Abstract:

Abstract: Pre-BCR signals are part of a checkpoint where early precursor (pre-) B cells with a pairing Ig muH chain (muHC) are clonally expanded before they differentiate into IgL-rearranging, resting pre-B cells. A pre-BCR consists of two muHCs, two surrogate L chains and the signal transducer Igalpha/Igbeta. The molecular circuits by which the pre-BCR controls proliferation and differentiation of pre-B cells are poorly characterized. Therefore, we identified the differential transcriptome by genome-wide expression profiling in progenitor (pro-) B cells from a Rag2-deficient mouse, in which the expression of a transgenic muHC and thus a pre-BCR as well as pre-BCR-mediated clonal expansion can be controlled by tetracycline (muHC-inducible mouse). This analysis revealed that pre-BCR signals upregulate components of the BCR signalosome, open the IgL chain (LC) locus and induce the krüppel-like transcription factor KLF2, a key regulator of quiescence and lymphocyte migration. Hence, pre-BCR signals establish the molecular network for BCR signaling even before the production of an IgLC and induce the expression of KLF2, a candidate for controlling clonal expansion and migration of functional pre-B cells.

Abstract: Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.

Abstract: Multiple myeloma is an incurable plasma cell neoplasia characterized by the production of large amounts of monoclonal immunoglobulins. The proteasome inhibitor bortezomib (PS-341, Velcade) induces apoptosis in various malignant cells and has been approved for treatment of refractory multiple myeloma. Inhibition of the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB) apparently contributes to the antitumor effects of bortezomib; however, this mechanism cannot fully explain the exceptional sensitivity of myeloma cells. Extensive protein synthesis as in myeloma cells is inherently accompanied by unfolded proteins, including defective ribosomal products (DRiPs), which need to be degraded by the ubiquitin-proteasome system. Therefore, we hypothesized that the proapoptotic effect of bortezomib in multiple myeloma is mainly due to the accumulation of unfolded proteins in cells with high protein biosynthesis. Using the IgG-secreting human myeloma cell line JK-6L and murine muH-chain-transfected Ag8.H myeloma cells, apoptosis induction upon proteasome inhibition was clearly correlated with the amount of immunoglobulin production. Preferentially in immunoglobulin-high myeloma cells, bortezomib triggered activation of caspases and induction of proapoptotic CHOP, a component of the terminal unfolded protein response induced by endoplasmic reticulum (ER) stress. In immunoglobulin-high cells, bortezomib increased the levels of proapoptotic Bax while reducing antiapoptotic Bcl-2. Finally, IgG-DRiPs were detected in proteasome inhibitor-treated cells. Hence, proteasome inhibitors induce apoptosis preferentially in cells with high synthesis rate of immunoglobulin associated with accumulation of unfolded proteins/DRiPs inducing ER stress. These findings further elucidate the antitumor activities of proteasome inhibitors and have important implications for optimizing clinical applications.

Abstract: The mammalian integration site 6 (INT6) protein has been implicated in breast carcinogenesis and characterized as the eIF3e non-core subunit of the translation initiation factor eIF3, but its role in this complex is not known. Here, we show that INT6 knockdown by RNA interference strongly inhibits nonsense-mediated messenger RNA decay (NMD), which triggers degradation of mRNAs with premature stop codons. In contrast to the eIF3b core subunit, which is required for both NMD and general translation, INT6 is only necessary for the former process. Consistent with such a role, immunoprecipitation experiments showed that INT6 co-purifies with CBP80 and the NMD factor UPF2. In addition, several transcripts known to be upregulated by UPF1 or UPF2 depletion were also found to be sensitive to INT6 suppression. From these observations, we propose that INT6, in association with eIF3, is involved in routing specific mRNAs for degradation.

Abstract: Survival of mature B cells is thought to depend on the BCR signaling (BCR) because ablation of either H chain (HC) expression or BCR signaling causes B cells to rapidly disappear. Whether a complete BCR is required for survival of mature B cells is not known. To address this question, we generated a mouse in which we can repress the expression of a transgenic Ig L chain (IgL) by doxycycline (IgL-repressible mouse). Repression of IgL abrogated expression. Surprisingly, however, IgL-negative B cells survived longer than 14 wk, expressed signal-competent HC on the cell's surface, and active unfolded protein response factors. Like postgerminal center B cells, IgL-negative B cells were small lymphocytes, not dividing and expressed Bcl-6. Our results indicate that expression of unpaired HC, as it may occur as a consequence of Ag ligation, somatic hypermutation, or receptor editing, facilitates the survival of cells either by inducing receptor signaling or by inducing unfolded protein response and/or the expression of survival genes such as Bcl-6.

