) (TRC-2) specific for human serum transferrin (Tf(h)) was developed. This antibody was depressive on cell growth in serum-free medium in the presence of limiting amounts of Tf(h), but it did not inhibit the binding of Tf(h)-alkaline phosphatase (AP) conjugate to the Tf-receptor (TfR) in a cellular enzyme-linked immunosorbent assay (CELISA) system. On the other hand, the immune complex Tf(h)-TRC-2 was implicated to bind to the receptor in indirect CELISA. Moreover, the detectability of Tf(h)-TfR on the cell surface via Tf-bound TRC-2 suggested that the antibody may inhibit the rapid internalization of this complex. To map the TRC-2-specific epitope, Tf(h) was subjected to proteolytic degradation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The treatment with trypsin gave rise to, among others, a fragment of about 42 kDa, which was reactive with TRC-2. Through sequence analysis by automated Edman degradation, the N-terminal sequence of the 42 kDa-tryptic fragment was aligned to the N-terminus of mature transferrin (VPDKTVR). The N-terminal sequence of an immunoreactive CNBr-fragment of about 13 kDa was, in turn, identical with the sequence (NQLRGKK) corresponding to the residues 110-116 on Tf(h).

Cross-reaction of anti-simian immunodeficiency virus envelope protein antibodies with human immunoglobulins.

It has been recently established that retroviral envelope proteins (REPs) have structural features similar to those of immunoglobulins (Igs). In this study, we asked whether anti-REP antibodies cross-react with human Igs (hIgs). To this end, murine monoclonal antibodies (mMoAbs) that had been raised against a simian immunodeficiency virus (SIV) envelope protein, SIVMac251gp120, were screened for their ability to react with human monoclonal Igs (HMIgs). We show that two HMIgs, RFSJ2 (a rheumatoid factor) and PAMLN6 (a human anti-hIg V region antibody), but not a number of other HMIgs, could be weakly, but consistently, bound by anti-SIVMac251gp120 mMoAbs KK17 and KK46, as judged by indirect enzyme-linked immunosorbent assay and a liquid-phase inhibition immunoassay. Both mMoAbs are specific to amino acid residues in the V3 loop of the SIVMac251gp120. The RFSJ2 Ig heavy-chain V region (VH) is coded in part by a human VH gene, VH3-30.3 and includes the idiotope 7B4 (NKYY), which was previously shown to be present in the gp120 protein of a number of HIV-2 and SIV strains. However, an entirely different VH gene codes the PAMLN6 VH region, opening the possibility that epitope(s) shared between SIVMac251gp120 and hIgs may not be limited to the 7B4 idiotope.

Immune response to progesterone immobilized on Cu2+-induced amphifilic polyelectrolyte-protein complex: antigen specificity and affinity of hybridoma clones.

Cu2+-mediated complex formation between copolymers of acrylic acid with N-isopropyl-acyrlamide (CP1) and negatively charged covalent conjugate of bovine serum albumin with progesterone (BSA.P) was studied in neutral water in the presence of Cu2+. It was shown that under conditions where CP and BSA.P are negatively charged and incapable of binding to one another, the divalent Cu2+ act as "fasteners" promoting the formation of relatively stable water-soluble ternary polycomplexes. The immunogenic properties of ternary mixtures BSA.P-Cu2+-CP1 and BSA.P+IFA were investigated and the production of monoclonal antibodies (MAbs) against progesterone hormone was analyzed. Fusion following the two different immunization procedures resulted in the growth of comparable numbers of progesterone-specific MAbs with apparently similar antigen affinities. Thus, immunizations using antigens in BSA.P-Cu2+-CP1 appear to provide an efficient alternative to incomplete Freund's adjuvant.

Enhancement of the immune response to hepatitis B virus vaccine by antigen specific IgM.

