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PubMed2003

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin.

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.

Amino Acid Substitution↗

PubMed1991

Localization of the dihydrolipoamide branched-chain transacylase gene (DBT) of the human branched-chain keto acid dehydrogenase complex to chromosome 1.

The gene coding for the transacylase subunit (DBT) of the human branched-chain keto acid dehydrogenase complex was localized to chromosome 1 by probing panels of human x mouse chromosome hybrids with an E2 cDNA amplified by the polymerase chain reaction. Additional data with two hybrids containing chromosome 1 fragments suggest that the DBT gene is located on the short arm (1pter----p21) of the chromosome.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1999

DNA molecules can drive the assembly of other DNA molecules into specific four-stranded structures.

Single-stranded DNA molecules containing clustered G-repeats can be assembled into various four-stranded structures linked by G-quartets. Here, we report that such molecules can also drive the assembly of other DNA molecules containing G-repeats into specific four-stranded structures. In these assays, the oligonucleotides 5'-CAGGCTGAGCAGGTACGGGGGAGCTGGGGTAGATTGGAATGTAG-3' (oligo D) and 5'-CGGGGGAGCTGGGGT-3' (oligo B), consisting of sequences found in immunoglobulin switch regions, were annealed in a buffer containing K+ and the annealing products were analyzed by polyacrylamide gel electrophoresis. This analysis revealed that whereas annealing of each oligo alone produced four-stranded structures designated D2 and B2, annealing of mixtures containing both oligos produced additional complexes designated D2* and B2*. D2* and B2* were found to contain only D molecules and only B molecules, respectively. The yield of D2* increased and the yield of B2* decreased, as the concentration ratio oligo B/oligo D was increased. These results indicated that B can drive the assembly of D into D2* and D can drive the assembly of B into B2*. Further studies revealed that while the assembly of D2 followed a second order kinetics, the B-driven assembly of D2* followed a first order kinetics. Dimethyl sulfate footprinting indicated that both D2 and D2* are four-stranded structures containing two parallel and two antiparallel chains. In addition, annealing of D mixed with various B mutants showed that only mutants containing two G-clusters can drive the assembly of D2*. Based on these data, we propose that in the process of D2* assembly, a four-stranded intermediate containing B and D is formed and then dissociates into D2* and B in a rate-limiting first order reaction. Driver mechanisms of this type may cause formation of specific four-stranded structures at G-rich chromosomal sites, thereby regulating processes such as recombination and telomere synthesis.

Base Sequence↗

PubMed1979

Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene.

Immunoglobulin kappa chain gene formation involves site-specific somatic recombination between one of several hundred germ-line variable region genes and a joining site (or "J segment") encoded close to the constant region gene. We have cloned and determined the nucleotide sequence of major portions of the recombination region of the mouse kappa gene and discovered a series of five such J segments spread out along a segment of DNA 2.4 kilobases from the kappa constant region gene. These J segments encode the 13 COOH-terminal amino acids of the variable region, probably including amino acids involved in the antigen combining site and in heavy/light chain contacts. The J segments also display striking sequence homology to one another in both their coding and immediately flanking sequences. Major elements of a short palindrome--CAC(TA)GTG--are preserved adjacent to the recombination sites of both variable and J region genes and constitute inverted repeats at both ends of the sequences to be joined. These palindromes can be written as a hypothetical stem structure that draws variable and J regions together, providing a possible molecular basis for the DNA joining event. Four of the J segments that we have discovered encode amino acid sequences already found in myeloma proteins. By altering the frame of recombination, we can account for additional light chain amino acid sequences, suggesting that the V/J joining event might generate antibody diversity somatically both by using different combinations of variable and J region genes and by using alternative joining frames.

Animals↗

PubMed1993

Structure of the apolipoprotein A-IV/lipid discoidal complexes: an attenuated total reflection polarized Fourier transform infrared spectroscopy study.