Abstract: B-cell receptor (BCR) signals are essential for B-cell differentiation, homeostasis and negative selection, which are regulated by the strength and quality of BCR signals. Recently, we identified a new adaptor protein, Swiprosin-1, in lipid rafts of B-cell lines that undergo apoptosis after BCR stimulation. During murine B-cell development, Swiprosin-1 exhibited highest expression in immature B cells of the bone marrow, but was also expressed in resting and activated splenic B cells and in non-lymphoid tissue, especially in the brain. Ectopic expression of Swiprosin-1 in the immature murine B-cell line WEHI231 enhanced spontaneous and BCR-induced apoptosis. In contrast, short hairpin RNA (shRNA)-mediated downregulation of Swiprosin-1 impaired specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle arrest and upregulation of the cell cycle inhibitor p27(Kip1). In accordance, Swiprosin-1 abundance regulated net cell growth of WEHI231 cell populations through reciprocal regulation of Bcl-xL, but not Bim, thereby controlling spontaneous apoptosis. Swiprosin-1-enhanced apoptosis was blocked through nuclear factor kappaB-activating stimuli, namely B-cell-activating factor of the TNF family, anti-CD40 and lipopolysaccharide (LPS). This correlated with enhanced BCR-induced IkappaB-alpha phosphorylation and degradation in cells expressing a Swiprosin-1-specific shRNA. Finally, ectopic Swiprosin-1 expression enhanced BCR-induced cell death in primary, LPS-stimulated splenic B cells. Hence, Swiprosin-1 may regulate lifespan and BCR signaling thresholds in immature B cells.

Abstract: Selective expansion of functional pre-B cells is accomplished by the assembly of a signaling-competent pre-B cell receptor (pre-BCR) consisting of immunoglobulin mu heavy chains (muHC), surrogate light chains (SLC) and Igalpha/Igbeta. Here, we review recent data showing that muHCs, in the absence of SLC, deliver autonomous differentiation signals. However, enhanced signaling necessary for pre-B cell expansion requires cross-linking of pre-BCRs via the non-immunoglobulin tail of SLC's subunit lambda5. We also discuss how SLC's ability to modulate the strength of pre-BCR signals is controlled by a muHC's idiotype and its affinity to the chaperone BiP. In this model, BiP in concert with SLC functions as a pre-selector of the antibody repertoire.

Abstract: Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic surveillance pathway that selectively degrades aberrant mRNAs with premature termination codons (PTCs). Although a small number of cases exist in mammals, where NMD controls levels of physiologic PTC transcripts, it is still unclear whether the engagement of NMD in posttranscriptional control of gene expression is a more prevalent phenomenon. To identify physiologic NMD substrates and to study how NMD silencing affects the overall dynamics of a cell, we stably down-regulated hUPF2, the human homolog of the yeast NMD factor UPF2, by RNA interference. As expected, hUPF2-silenced HeLa cells were impaired in their ability to recognize ectopically expressed aberrant PTC transcripts. Surprisingly, hUPF2 silencing did not affect cell growth and viability but clearly diminished phosphorylation of hUPF1, suggesting a role of hUPF2 in modulating NMD activity through phosphorylation of hUPF1. Genome-wide DNA microarray expression profiling identified 37 novel up-regulated and 57 down-regulated transcripts in hUPF2-silenced cells. About 60% of the up-regulated mRNAs carry typical NMD motifs. Hence, NMD is important not only for maintaining the transcriptome integrity by removing nonfunctional and aberrant PTC-bearing transcripts but also for posttranscriptional control of selected physiologic transcripts with NMD features.

