Quantification of illegitimate V(D)J recombinase-mediated mutations in lymphocytes of newborns and adults

We used a direct polymerase chain reaction (PCR) method for quantification of HPRT exons 2+3 deletions and t(14;18) translocations as a measure of illegitimate V(D)J recombination. We determined the baseline frequencies of these two mutations in mononuclear leukocyte DNA from the umbilical cord blood of newborns and from the peripheral blood of adults. In an initial group of 21 newborns, no t(14;18) translocations were detected (<0.049×10⁻⁷). The frequency of HPRT exons 2+3 deletions was 0.10×10⁻⁷ per mononuclear leukocyte, lower than expected based on the T-cell proportion of this cell fraction (55%–70%) and previous results using the T-cell cloning assay (2–3×10⁻⁷ per clonable T-cell). Phytohemagglutinin (PHA), as used in the T-cell cloning assay, was examined for its effect on the frequencies of these mutation events in mononuclear leukocytes from an additional 11 newborns and from 12 adults. There was no significant effect of PHA on t(14;18) translocations which were rare among the newborns (1 detected among 2.7×10⁸ leukocytes analyzed), and which occurred at frequencies from <1×10⁻⁷ (undetected) to 1.6×10⁻⁴ among the adults. The extremely high frequencies of t(14;18)-bearing cells in three adults were due mainly to in vivo expansion of two to six clones. However, PHA appeared to stimulate a modest (although not significant) increase in the frequency of HPRT exons 2+3 deletions in the leukocytes of the newborns, from 0.07×10⁻⁷ to 0.23×10⁻⁷. We show that both the direct PCR assay and the T-cell cloning assay detect similar frequencies of HPRT exons 2+3 deletions when calculations are normalized to blood volume, indicating that the apparent discrepancy is probably due to the different population of cells used in the assays. This direct PCR assay may have utility in characterizing the effects of environmental genotoxic agents on this clinically important recombination mechanism.     

MHC class I genes of the channel catfish: sequence analysis and expression

 Four cDNAs encoding the major histocompatibility complex (MHC) class I α chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I α chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I α and β₂-microglobulin (β₂m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 M _r glycoprotein (presumably class I α) was associated with β₂m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of β₂m with the MHC class I α chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts. Received: 25 March 1998 / Revised: 28 July 1998     

Highly conserved antigen-presenting function of CD1d molecules

 The lack of polymorphism of nonclassical antigen-presenting molecules has led to the proposal that they may carry out some conserved and essential antigen-presenting function. Although this is a plausible hypothesis, the major histocompatibility complex has undergone dramatic expansions and contractions through evolution, and there is surprisingly little evidence for interspecies conservation of nonclassical class I molecules. The CD1d molecule, by contrast, shows an extremely high degree of functional conservation between mice and humans, with regard to its interaction with the relatively invariant TCRs that are expressed by NK T cells. This conservation for CD1d recognition is observed either in the absence of exogenous Ag or together with a lipoglycan antigen. The close functional and phenotypic conservation of NK T cells, in mammalian species separated by approximately 50 million years, suggests an essential role in the immune system for CD1d recognition by NK T cells.     

Integration of the Mitochondrial-Processing Peptidase into the Cytochrome bc 1 Complex in Plants

The plant mitochondrial cytochrome bc ₁ complex, like nonplant mitochondrial complexes,consists of cytochromes b and c ₁, the Rieske iron–sulfur protein, two Core proteins, and fivelow-molecular mass subunits. However, in contrast to nonplant sources, the two Core proteinsare identical to subunits of the general mitochondrial processing peptidase (MPP). The MPPis a fascinating enzyme that catalyzes the specific cleavage of the diverse presequence peptidesfrom hundreds of the nuclear-encoded mitochondrial precursor proteins that are synthesizedin the cytosol and imported into the mitochondrion. Integration of the MPP into the bc ₁complex renders the bc ₁ complex in plants bifunctional, being involved both in electrontransport and in protein processing. Despite the integration of MPP into the bc ₁ complex,electron transfer as well as translocation of the precursor through the import channel areindependent of the protein-processing activity. Recognition of the processing site by MPPoccurs via the recognition of higher-order structural elements in combination with charge andcleavage-site properties. Elucidation of the three-dimensional (3-D) structure of the mammaliancytochrome bc ₁ complex is highly useful for understanding of the mechanism of action of MPP.In memory of my teacher—an insightful, devoted, and enthusiastic scientist and an amiable and kind-hearted human being—Lars Ernster     

Structural Basis of Multifunctional Bovine Mitochondrial Cytochrome bc 1 Complex

The mitochondrial cytochrome bc ₁ complex is a multifunctional membrane protein complex. Itcatalyzes electron transfer, proton translocation, peptide processing, and superoxide generation.Crystal structure data at 2.9 Å resolution not only establishes the location of the redox centersand inhibitor binding sites, but also suggests a movement of the head domain of the iron–sulfurprotein (ISP) during bc ₁ catalysis and inhibition of peptide-processing activity during complexmaturation. The functional importance of the movement of extramembrane (head) domain ofISP in the bc ₁ complex is confirmed by analysis of the Rhodobacter sphaeroides bc ₁ complexmutants with increased rigidity in the ISP neck and by the determination of rate constants foracid/base-induced intramolecular electron transfer between [2Fe–2S] and heme c ₁ in nativeand inhibitor-loaded beef complexes. The peptide-processing activity is activated in bovineheart mitochondrial bc ₁ complex by nonionic detergent at concentrations that inactivate electrontransfer activity. This peptide-processing activity is shown to be associated with subunits Iand II by cloning, overexpression and in vitro reconstitution. The superoxide-generation siteof the cytochrome bc ₁ complex is located at reduced b _L and Q^–. The reaction is membranepotential-, and cytochrome c-dependent.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Molecular study of the rat liver NADH: Cytochrome c oxidoreductase complex during development and aging