In the present study, modulation of antibody response induced by Hepatitis B virus vaccine-IgM complex was investigated. Purified IgM-type anti HBv monoclonal antibody (1B11) was complexed to commercially available HBv vaccine (GenHevac B Pasteur, France) at varying concentrations of HBsAg (0.5, 1, 1.5 microg of HBsAg) and used to immunize BALB/c mice. An enhanced humoral immune response was obtained with the HBv vaccine-IgM complex at all the doses compared with those immunized by vaccine alone and increased antibody levels were observed with increased concentrations of HBsAg in vaccine formulation. Immunization with HBv vaccine-IgM complex mostly generated IgG-type antibodies in the sera of mice, and also gave rise to the development of hybrid cells which predominantly produced IgG-type monoclonal antibodies. Hence, results from this study indicate that 1B11 can be effectively used to obtain a better immune response to HBv vaccine.

No association between low- and high-activity catecholamine-methyl-transferase (COMT) and attention deficit hyperactivity disorder (ADHD) in a sample of Turkish children.

Biochemical and genetic studies of attention deficit hyperactivity disorder (ADHD) suggest that regulation of catecholamine neurotransmission is a key factor in the aetiology of the disorder. In particular, it is postulated that an underactive dopamine system is associated with the disorder. In this study we have tested this hypothesis by screening a clinical sample of Turkish children with the combined subtype of ADHD with a functional variant of catecholamine-methyl-transferase (COMT) that codes for high- and low-activity variants of the enzyme. Using within-family tests of association and linkage in a sample of 72 children, we found no evidence for a genetic association or linkage. We conclude that altered regulation of catecholamines due to this polymorphism does not have a significant main effect on the risk for ADHD in this population. However, it remains feasible that more minor effects or interacting effects with other genes or environment exist.

Association and linkage of DRD4 and DRD5 with attention deficit hyperactivity disorder (ADHD) in a sample of Turkish children.

The search for genetic factors predisposing to Attention Deficit Hyperactivity Disorder (ADHD) has focused on genes that regulate dopaminergic pathways such as dopamine receptors and enzymes that regulate levels of dopamine in the synapse. There have been several reports of association between ADHD and polymorphic variants within or near DRD4, DRD5, DAT1, DBH and COMT. In this study we set out to investigate specific alleles of DRD4 and DRD5, previously reported to be associated with ADHD, in a sample of Turkish children with DSM-IV ADHD children, as well as their relation to methylphenidate response and dimensional measures of symptom domains. One hundred and four independent trios and seven dyads were analysed using the transmission disequilibrium test (TDT). We found increased transmission of the DRD4 7-repeat allele (DRD4*7) (TDT chi2 = 2.79, P = 0.047). Given that we were testing specific a priori hypotheses regarding the associated alleles, we have used one-tailed P-values throughout. There was evidence of an interaction with methlyphenidate (MPH) response and analysis of the sample excluding non-responders revealed more significant evidence for the association (TDT chi2 = 4.48, P = 0.017). We also detected a trend for linkage and association in the DRD5 polymorphism (TDT chi2 = 2. 38, P = 0.06). Similar findings were obtained in relation to MPH response as analysis of MPH responders alone gave rise to a more significant association than that of the group as a whole (TDT chi2 = 4.9, P = 0.013). t-Test and logistic regression TDT analyses of DRD4*7 transmission with respect to dimensional rating scales of hyperactivity and impulsivity showed an inverse relation suggesting that in this sample DRD4*7 is associated with a lower level of ADHD symptomatology. While this may be due to stratification along a dimension of severity such that severe cases belong to a more extreme group with other specific genetic and environmental causes, similar to the model for low cognitive ability, it is more likely the result of a chance selection bias in this sample.

Detection and typing of human papillomavirus in cervical specimens of Turkish women.

