B lymphocyte differentiation and the control of IgM μ chain expression

The biosynthesis of IgM μ polypeptides was studied in isolated populations of normal B lymphocytes and in various IgM-producing cell lines. Membrane and secretory μ were found to be distinct polypeptide species, with separate biosynthetic intermediates from the translation stage onwards. Various B cell populations express different portions of the two biosynthetic μ pathways. Normal, resting small B lymphocytes do not secrete detectable μ and lack the later intermediate forms of secretory μ. However, they apparently possess, and translate, secretory μ mRNA, and show earlier secretory μ intermediate protein forms. Resting B cells thus exert posttranslational control over secretory μ expression. Since the later intermediate forms of secretory μ, which are lacking in small B cells, are due to carbohydrate modifications of the μ chain, it is suggested that the carbohydrate portion may be involved in regulating the expression of the secretory μ glycoprotein. In contrast to small B cells, highly differentiated IgM-secreting cells control the expression of membrane μ by a pretranslational mechanism.     

Molecular mechanisms of allosteric probe dependence in μ opioid receptor

Allostery is one of the most important features of proteins. It greatly contributes to the complexity of life, since it enables possibility of precise tuning of protein function, as well as performing more than one function per protein. Probe dependence is one of the unique features of allostery. It allows a protein to respond differently to the same allosteric modulator when different drugs or transmitters are bound. Unfortunately, allosteric mechanisms are difficult to investigate experimentally. Instead, they can be reproduced artificially in simulations. We simulated in silico a native-like cell membrane fragment with an active-state human μ opioid receptor (MOR) in order to investigate diverse effects of a receptor’s positive allosteric modulator on various agonists. Particular emphasis on native-likeness of the environment was put. We managed to reproduce the experimentally observed effects, which allowed us to take deeper insight into their underlying mechanisms. We found an allosteric pathway in the receptor, leading from the ligand binding site to the intracellular, effector site. We observed that the modulator affected the pathway, inducing different resultant responses for full and partial agonists.     

Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase μ (polμ), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polμ and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polμ. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polμ expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polμ is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polμ in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polμ provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polμ themselves.     

Inactivation of the Purified Bovine μ Opioid Receptor by Sulfhydryl Reagents

We have investigated the role of cysteine residues in a highly purified opioid receptor protein (ORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of ORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([³H]DAMGO) was much more sensitive to inactivation than antagonist binding ([³H]bremazocine). Prebinding ORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.     

Ligand recognition in μ opioid receptor: experimentally based modeling of μ opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the μ opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to ‘second messenger’ effector molecules, and in identifying specific ligand-binding contacts with the μ opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.     

Core fucosylation of μ heavy chains regulates assembly and intracellular signaling of precursor B cell receptors

α1,6-Fucosyltransferase (Fut8) knock-out (Fut8(-/-)) mice showed an abnormality in pre-B cell generation. Membrane assembly of pre-BCR is a crucial checkpoint for pre-B cell differentiation and proliferation in both humans and mice. The assembly of pre-BCR on the cell surface was substantially blocked in the Fut8-knockdown pre-B cell line, 70Z/3-KD cells, and then completely restored by re-introduction of the Fut8 gene to 70Z/3-KD (70Z/3-KD-re) cells. Moreover, loss of α1,6-fucosylation (also called core fucosylation) of μHC was associated with the suppression of the interaction between μHC and λ5. In contrast to Fut8(+/+) CD19(+)CD43(-) cells, the subpopulation expressing the μHC·λ5 complex in the Fut8(-/-) CD19(+)CD43(-) cell fraction was decreased. The pre-BCR-mediated tyrosine phosphorylation of CD79a and activation of Btk were attenuated in Fut8-KD cells, and restored in 70Z/3-KD-re cells. The frequency of CD19(low)CD43(-) cells (pre-B cell enriched fraction) was also reduced in Fut8(-/-) bone marrow cells, and then the levels of IgM, IgG, and IgA of 12-week-old Fut8(-/-) mice sera were significantly lower than those of Fut8(+/+) mice. Our results suggest that the core fucosylation of μHC mediates the assembly of pre-BCR to regulate pre-BCR intracellular signaling and pre-B cell proliferation.     