The mechanisms involved in ageing are yet to be fully understood but it is thought that changes produced in energy transfer pathways occurring in the mitochondria may be responsible for the lack of energy typical of the later stages of life. The aim of the present investigation was to determine the enzymatic activity of the liver NADH cytochrome c oxidorectuctase complex (Complex I-III) in mitochondria isolated from the liver of rats of 3 different age groups: lactating, animals (15-17 days), adult females (3-5 months) and old animals (26-30 months). The activities of the unbound Complexes I and III were also determined.An increase in Complex I-III activity was detected during development (142 ± 10 vs. 447 ± 23 mol cyt. c/mg/min, p < 0.001) ang ageing (447 ± 23 vs. 713 ± 45 mol cyt. c/mg//min, p < 0.001). However, unbound Complex I showed a reduction in activity during the ageing period whilst Complex III activity moderately increased. Immunological studies indicated only a moderate increase in the amount of Complex I-III and studies on the purified complex suggested that the increase in activity was due to effects other than an increase in enzyme quantity. The analysis of protein bands and the quantification of prosthetic groups showed particular reductions in the relative concentrations of Complex I subunits including the 51 kDa unit, which binds FMN, confirmed by a similar reduction in levels of the nucleotide. In contrast, 4 of the 5 subunits which increased during the lifetime of the animals corresponded to those of Complex III. These subunits are responsible for the binding of catalytic groups. The results suggest that, in addition to the increase in the amount of enzyme, binding factors between Complexes I and III may also play an important role in the observed increase in Complex I-III activity.     

Effect of endurance training on the phospholipid content of skeletal muscles in the rat

Only few data are available on the effect of training on phospholipid metabolism in skeletal muscles. The aim of the present study was to examine the effect of 6 weeks of endurance training on the content of particular phospholipid fractions and on the incorporation of blood-borne [14C]-palmitic acid into the phospholipids in different skeletal muscles (white and red sections of the gastrocnemius, the soleus and the diaphragm) of the rat. Lipids were extracted from the muscles and separated using thin-layer chromatography into the following fractions: sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, cardiolipin and neutral lipids (this fraction being composed mostly of triacylglycerols). It was found that training did not affect the content of any phospholipid fraction in soleus muscle. It increased the content of sphingomyelin in white gastrocnemius muscle, cardiolipin and phosphatidylethanolamine in red gastrocnemius muscle and phosphatidylinositol in white gastrocnemius muscle and diaphragm. The total phospholipid content in red gastrocnemius muscle of the trained group was higher than in the control group. Training reduced the specific activity of sphingomyelin and cardiolipin in all muscles, phosphatidylcholine in soleus, red, and white gastrocnemius muscles, phosphatidylserine in all muscles, phosphatidylinositol in all except the soleus muscle, and phosphatidylethanolamine in hindleg muscles, but not in the diaphragm compared to the corresponding values in the sedentary group. It was concluded that endurance training affects skeletal muscle phospholipid content and the rate of incorporation of the blood-borne [14C]palmitic acid into the phospholipid moieties.     

Modulation of CD4 cell cytokine production by colon cancer-associated mucin

Mucins have been implicated in tumor-associated immunosuppression. The possibility that colon cancer mucin (CCM) may modulate T-helper 1 (TH1) activity was evaluated by investigating its effect on the production of interleukin-2 (IL-2) by CD4⁺ cells, a process that requires antigen-specific and costimulatory signals. Methods: CCM was purified from human colorectal cancer cells by gel-exclusion fast-pressure liquid chromatography. Cytokine production of purified CD4⁺ cells was evaluated at the protein and gene level in the presence of a phorbol ester or an anti-CD3 monoclonal antibody (mAb) plus mAb against the CD28 costimulatory receptor to mimic two-signal activation. Results: Soluble CCM, which contains mucins MUC2 as well as MUC1, inhibited IL-2 mRNA expression and secretion of CD4⁺ stimulated with a phorbol ester or an anti-CD3 mAb plus anti-CD28 mAb. Pretreatment of CD4⁺ cells with anti-CD28 mAb abrogated the suppressive effects of CCM on IL-2 production, and flow cytometry showed decreased binding of anti-CD28 mAb to its receptor in the presence of mucin. In addition, Ca²⁺ mobilization after T cell receptor cross-linking with anti-CD3 mAb was maintained in the presence of CCM. Although interferon γ production was also diminished, CCM did not induce a general inhibition of cytokine production, nor did it decrease cell viability. Macrophage inflammatory protein 1α production was up-regulated; the production of IL-10 and transforming growth factor β was unchanged. Conclusions: The results indicate that CCM can alter TH1 activity and suggest that the modulation of costimulatory interactions is involved. They provide another mechanism of immunosuppression mediated by these highly expressed tumor products. Received: 23 March 1999 / Accepted: 3 August 1999