The DNA in situ hybridization (DISH) and conventional solution phase polymerase chain reaction (PCR) were applied to identify human papillomavirus (HPV) DNA in cervical specimens of Turkish women. Samples consisted of 21 cervical brushings from pregnant women and 20 paraffin-embedded biopsies from women with condylomatous or dysplasic lesions. It was found that two out of 21 (9.5%) pregnant women were harbouring HPV-DNA detected by PCR. One woman was infected with HPV 16/30's and the other with an unidentified type. As for the biopsy specimens, the rate of HPV-DNA positivity was 30% and 45% by DISH and PCR, respectively. A double infection was observed in more than 50% of the positive cases. Moreover, HPV 18 was never detected. The results indicated that HPV-DNA is rarely present in cytomorphologically normal smears from pregnant women. The PCR method was successfully adapted for HPV typing in clinical lesions which simultaneously contained different HPV sequences.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and bovine serum albumin.

Cu(2+)-mediated complex formation between polyacrylic acid (PAA) and negatively charged bovine serum albumin (BSA) was studied in neutral water in the presence of Cu2+. Depending on the concentration of Cu2+, the reaction between PAA-Cu2+ complexes and BSA appeared to follow one of two possible paths. At low Cu2+ concentrations (nCu/nAA < 0.15), a further increase in BSA concentration led to the breakdown of the complex as in mechanism I: [formula: see text] At higher Cu2+ concentrations (nCu/nAA > 0.15), a further increase in BSA concentration led to the formation of non-stoichiometric polycomplexes (mechanism II): [formula: see text] The immunogenic properties of ternary mixtures of BSA-Cu(2+)-PAA were investigated and the relationship between immunogenicity and complex formation in solution was analyzed. The addition of Cu2+ to solutions of PAA with BSA gave rise to a considerable increase in BSA-specific immunogenicity. Data obtained from the analysis of the immunogenicity of BSA-Cu(2+)-PAA mixtures formed using different ratios of the components suggested that (1) the highest immunogenic activity is exhibited by stable ternary complexs, and (2) immunoactive polyelectrolyte complexes have a non-stoichiometric composition. We thus propose a novel method, based on Cu2+ mediated complex formation, to enhance protein-specific antibody responses.

Immune response to progesterone involved in Cu(2+)-mediated polyanion-protein complex--antigen specificity and affinity of hybridoma clones.

The immunogenic properties of water soluble (PAA-Cu(2+)-BSA) and colloidal (PAA-Cu(2+)-BSA.P) polycomplexes were investigated, and the specificity of antibodies produced was analyzed. Polycomplexes containing progesterone appeared to possess a high steroid-specific immunogenic activity. A comparative study of immunogenic properties of polycomplexes versus BSA.P + incomplete Freund's adjuvant (IFA) mixtures revealed differences in regards to the specificity of antibody production. In contrast to the IFA system, polycomplexes were able to generate P- as well as BSA-specific antibodies. Such a response is determined, possibly, by increases in the immunogenicity of weak antigenic determinants on the surface of protein globules and or by the representation of dormant determinants existing in the miner site upon complex formation with polyelectrolytes. Finally, using a short immunization procedure based on use of PAA-Cu(2+)-BSA polycomplexes, we produced seven monoclonal antibodies against progesterone included in polyelectrolyte complexes with affinities Kd ranging between 1.3 x 10 (-5) and 9 x 10(-8) M.

Immune response to 17 beta-estradiol in polyelectrolyte complex: antigen specificity and affinity of hybridoma clones.