DNA polymerase μ is a global player in the repair of non-homologous end-joining substrates

The specialized DNA polymerase μ (pol μ) intervenes in the repair mechanism non-homologous end-joining (NHEJ) as an end-processing factor but its role has not been fully elucidated. Pol μ has been shown to participate in DNA synthesis at junctions in vitro, including on unpaired substrates, and to promote annealing. However, the phenotypes observed in vivo poorly recapitulate the functions of pol μ reported in vitro. We analysed the repair of DNA double-strand breaks (DSBs) in a cellular context using improved NHEJ substrates. These substrates do not replicate in mammalian cells, thereby result in clonal repair events, which allows the measure of the efficiency of repair. We validated this paradigm by comparing the repair of NHEJ substrates to the repair reported for chromosome DSBs in mouse cells. Molecular analysis and, in most cases sequencing of more than 1500 repair events on a variety of NHEJ substrates in wild type and pol μ(-/-) mouse embryonic fibroblasts shows that, unexpectedly, the absence of pol μ decreases the efficiency of joining of all types of DSBs, including those that do not undergo end-processing. Importantly, by reducing the efficiency of accurate events, lack of pol μ also affects the overall fidelity of the repair process. We also show that, although pol μ does not help protect DNA ends from resection, the efficiency of repair of resected ends is reduced in the absence of pol μ. Interestingly, the DNA synthesis activity of pol μ, including on non-aligned substrates, appears negligible at least in a cellular context. Our data point to a critical role for pol μ as a global repair player that increases the efficiency and the fidelity of NHEJ.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

δ versus μ receptors: cardiovascular and respiratory effects of opiate agonists microinjected into nucleus tractus solitarius of cats

The cardiovascular and respiratory responses to relatively specific μ or δ agonists microinjected (0.5 μl/kg) into the region of the nucleus of tractus solitarius (NTS) were examined in anesthetized cats. Blood pressure, heart rate, and respiratory rate were monitored for 30 min after the microinjection of opioid compounds or saline vehicle. The δ agonist, (d-Ala²,d-Leu⁵)-enkephalin (10–100 nmol/kg) elicited dose-dependent decreases in blood pressure, heart rate, and respiratory rate which were naloxone reversible. In contrast the μ agonists, morphine (10–54 nmol/kg) and morphiceptin (100–320 nmol/kg) had no effect on blood pressure or respiratory rate; yet, naloxone elicited pressor responses in animals pretreated with these μ agonists. A receptor-binding assay demonstrated a predominance of μ sites in the NTS. These data show that the δ opiate agonist is more effective than μ agonists in modifying cardiovascular variables in the NTS; we suggest caution in relating specific cardiovascular function to receptor subtypes defined by binding assays.     

Lack of DNA polymerase μ affects the kinetics of DNA double-strand break repair and impacts on cellular senescence

The specialised DNA polymerase μ (pol μ) affects a sub-class of immunoglobulin genes rearrangements and haematopoietic development in vivo. These effects appear linked to double-strand breaks (DSBs) repair, but it is still unclear how and to what extent pol μ intervenes in this process. Using high-resolution quantitative imaging of DNA damage in irradiated wild-type and pol μ^?/? mouse embryonic fibroblasts (MEFs) we show that lack of pol μ results in delayed DSB repair kinetics and in persistent DNA damage. DNA damage triggers cellular senescence, and this response is thought to suppress cancer. Independent investigations either report or not a proliferative decline for MEFs lacking pol μ. Here we show pronounced senescence in pol μ^?/? MEFs, associated with high levels of the tumor-suppressor p16^INK4A and the DNA damage response kinase CHK2. Importantly, cellular senescence is induced by culture stress and exacerbated by low doses of irradiation in pol μ^?/? MEFs. We also found that low doses of irradiation provoke delayed immortalisation in MEFs lacking pol μ. Pol μ^?/? MEFs thus exhibit a robust anti-proliferative defence in response to irreparable DNA damage. These findings indicate that sub-optimal DSB repair, due to the absence of an auxiliary DNA damage repair factor, can impact on cell fitness and thereby on cell fate.     