Abstract: BACKGROUND: The amount of a particular protein, and not just its presence or absence, frequently determines the outcome of a developmental process or disease progression. These dosage effects can be studied by conditionally expressing such proteins at different levels. With typical gene regulation systems like the Tet-On system, intermediate expression levels can be obtained by varying the effector concentration. However, this strategy is limited to situations in which these concentrations can be precisely controlled and, thus, not suited for animal models or gene therapy approaches. Here, we present a Tet transregulator setup that allows establishment of four levels of promoter activity largely independent of effector concentration. METHODS: A newly introduced transsilencer is combined with a reverse transactivator. As the regulators respond differentially to tetracycline derivatives, four expression levels are obtained by adding different effectors. To facilitate integration of the components, we generated versatile all-in-one vectors. Apart from a cassette expressing the transregulators and a selection marker, these vectors encode a bidirectional, regulated promoter driving expression of GFP and the gene of interest. The features of this stepwise regulation system were analyzed by transient and stable transfections of human cell lines. RESULTS: We demonstrate in a variety of experimental settings that coexpression of these transregulators leads to robust stepwise regulation. Depending on the respective effectors, four expression levels are achieved with different responsive promoters, cell lines and target genes. CONCLUSIONS: This system shows that a promoter can be adjusted to different activities and provides an excellent strategy to investigate protein dosage effects.

Abstract: Immunoglobulin beta (Ig-beta) is a critical signal transducer of precursor B cell and B cell receptors. B29, the gene coding for Ig-beta, is switched on in progenitor B cells and expressed until the terminal stage of antibody-producing plasma cells. Although several cis-acting elements and transcription factors required for B29 expression have been characterized in cell lines, the in vivo significance of individual motifs located in the 1.2-kb promoter region remained unclear. To address whether this region drives B lineage-specific expression in mice as efficiently as in transfected cell lines, we established transgenic animals carrying the B29 promoter fused to either enhanced green fluorescent protein (EGFP) or the precursor B cell receptor component lambda5. Surprisingly, only minimal levels of B29-derived transcripts were produced in B lymphoid tissues of several independent transgenic lines, and the respective proteins were below the detection limit. In addition, transgenic transcripts were found in testis, kidney and brain. Hence, the 1.2-kb-sized B29 promoter does not define a strong, B lineage-restricted expression unit when randomly integrated into the genome and passed through the murine germ line. Therefore, yet unidentified genomic locus control elements are required to efficiently drive B29 expression in B lymphocytes.

Abstract: The transit of T cell-activated B cells through the germinal center (GC) is controlled by sequential activation and repression of key transcription factors, executing the pre- and post-GC B cell program. B cell lymphoma (BCL) 6 and IFN regulatory factor (IRF) 8 are necessary for GC formation and for its molecular activity in Pax5+PU.1+ B cells. IRF4, which is highly expressed in BCL6- GC B cells, is necessary for class switch recombination and the plasma cell differentiation at exit from the GC. In this study, we show at the single-cell level broad coexpression of IRF4 with BCL6, Pax5, IRF8, and PU.1 in pre- and post-GC B cells in human and mouse. IRF4 is down-regulated in BCL6+ human GC founder cells (IgD+CD38+), is absent in GC centroblasts, and is re-expressed in positive regulatory domain 1-positive centrocytes, which are negative for all the B cell transcription factors. Activated (CD30+) and activation-induced cytidine deaminase-positive extrafollicular blasts coexpress Pax5 and IRF4. PU.1-negative plasma cells and CD30+ blasts uniquely display the conformational epitope of IRF4 recognized by the MUM1 Ab, an epitope that is absent from any other IRF4+PU.1+ lymphoid and hemopoietic subsets. Low grade B cell lymphomas, representing the malignant counterpart of pre- and post-GC B cells, accordingly express IRF4. However, a fraction of BCL6+ diffuse large B cell lymphomas express IRF4 bearing the MUM1 epitope, indicative of a posttranscriptional modification of IRF4 not seen in the normal counterpart.

Abstract: Inaccurate VDJ rearrangements generate a large number of progenitor (pro)-B cells with two nonproductive IgH alleles. Such cells lack essential survival signals mediated by surface IgM heavy chain (muH chain) expression and are normally eliminated. However, secondary rearrangements of upstream VH gene segments into assembled VDJ exons have been described in mice transgenic for productive muH chains, a process known as VH replacement. If VH replacement was independent of muH chain signals, it could also modify nonproductive VDJ exons and thus rescue pro-B cells with unsuccessful rearrangements on both alleles. To test this hypothesis, we homologously replaced the JH cluster of a mouse with a nonproductive VDJ exon. Surprisingly, B cell development in IgHVDJ-/VDJ- mice was only slightly impaired and significant numbers of IgM-positive B cells were produced. DNA sequencing confirmed that all VDJ sequences from muH chain-positive B lymphoid cells were generated by VH replacement in a RAG-dependent manner. Another unique feature of our transgenic mice was the presence of IgH chains with unusually long CDR3-H regions. Such IgH chains were functional and only modestly counter-selected, arguing against a strict length constraint for CDR3-H regions. In conclusion, VH replacement can occur in the absence of a muH chain signal and provides a potential rescue mechanism for pro-B cells with two nonproductive IgH alleles.