Discoidal lipid particles were prepared from a reaction mixture containing apo A-IV and dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) in the molar ratio of 185:1 (lipid/protein). The complexes were isolated by gel filtration and characterized in terms of composition and size. Infrared attenuated total reflection spectroscopy was used to estimate the secondary structure of apolipoprotein A-IV and the orientation of its amphipathic alpha-helices with respect to the lipid hydrocarbon chains. In addition, infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the particles. This approach has been applied successfully to reconstituted HDL particles prepared from a reaction mixture containing DPPC and apo A-I in the molar ratio of 150:1 (Wald, J.H., Goormaghtigh, E., De Meutter, J., Ruysschaert, J.M. and Jonas, A. (1990) J. Biol. Chem. 265, 20044-20050). Apo A-IV helicity increased for the protein bound to DMPC or DPPC but the increase was more pronounced for the apo A-IV/DMPC particles. In both complexes, the alpha helical amphipathic segments of the protein were parallel to the lipid acyl chains and no significant modification of the overall organization of the lipid molecules in the lipid bilayer was observed. The presence of apo A-IV seems only to affect the conformation of the lipid hydrocarbon chains in close contact with the protein in the discoidal particles.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1999

The use of monoclonal antibody R92F6 and polymerase chain reaction to confirm the presence of parvovirus B19 in bone marrow specimens of patients with acquired immunodeficiency syndrome.

BACKGROUND: Parvovirus B19 infection is a cause of chronic anemia and red cell aplasia in patients with acquired immunodeficiency syndrome (AIDS) and in other immunocompromised hosts. Anemia in AIDS patients has a multifactorial etiology, with parvovirus B19 infection being an infrequent but nevertheless treatable cause. Therapy with intravenous immune globulin can result in rapid improvement of parvovirus-induced anemia. This treatment is expensive, therefore accurate and rapid confirmation of parvovirus infection is important in providing appropriate and cost-effective therapy. METHODS: Bone marrow samples from 2 AIDS patients with severe anemia and reticulocytopenia were studied. Bone marrow morphology and serologic studies were evaluated for parvovirus B19 infection. An immunohistochemical method using a monoclonal antibody, R92F6, to B19 capsid proteins was utilized on decalcified, B5-fixed, paraffin-embedded bone marrow biopsies. Bone marrow aspirate cells were examined by electron microscopy for evidence of viral particles. In addition, polymerase chain reaction (PCR) studies using a nested PCR assay to the parvovirus B19 viral genome were performed in a case for which fresh cells were available. RESULTS: Bone marrow findings included marked erythroid hypoplasia with characteristic giant pronormoblasts and intranuclear inclusions. Serologic studies were negative in one case, while the second case showed positive parvovirus B19 immunoglobulin M antibody. Immunohistochemical studies for parvovirus B19 were positive in both cases. The presence of intranuclear virions was demonstrated by electron microscopy and was confirmed by PCR analysis. Both patients were treated with intravenous immune globulin, and subsequent improvement was noted. CONCLUSIONS: Both immunohistochemistry and PCR studies on bone marrow specimens from AIDS patients with anemia are rapid and sensitive methods for the confirmation of parvovirus B19 infection. They are valuable tools, particularly when serologic studies are negative. When PCR is not available, immunohistochemical methods can be useful. The rapid confirmation of parvovirus B19 infection will allow for early and cost-effective therapy.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Interaction of a macrocyclic bisacridine with DNA.

The binding of the macrocycle SDM to DNA was investigated by visible spectroscopy, stopped-flow kinetics, and NMR spectroscopy. SDM is composed of two 9-aminoacridines linked via the amino groups by a spermine side chain and via the 4-positions by a N,N'-[(methylthio)ethyl]succinamide side chain [Zimmerman, S. C., Lamberson, C. R., Cory, M., & Fairley, T. A. (1989) J. Am. Chem. Soc. 111, 6805-6809]. The visible spectrum of SDM bound to poly[d(A-T)]2 or poly[d(G-C)]2 is red-shifted relative to the spectrum of SDM alone and displays considerable hypochromicity. Results from titrations of SDM with polymer indicate a binding site size of three base pairs per macrocycle. The dissociation constant for SDM bound to either poly[d(A-T)]2 or poly[d(G-C)]2 is an order of magnitude lower than that for a similar bisacridine linked only by a spermine side chain. In addition, the dependence of the dissociation constant on ionic strength is significantly reduced. NMR studies of SDM complexes with poly[d(A-T)]2 or a tetramer, d(CGCG)2, show that intercalation is the mode of binding. The magnitudes of the chemical shift differences for SDM aromatic protons in the free and bound states support intercalation with the acridine ring systems essentially parallel to the long axis of the base pairs. Cross peaks from NOESY spectra of the SDM complex with d(CGCG)2 further support this mode of binding and provide information on the structure of the complex. The results are analyzed for consistency with each of three binding models: (i) bisintercalation with the two side chains in the same groove; (ii) bisintercalation according to the neighbor-exclusion principle with the two side chains in opposite grooves; and (iii) bisintercalation with two side chains in opposite grooves but with violation of the neighbor-exclusion principle. Model i is found to be unlikely on the basis of all evidence obtained, including preliminary modeling studies. Both models ii and iii can be reconciled with the experimental evidence and from a modeling standpoint are energetically feasible.