Complex formation between synthetic polyelectrolytes (PE) [poly-4-vinyl-N-ethyl (cetyl), pyridine bromides-PVP(R2, R16)], bovine serum albumin (BSA), or 17 beta-estradiol-BSA conjugate (BSA.E) was studied in neutral water. Weakly water-soluble (colloidal) complex was formed upon addition of BSA.E to PVP (R2, R16) solution at pH 7. A nonrandom distribution of the protein molecules between the coils of polycations and self-assembly in the nonstochiometric polycomplex particles took place. The immunogenic properties of PVP (R2, R16)-BSA.E polycomplex were investigated and the specificities of produced antibodies analyzed. 17 beta-Estradiol introduced in polyelectrolyte complexes (PE-BSA) was found to invoke considerable increases in the steroid-specific immunoresponse. However, a comparative study of immunogenic activity of polycomplexes versus BSA.E+incomplete Freund's adjuvant (IFA) mixtures revealed some differences in regards to the specificity of antibody production. In contrast to IFA+BSA.E systems, polycomplexes were able to generate estradiol-as well as BSA-specific antibodies. Such a carrier-directed response may be determined by increase in immunogenicity of weak antigenic determinants and/or by the exposure of internally located determinants upon complex formation with polyelectrolytes. Fusions following the two different immunization procedures resulted in the growth of comparable numbers of estradiol-specific monoclonal antibodies with apparently similar antigen affinities. Thus, immunizations using antigens in PEC appear to provide an efficient alternative to IFA.

Apolipoprotein E Genotyping in Turkish Myocardial Infarction Survivors and Healthy Controls.

The apolipoprotein (Apo) E gene is known to be polymorphic. Three common alleles determine six phenotypes which can easily be detected by restriction fragment length polymorphism. We performed apo E genotyping in myocardial infarction survivors and healthy controls for the first time in the Turkish population. DNA was amplified by polymerase chain reaction (PCR) and the PCR product was digested with restriction enzymes HhaI to detect apo E2, E3, E4 and with TaqI to detect apo E1. Relative allele frequency for the patient group was found to be 0.91 for E3, 0.07 for E2, 0.02 for E4 and for the control group 0.875 for E3, 0.067 for E2, 0.058 for E4. Copyright 1995 S. Karger AG, Basel

Regional distributions of beta-thalassemia mutations in Turkey.

The distributions of twelve beta-thalassemic mutations in samples (n = 139 chromosomal samples) from four regions of Turkey were determined. The frequencies of these mutations did not reveal a notable region specific heterogeneity. In particular, the four mutations, IVS.1/nt.110(G/A), IVS.1/nt.6(T/C), IVS.1/nt.1(G/A) and nonsense codon.39(C/T), with country-scale frequencies of 35.9%, 21.6%, 13.0% and 7.2%, respectively, were found to be distributed with rather similar frequencies also on a regional scale.

Leucyl-tRNA and lysyl-tRNA synthetases, derived from the high-Mr complex of sheep liver, are hydrophobic proteins.

The leucyl-tRNA and lysyl-tRNA synthetase components of the multienzyme complex from sheep liver were selectively dissociated by hydrophobic interaction chromatography on hexyl-agarose and purified to homogeneity. Conservation of activities during the purification required the presence of Triton X-100. The homogeneous enzymes corresponded to a monomer of Mr 129000 and a dimer of Mr 2 X 79000, respectively. Both were strongly adsorbed to the hydrophobic support phenyl-Sepharose, in conditions where the corresponding purified enzymes from yeast and Escherichia coli were not bound. Moreover, like the corresponding enzymes from yeast but unlike those of prokaryotic origin, the purified leucyl-tRNA and lysyl-tRNA synthetases derived from the complex displayed affinity for polyanionic supports. It is shown that proteolytic conversion of lysyl-tRNA synthetase to a fully active dimer of Mr 2 X 64000, leads to loss of both the hydrophobic and the polyanion-binding properties. These results support the view that each subunit of lysyl-tRNA synthetase is composed of a major catalytic domain, similar in size to the subunit of the prokaryotic enzyme, contiguous to a chain extension which carries both cationic charges and hydrophobic residues. The implications of these findings on the structural organization of the complex are discussed in relation to its other known properties.

Multiple forms of arginyl- and lysyl-tRNA synthetases in rat liver: a re-evaluation.