Abstract: Lipid rafts serve as platforms for BCR signal transduction. To better define the molecular basis of these membrane microdomains, we used two-dimensional gel electrophoresis and mass spectrometry to characterize lipid raft proteins from mature as well as immature B cell lines. Of 51 specific raft proteins, we identified a total of 18 proteins by peptide mass fingerprinting. Among them, we found vacuolar ATPase subunits alpha-1 and beta-2, vimentin, gamma-actin, mitofilin, and prohibitin. None of these has previously been reported in lipid rafts of B cells. The differential raft association of three proteins, including a novel potential signaling molecule designated swiprosin-1, correlated with the stage-specific sensitivity of B cells to BCR-induced apoptosis. In addition, MHC class II molecules were detected in lipid rafts of mature, but not immature B cells. This intriguing finding points to a role for lipid rafts in regulating Ag presentation during B cell maturation. Finally, a fraction of the BCR in the B cell line CH27 was constitutively present in lipid rafts. Surprisingly, this fraction was neither expressed at the cell surface nor fully O-glycosylated. Thus, we conclude that partitioning the BCR into lipid rafts occurs in the endoplasmic reticulum/cis-Golgi compartment and may represent a control mechanism for surface transport.

Abstract: Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) show genotypic features of germinal centre-derived B-cells in most cases. Nevertheless, these cells typically lack expression of B-cell antigens. Previous studies have suggested that plasma cell differentiation may occur in HRS cells and that this may account for the down-regulation of B-cell antigens. However, these results are controversial. We have addressed this question using immunohistochemistry and a panel of antibodies directed against antigens which are differentially expressed during terminal B-cell differentiation. Pax-5, a transcription factor required for B-lineage commitment, and IRF4/Mum1, which is physiologically expressed in germinal centre cells and plasma cells, were consistently detectable in HRS cells. Bcl-6, a transcription factor expressed in germinal centre B-cells, was present in HRS cells of approximately 25% of cHL cases. Expression of the B-lymphocyte-induced maturation protein-1 (Blimp-1), a key regulator of plasma cell differentiation, was observed in HRS cells of 23% of cHL cases. In these cases, Blimp-1 expression was restricted to a small proportion of HRS cells. HRS cells were consistently negative for the plasma cell marker CD138. These results suggest that plasma cell differentiation may be initiated in a small subset of HRS cells but remains abortive. Thus, terminal differentiation is unlikely to explain the lack of B-cell antigen expression in HRS cells.

Abstract: Signals delivered by the immunoglobulin (Ig)-like pre-B-cell receptor (pre-BCR) are critical for efficient maturation of early precursor B (pre-B) cells. A pre-BCR contains two immunoglobulin mu-heavy chains (muHC), two surrogate light chains (SLC) consisting of the non-covalently associated polypeptides, VpreB and lambda5, and the heterodimeric signaling transducer Igalpha/beta. Although, it is generally accepted that signals initiated from the pre-BCR are required for efficient expansion and differentiation of pre-B cells, the three-dimensional structure of this receptor has not yet been determined by either NMR or X-ray spectroscopy. Therefore, we used indirect computer-assisted molecular modeling techniques to predict for the first time three-dimensional coordinates of the pre-BCR, the conformation of the SLC components, VpreB and lambda5, and the position and flexibility of the so-called non-Ig-like unique tails at the C-terminus of VpreB and the N-terminus of lambda5. Structure prediction revealed that these unique tails of VpreB and lambda5 protrude from the SLC at the position where the CDR3 of a conventional IgLchain would be located. Thus, the unique tails are accessible for ligand binding, which supports the recent finding that the lambda5 unique tail is required for pre-BCR/stroma cell interaction. Further, the non-covalent interaction of the extra beta-strand of lambda5 (beta8) with VpreB is predicted to result in a stabilization of the tertiary structure of VpreB. In summary, three-dimensional computer modeling suggests that the structure of a pre-BCR resembles that of a conventional B-cell receptor (BCR) and that the lambda5 unique tail could be a major binding site for pre-BCR ligands.