Aminoacridines↗

PubMed1992

Structure and organization of the human S-adenosylmethionine decarboxylase gene.

Genomic clones for the S-adenosylmethionine (AdoMet) decarboxylase gene were isolated from a human chromosome 6 DNA library. In addition, polymerase chain reaction and specific primers were used to amplify fragments from chromosomal DNA covering exonic regions not found in the screening of DNA libraries with AdoMet decarboxylase cDNA. The gene encompasses at least 22 kilobases of chromosome 6 DNA and comprises nine exons and eight introns, in contrast to the corresponding rat gene that has only eight exons (Pulkka, A., Ihalainen, R., Aatsinki, J., and Pajunen, A. (1991) FEBS Lett. 291, 289-295). Exon-intron junctions in the human and rat AdoMet decarboxylase genes were in identical positions except that exons 6 and 7 of the human gene formed a single exon in the rat gene. Alu-like sequences are present in four introns and the 5'-flanking region of the human gene. The promoter region contains a TATA box adjacent to the cap site; in addition, DNA elements for binding of transcription factors AP-1, AP-2, CREB, SP-1, and multiple steroid receptors are present between position -3,158 and the transcription start site. Two AdoMet decarboxylase promoter-reporter gene constructs with about 170 and 1,500 nucleotides of the 5'-flanking DNA were used in transient expression studies. AdoMet decarboxylase promoter was capable of driving reporter gene expression, but it was less active than the murine ornithine decarboxylase promoter. There are at least three potential polyadenylation signals at the 3'-end of the gene, and utilization of the first two results in the formation of the 2.0- and 3.6-kilobase AdoMet decarboxylase mRNA species present in human tissues and cell lines. AdoMet decarboxylase gene-related sequences were also present in a human X chromosome-specific DNA library. Partial nucleotide sequencing of this DNA revealed a lack of introns present in the gene located on chromosome 6, suggesting that the locus on the X chromosome contains a processed AdoMet decarboxylase pseudogene.

3T3 Cells↗

PubMed1992

Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.

Animals↗

PubMed2006

Short RNAs repress translation after initiation in mammalian cells.

MicroRNAs (miRNAs) are predicted to regulate 30% of mammalian protein-encoding genes by interactions with their 3' untranslated regions (UTRs). We use partially complementary siRNAs to investigate the mechanism by which miRNAs mediate translational repression in human cells. Repressed mRNAs are associated with polyribosomes that are engaged in translation elongation, as shown by puromycin sensitivity. The inhibition appears to be postinitiation because translation driven by the cap-independent processes of HCV IRES and CrPV IRES is repressed by short RNAs. Further, metabolic labeling suggests that silencing occurs before completion of the nascent polypeptide chain. In addition, silencing by short RNAs causes a decrease in translational readthrough at a stop codon, and ribosomes on repressed mRNAs dissociate more rapidly after a block of initiation of translation than those on control mRNAs. These results suggest that repression by short RNAs, and thus probably miRNAs, is primarily due to ribosome drop off during elongation of translation.

Animals↗

PubMed2005

Evaluation of the field-adapted ADMA approach: absolute and relative energies of crambin and derivatives.