SAR and biological evaluation of 3-azabicyclo[3.1.0]hexane derivatives as μ opioid ligands

3-Azabicyclo[3.1.0]hexane compounds were designed as novel achiral μ opioid receptor ligands for the treatment of pruritus in dogs. In this paper, we describe the SAR of this class of opioid ligand, highlighting changes to the lead structure which led to compounds having picomolar binding affinity, selective for the μ receptor over δ and κ subtypes. Some subtleties of functional activity will also be described.     

A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJ_H joints and compare them with those present in later life. The embryo DJ_H joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ^−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.The adaptive immune system is characterized by the great diversity of its antigen receptors, which result from the activities of enzymatic complexes that cut and paste the genomic DNA of antigen receptor loci. The nonhomologous end-joining (NHEJ) machinery is then recruited to repair the double-strand DNA breaks (DSBs) inflicted by the products of the recombination-activating genes (RAGs) (45, 65). Within B cells, each immunoglobulin (Ig) receptor represents a singular shuffling of two heavy (H) and two light (L) chains, which are derived from the recombination of V, D, and J gene segments of the IgH locus and of V and J for IgL (71). Besides these combinatorial possibilities, most Ig variability derives from extensive processing of the coding ends, including exonucleolytic trimming of DNA ends, together with the addition of palindromic (P) nucleotides templated by the adjacent germ line sequence and of nontemplated (N) nucleotides secondary to the activity of the terminal deoxynucleotidyl transferase (TdT), a lymphoid-specific member of family X of DNA polymerases (reviewed in reference 56). During B-lineage differentiation, IgH rearrangements occur before those of the IgL locus, and D-to-J_H rearrangements precede V-to-DJ_H rearrangements (62). DJ_H joints are formed in any of the three open reading frames (ORFs). ORF1 is predominantly used in mature Igs, ORF2 is transcribed as a Dμ protein that provides negative signals to the B-cell precursors, and ORF3 frequently leads to stop codons (32, 33, 37). Germ line V, D, and J gene segments display short stretches of mutually homologous nucleotides (SSH), which are frequently used in gene rearrangements during perinatal periods, when N additions are absent (27, 32, 55, 57). The actual Ig V-region repertoires represent both the results of the NHEJ process associated with genomic VDJ recombination and those of antigen-independent and -dependent selection events. Although the core NHEJ components (Ku-Artemis-DNA-PK and XLF-XRCC4-DNA ligase IV) are by themselves able to join RAG-induced, incompatible DNA ends, family X DNA polymerases can be recruited to fill gaps created by imprecise coding ends with 3′ overhangs (DNA polymerase μ [Polμ] and Polλ) and/or to promote diversity through the addition of N nucleotides (TdT) (34, 56).The lymphoid differentiation pathways and clonotypic repertoires are developmentally regulated and differ between the embryo-fetal and adult periods (2, 44, 68). The perinatal B cells result from a wave of B lymphopoiesis occurring during the last third of mouse gestation (13, 14, 21, 70). Perinatal V_H gene usage differs from that predominating in the adult (1, 69), and the former VDJ joints rarely display N additions, leading to V-region repertoires enriched in multi- and self-reactive specificities (36, 40). The program of B-cell differentiation starts at embryonic days 10 to 11 (E10 to E11) in embryo hematopoietic sites, after the emergence of multipotent progenitors (at E8.5 to E9.5) (18, 19, 23, 31, 51, 73). DJ_H rearrangements were detected in these early embryos, whereas full VDJ_H sequences were not observed before E14 (14, 18, 51, 66), when VJκ rearrangements were also found (63). The earliest mouse DJ_H/VDJ_H Ig sequences analyzed to date corresponded to late fetuses (E16) (14, 53). We reasoned that the true baseline of the Ig rearrangement process occurs in midgestation embryos, when the first DJ_Hs are not yet transcribed and, consequently, not subjected to selection and are conditioned only for the evolutionarily established and developmentally regulated usage of distinct NHEJ machineries.We report here the sequence profiles of the earliest embryo E10 to E12 DJ_H joints. Unexpected frequencies of embryonic DJ_H joints bearing N nucleotides, in the absence of detectable TdT expression, were found. Moreover, the embryo DJ_H joints lacking N nucleotides (N⁻) used fewer SSH to recombine than newborn DJ_Hs, and these SSH were widely dispersed along the embryo D sequences, in contrast to the most joint-proximal ones, which predominated in newborn DJ_Hs. Considering that Polμ is the closest relative of TdT (42% amino acid identity) (22), which is able to introduce N nucleotides in vitro (4, 22, 34, 39, 49) and to join DNA ends with minimal or even null complementarity (17, 58), and that it is expressed in early-embryo organs, we decided to investigate its putative contribution to the first embryo DJ_H joints. The DJ_H joints obtained from Polμ^−/− embryos (48) showed a significant reduction of N nucleotides compared to wild-type (WT) embryos. Moreover, highly preserved DJ_H joints (with <3 deleted nucleotides) were selectively depleted in the Polμ^−/− mouse embryos, while the remaining DJ_Hs preferentially relied upon longer stretches of homology for end ligation. These findings support the idea that Polμ is active during early-embryo DJ_H rearrangements and that both its template-dependent and -independent ambivalent functions may be used to fill in small nucleotide gaps generated after asymmetric hairpin nicking and also to extend coding ends via a limited TdT-like activity.     