Abstract: Several studies have implicated a role for the cell surface glycoprotein CD44 in lymphocyte differentiation, adhesion to the matrix, homing, activation and apoptosis. However, the requirement of CD44 in B cell maturation remains elusive. To address this point, we analyzed the generation and activation of B lymphocytes in CD44-deficient mice. Unexpectedly, both maturation and autoreconstitution of early bone marrow B cell progenitors after sublethal irradiation as well as frequencies of splenic B cell subsets are indistinguishable in wild-type and CD44-deficient mice. Furthermore, splenic CD44-deficient B lymphocytes show a normal activation pattern after stimulation with LPS, anti-IgM/IL-4 or anti-CD40/IL-4. These results show for the first time that CD44 is clearly dispensable for development, reconstitution and activation of B lymphocytes.

Abstract: The development of acute renal failure (ARF) in the ICU setting carries a high morbidity and mortality. To assess the outcomes and its predictive factors in our ICU, we analyzed the data of patients with ARF treated during 18 months. The 33 patients included 21 men and 12 women of mean age 51 +/- 21.7 years (13 to 87). Sepsis with multi-organ dysfunction (MOD) was the leading cause of ARF (58%). Comorbid conditions were malignancy in 30% of patients, diabetes mellitus in 24%, hypertension in 21%, ischemic heart disease in 21%, liver disease in 15%, and chronic renal failure in 15%. Predisposing factors were hypotension in 67% of cases, dehydration in 36%, drug related in 33%, congestive heart failure in 24%, and liver cirrhosis in 6%. Twenty-five (76%) patients needed mechanical ventilation, 22 (67%) were anuric, 18 (55%) had MODS, and 15 (45%) needed inotropic support. Length of stay in hospital was 27.2 +/- 28.0 days (2 to 94). Nineteen patients (58%) were managed conservatively and 14 (42%) by renal replacement therapy. Patient mortality was 67% and renal mortality 52%. The impact of the following factor: was assessed on patient and renal outcome was assessed ventilation support, presence of oliguria, need for inotropes, and presence of MOD. Patient mortality was significantly influenced by an elevated odds ratios (OR) (95% CI): mechanical ventilation [OR = 34 (95% CI 1.95 to 538)], and presence of MODS [OR = 12.3 (95% CI 2 to 75)]. Renal mortality was influenced by mechanical ventilation [OR = 12.3 (95% CI 1.6 to 119)], oliguria [OR = 12 (95% CI 2 to 72)], inotrope support [OR = 10 (95% CI 2 to 52), and MOD [OR = 35 (95% CI 3.5 to 35.0)]. This study confirms the high patient and renal mortality of ARF among patients to ICU. The four parameters were excellent predictors of renal outcome, while only the need for mechanical ventilation and the presence of MOD were predictors for patient survival.

Abstract: The transmembrane receptor Notch1 plays a crucial role in differentiation and apoptosis of hematopoietic cells. To investigate the influence of Notch1 on apoptosis and cell growth of mature murine B cells, we transduced the murine B-lymphoma line NYC 31.1 with a constitutively active, intracellular form of human Notch1 (Notch1-ICT). NYC cells represent mature activated B cells that can be induced to undergo apoptosis by crosslinking of the B-cell receptor (BCR). In contrast to investigations in immature chicken B-cell lines, transduced Notch1-ICT did not affect cell cycle progression, cell growth or surface IgM levels in NYC cells and resulted only in a slight induction of apoptosis. However, BCR-crosslinking enhanced apoptosis, but did not influence cell cycle progression in Notch1-ICT-positive NYC cells. These data imply a distinct function of Notch1 in mature murine B-cells as compared to immature chicken B cells and provide further evidence for Notch1's involvement in B-cell differentiation and development.