The size distribution of lysyl- and arginyl-tRNA synthetases in crude extracts from rat liver was re-examined by gel filtration. It is shown that irrespective of the addition or not of several proteinase inhibitors, lysyl-tRNA synthetase was present exclusively as a high-Mr entity, while arginyl-tRNA synthetase occurred as high- and low-Mr forms, in the constant proportions of 2:1, respectively. The polypeptide molecular weights of the arginyl-tRNA synthetase in these two forms were 74000 and 60000, respectively. The high-Mr forms of lysyl- and arginyl-tRNA synthetases were co-purified to yield a multienzyme complex, the polypeptide composition of which was virtually identical to that of the complexes from rabbit liver and from cultured Chinese hamster ovary cells. Of the nine aminoacyl-tRNA synthetases, specific for lysine, arginine, methionine, leucine, isoleucine, glutamine, glutamic and aspartic acids and proline, which characterize the purified complex, each, except prolyl-tRNA synthetase, was assigned to the constituent polypeptides by the protein-blotting procedure, using the previously characterized antibodies to the aminoacyl-tRNA synthetase components of the corresponding complex from sheep liver.

Do yeast aminoacyl-tRNA synthetases exist as soluble enzymes within the cytoplasm?

The aminoacyl-tRNA synthetases from a crude extract of yeast were shown to bind to heparin-Ultrogel through ionic interactions, in conditions where the corresponding enzymes from Escherichia coli did not. The behaviour of purified lysyl-tRNA synthetases from yeast and E. coli was examined in detail. The native dimeric enzyme from yeast (Mr 2 X 73000) strongly interacted with immobilized heparin or tRNA, as well as with negatively charged liposomes, in conditions where the corresponding native enzyme from E. coli (Mr 2 X 65000) displayed no affinity for these supports. Moreover, the aptitude of the native enzyme from yeast to interact with polyanionic carriers was lost on proteolytic conversion to a fully active modified dimer of Mr 2 X 65500. A structural model is proposed, according to which each subunit of yeast lysyl-tRNA synthetase is composed of a functional domain similar in size to that of the prokaryotic enzyme, contiguous to a 'binding' domain responsible for association to negatively charged carriers. The evolutionary acquisition of this property by lower eukaryotic aminoacyl-tRNA synthetases suggests that it fulfils an important function in vivo, unrelated to catalysis. We propose that it promotes the compartmentalization of these enzymes within the cytoplasm, through associations with as yet unidentified, negatively charged components, by electrostatic interactions too fragile to withstand the usual extraction conditions.

Seven mammalian aminoacyl-tRNA synthetases associated within the same complex are functionally independent.

A heterotypic multienzyme complex from sheep liver containing seven aminoacyl-tRNA synthetases specific for isoleucine, leucine, methionine, glutamine, glutamic acid, lysine and arginine was subjected to kinetic analyses to examine the possibility that association of these enzymes may impart kinetic properties which differ from those of their unassociated counterparts. The evidence obtained by two different approaches leads to the conclusion that the associated enzymes are functionally independent. Firstly, the kinetic constants of the methionyl-tRNA and lysyl-tRNA synthetase components of the complex do not differ significantly from those of their unassociated counterparts obtained after controlled proteolysis of the complex. Secondly, the methionyl-tRNA synthetase component of the complex displays identical kinetic constants, whether assayed in the presence of [14C]methionine, ATP and highly enriched tRNAMet alone, or in the additional presence of the substrates required for unlabeled aminoacyl-tRNA formation by each of the other six enzymes. Similarly, the initial rates of [14C]aminoacyl-tRNA formation catalyzed by any of the six other enzymes was unaffected by the concomitant functioning of the other aminoacyl-tRNA synthetases. The sedimentation behaviour of the aminoacyl-tRNA synthetase components of the complex under conditions prevailing in the tRNA aminoacylation assay indicates that they remain associated under these conditions. The implications of these findings on the structural organization of the enzymes within the complex are discussed.