A large number of conformations and chemically modified variants of the protein crambin were used to extensively test the field-adapted adjustable density matrix assembler (FA-ADMA) method developed for ab initio quality quantum chemistry computations of proteins and other macromolecules, introduced in an earlier publication. In this method, the fuzzy density matrix fragmentation scheme of the original adjustable density matrix assembler (ADMA) method has been made more efficient by combining it with an approach of using point charges to approximate the effects of additional, distant parts of a given macromolecule in the quantum chemical calculation of each fragment. In this way, smaller parent molecules can be used for fragment generation, while achieving accuracy that can be obtained only with large parent molecules in the original ADMA method. Whereas in both methods the error relative to the Hartree-Fock result can be reduced below any threshold by choosing large enough parent molecules, this can be done more efficiently with the new method. In order to obtain reliable test results for the accuracy obtainable by the new method when compared to conventional Hartree-Fock calculations, we performed a large number of energy calculations for the protein crambin using various conformations available in the Protein Data Bank, various protonation states, and side chain mutations. Additionally, in order to test the performance of the method for protein-solvent interaction studies, the energy changes due to the formation of complexes with ethanol and single and multiple water molecules were investigated.

Chemical Phenomena↗

PubMed1988

Synthesis and antiherpetic activity of (+/-)-9-[[(Z)-2-(hydroxymethyl)cyclopropyl]methyl]guanine and related compounds.

A series of analogues of acyclovir and ganciclovir were prepared in which conformational constraints were imposed by incorporation of a cyclopropane ring or unsaturation into the side chain. In addition, several related base-modified compounds were synthesized. These acyclonucleosides were evaluated for enzymatic phosphorylation and DNA polymerase inhibition in a staggered assay and for inhibitory activity against herpes simplex virus types 1 and 2 in vitro. Certain of the guanine or 8-azaguanine derivatives were good substrates for the viral thymidine kinase and were further converted to triphosphate, but none was a potent inhibitor of the viral DNA polymerase. Nevertheless, one member of this group, (+/-)-9-[[(Z)-2-(hydroxymethyl)cyclopropyl]methyl]guanine (3a), displayed significant antiherpetic activity in vitro, superior to that of the corresponding cis olefin 4a. Another group, typified by (+/-)-9-[[(E)-2-(hydroxymethyl)cyclopropyl]methyl]adenine (17b), possessed modest antiviral activity despite an apparent inability to be enzymatically phosphorylated. The relationship of side-chain conformation and flexibility to biological activity in this series is discussed.

Acyclovir↗

PubMed1991

Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids.

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

Amino Acid Sequence↗

PubMed1993

A selective defect in IgG2b switching as a result of targeted mutation of the I gamma 2b promoter and exon.

LPS stimulation of B lymphocytes induces germline transcription of and subsequent switching to the gamma 2b gene. Mature germline transcripts contain an I exon (non-coding) spliced to the C gamma 2b exons. To investigate the role of germline transcription and/or transcripts in heavy chain class switching, we have replaced the germline I gamma 2b promoter and I exon in ES cells with an expressed neomycin resistance gene. The mutated chromosome retains the downstream target sequence for switch recombination (S regions) and all sequences necessary for expression of a switched gamma 2b gene. Wild-type or mutant ES cells were injected into RAG-2 deficient blastocysts to generate somatic chimeras in which all lymphocytes were ES-cell derived. Chimeras derived from injection of heterozygous mutant ES cells had normal levels of serum IgG2b, but their splenic B cells showed a partial decrease in ability to switch to gamma 2b. Strikingly, B lymphocytes from chimeras derived by injection of homozygous mutant ES cells were deficient in IgG2b production both in vivo and in vitro, but normal with respect to production of other Ig heavy chain isotypes. Additional studies demonstrated that lack of ability to produce IgG2b by the mutant B cells correlated with lack of germline transcription and resulted from a specific defect in class-switch recombination to S gamma 2b. Together, these studies demonstrate that the I region is an important regulatory element for control of class-switch recombination.

Animals↗

PubMed1999

Evidence for the presence of hepatocyte growth factor expression in human ovarian follicles.