Design,synthesis and biological evaluation of a bivalent μ opiate and adenosine A1 receptor antagonist

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A₁ adenosine and μ opiate receptors with a K_i of 0.8 ± 0.05 and 0.7 ± 0.03 μM, respectively. This hybrid molecule increases cAMP production in cells that over express the μ receptor as well as those over expressing the A₁ adenosine receptor and reverses the antalgic effects of μ and A₁ adenosine receptor agonists in animals.     

Glutathione S-transferase class μ in French alcoholic cirrhotic patients

Summary The lack of glutathione S-transferase (GST) was examined in 45 healthy French Caucasians and 45 alcoholic cirrhotic French Caucasians: microsamples of blood were taken and DNA amplified by the polymerase chain reaction. We have concluded that there is no relationship between this genotype and the development of alcoholic cirrhosis in these heavy consumers of ethanol.     

Co-localization of μ and δ opioid receptors on SK-N-SH cells detected by fluorescence microscopy using labeled anti-idiotypic antibodies

Selective fluorescence labeling of opioid receptor subclasses on SK-N-SH cultured cells has been accomplished using labeled polyclonal anti-idiotypic antibodies along with subclass-selective opioid agonists (DPDPE, δ-selective; DAMGO, μ-selective) as blocking reagents. Labeling of the cells was examined using conventional fluorescence microscopy. Co-localization of μ- and δ- opioid receptors on SK-N-SH cells has been studied by double labeling fluorescence experiments. In agreement with our own, and other workers', previous observations on NG108-15 cells, a subpopulation of viable cells in asynchronous cultures are labeled. Amon those SK-N-SH cells that are labeled, both subclasses of receptors are seen. On the basis of sequential blocking experiments we interpret our combined results to be consistent with a model where μ- and δ- binding sites reside on different subunits of a multimeric complex.     

Differential Expressions of the Alternatively Spliced Variant mRNAs of the μ Opioid Receptor Gene,OPRM1, in Brain Regions of Four Inbred Mouse Strains

The µ opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing in rodents and humans, with dozens of alternatively spliced variants of the OPRM1 gene. The present studies establish a SYBR green quantitative PCR (qPCR) assay to more accurately quantify mouse OPRM1 splice variant mRNAs. Using these qPCR assays, we examined the expression of OPRM1 splice variant mRNAs in selected brain regions of four inbred mouse strains displaying differences in µ opioid-induced tolerance and physical dependence: C56BL/6J, 129P3/J, SJL/J and SWR/J. The complete mRNA expression profiles of the OPRM1 splice variants reveal marked differences of the variant mRNA expression among the brain regions in each mouse strain, suggesting region-specific alternative splicing of the OPRM1 gene. The expression of many variants was also strain-specific, implying a genetic influence on OPRM1 alternative splicing. The expression levels of a number of the variant mRNAs in certain brain regions appear to correlate with strain sensitivities to morphine analgesia, tolerance and physical dependence in four mouse strains.