The presence of hepatocyte growth factor (HGF) in follicular fluid (FF) relative to concentrations of sex steroid hormones and human chorionic gonadotrophin (HCG) was investigated. A total of 69 FF samples were obtained during oocyte retrieval for in-vitro fertilization (IVF) from 11 patients with no apparent endocrine disorders. The concentrations of HGF, oestradiol, progesterone, HCG and testosterone in FF samples were measured by enzyme-linked immunosorbent assay. Transcription of HGF and its receptor, c-met, was detected by reverse transcription-polymerase chain reaction (RT-PCR). Human FF samples contained approximately 90-fold higher amounts of HGF (24.2 +/- 1.2 ng/ml), compared with those of serum (0. 28 +/- 0.04 ng/ml). Concentrations of HGF in FF were positively correlated with those of progesterone (r = 0.649, P < 0.0001) and HCG (r = 0.264, P = 0.026) concentrations in FF. However, HGF concentrations were not significantly correlated with oestradiol and testosterone. HGF in FF was detected by Western blotting, as a single 90 kDa band, corresponding to a single chain form. Additionally, mRNA for both HGF and its receptor were detected in a crude granulosa cell preparation from the pre-ovulatory follicles. These findings suggest that HGF is produced locally in human ovarian follicles and may have a physiological role as an autocrine/paracrine factor.

Adult↗

PubMed1983

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: localization and characterization of idiotypic determinants.

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (Ka values ranging from 5 X 10(6) to 10(10) M-1) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (gamma, kappa) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15-20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained gamma 1 heavy (H) chains and 4-6-10 contained both gamma 1 and gamma 2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which defined binding mechanism diversity. The location of determinants recognized by anti-idiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotype by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (approximately 25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the VH- and VL-chain interaction must exist for the expression of these idiotypes.

Animals↗

PubMed2002

Differential patterns of methylation of the IFN-gamma promoter at CpG and non-CpG sites underlie differences in IFN-gamma gene expression between human neonatal and adult CD45RO- T cells.

IFN-gamma is a potent pleiotropic Th1 cytokine, the production of which is tightly regulated during fetal development. Negative control of fetal/neonatal IFN-gamma production is generally attributed to the Th1-antagonistic effect of mediators produced by the placenta, but evidence exists of additional and more direct transcriptional regulation. We report that neonatal (cord blood) CD3(+)/CD45RO(-) T cells, in particular the CD4(+)/CD45RO(-) subset, are hypermethylated at CpG and non-CpG (CpA and CpT) sites within and adjacent to the IFN-gamma promoter. In contrast, CpG methylation patterns in cord blood IFN-gamma-producing CD8(+)/CD45RO(-) T cells and CD56(+)/CD16(+)/CD3(-) NK cells did not differ significantly from those in their adult counterparts. Consistent with this finding, IFN-gamma production by stimulated naive cord blood CD4(+) T cells is reduced 5- to 10-fold relative to adult CD4(+) T cells, whereas production levels in neonatal and adult CD8(+) T cells are of a similar order. Evidence of significant CpA and CpT methylation was not discovered in promoter sequence from other cytokines (IL-4, TNF-alpha, or IFN-gammaR alpha-chain). We additionally demonstrate that overexpression of DNA methyltransferase 3a in embryonic kidney carcinoma cells is accompanied by CpA methylation of the IFN-gamma promoter.

5-Methylcytosine↗

PubMedDate not supplied

Two different subpopulations of Ig-bearing cells in lymphoid organs of rainbow trout.

In a previous report we described the existence of two structural and antigenic variants of light chains in serum trout Igs, defined by mAbs 2H9 and 2A1. In this report, we analyse the cell distribution, the "in vitro" kinetics of production, and the ontogenic appearance of these two variants. Both variants were already present at day 8 before hatching and their kinetics of appearance correlated with that of total Ig, showing a peak around hatching. These mAbs stained two distinct lymphoid cell populations in the pronephros, mesonephros, spleen, and peripheral blood. Both mAbs reacted with approximately 1% of thymocytes. The ratio between these two cell populations (2A1+/2H9+) was about 2/1 in the lymphoid organs analysed. No differences were found between the profiles of the "in vitro" production of these variants in pronephric cell cultures stimulated with LPS. In these cultures, the 2A1 and 2H9 Igs together accounted for 10-70% of the total trout Ig produced, suggesting the existence of at least one additional L chain variant.

Animals↗