Abstract: Efficient clonal expansion of early precursor B (pre-B) cells requires signals delivered by an Ig-like integral membrane complex, the so-called pre-B cell receptor (pre-BCR). A pre-BCR consists of two membrane micro H chains, two covalently associated surrogate L chains, and the heterodimeric signaling transducer Igalphabeta. In contrast to a conventional Ig L chain, the surrogate L chain is a heterodimer composed of the invariant polypeptides VpreB and lambda5. Although it is still unclear how pre-BCR signals are initiated, two recent findings support a ligand-dependent initiation of pre-BCR signals: 1) a pre-BCR/galectin-1 interaction is required to induce phosphorylation of Igalphabeta in a human precursor B line, and 2) soluble murine as well as human pre-BCR molecules bind to stroma and other adherent cells. In this study, we show that efficient binding of a soluble murine pre-BCR to stroma cells requires the non-Ig-like unique tail of lambda5. Surprisingly however, a murine pre-BCR, in contrast to its human counterpart, does not interact with galectin-1, as revealed by lactose blocking, RNA interference, and immunoprecipitation assays. Finally, the binding of a murine pre-BCR to stroma cells can be blocked either with heparin or by pretreatment of stroma cells with heparitinase or a sulfation inhibitor. Hence, efficient binding of a murine pre-BCR to stroma cells requires the unique tail of lambda5 and stroma cell-associated heparan sulfate. These findings not only identified heparan sulfate as potential pre-BCR ligands, but will also facilitate the development of appropriate animal models to determine whether a pre-BCR/heparan sulfate interaction is involved in early B cell maturation.

Abstract: Pre-B cell receptor (pre-BCR) signals are essential for pro-B cells to mature efficiently into pre-B cells. The pre-BCR is an Ig-like transmembrane complex that is assembled from two mu H chains (mu HC) and two surrogate L chains consisting of the non-covalently associated polypeptides VpreB and lambda5. In lambda5(-/-) mice, pro-B cell maturation is impaired, but not completely blocked, implying that a mu HC induces differentiation signals in the absence of lambda5. Using a mouse model, in which transgenic mu HC expression can be controlled by tetracycline, we show that in the absence of lambda5, the transgenic mu HC promotes in vivo differentiation of pro-B cells, induces IL-7-dependent cell growth, and is expressed on the surface of pre-B cells. Our findings not only show that an incomplete pre-BCR can initiate signals, but also challenge the paradigm that an IgHC must associate with an IgLC or a SLC to gain transport and signaling competency.

Abstract: Although it is generally accepted that Ig heavy chains (HC) are selected at the pre-B cell receptor (pre-BCR) checkpoint, the characteristics of a functional HC and the role of pre-BCR assembly in their selection have remained elusive. We determined the characteristics of HCs that successfully passed the pre-BCR checkpoint by examining transcripts harboring V(H)81X and J(H)4 gene segments from J(H)(+/-) and lambda5(-/-)mice. V(H)81X-J(H)4-HC transcripts isolated from cells before or in the absence of pre-BCR assembly had no distinguishing complementarity-determining region 3 traits. In contrast, transcripts isolated subsequent to passage through the pre-BCR checkpoint had distinctive complementarity-determining regions 3 of nine amino acids in length (49%) and a histidine at position 1 (73%). Hence, our data define specific structural requirements for a functional HC, which is instrumental in shaping the diverse B cell repertoire.

Abstract: Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.

Abstract: Delta helicase is a 5' to 3' DNA helicase that partially co-purifies with DNA polymerase delta (pol delta) from fetal bovine thymus tissue. We describe the resolution of delta helicase from pol delta on heparin-agarose chromatography and its purification to apparent homogeneity by affinity purification on single-stranded DNA-cellulose chromatography, unique-sequence RNA-agarose chromatography, and ceramic hydroxyapatite chromatography. Delta helicase isolated from fetal bovine thymus had an apparent M(r) of 115 kDa in SDS-PAGE, and photo-crosslinked to [alpha-32P]ATP. Tandem mass spectrometry peptide mass data derived from the bovine polypeptide matched to human UPF1 (HUPF1), a 5' to 3' RNA and DNA helicase, and a requisite component of the mRNA surveillance complex. Antisera against HUPF1 cross-reacted with delta helicase on western analysis, and delta helicase activity was immunoinactivated by pre-incubation with antibodies to HUPF1, suggesting that delta helicase is the bovine homolog of HUPF1. Immunoprecipitation experiments demonstrated that HUPF1 interacts with the 66-kDa third subunit of pol delta in vivo.

Abstract: The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.

Abstract: The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig mu heavy chain (mu H-chain), the surrogate light (SL) chain, and the Ig alpha/beta dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which mu H-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of mu H-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of mu H-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.

Abstract: Signals initiated by the precursor B cell receptor (pre-BCR) are critical for B cell progenitors to mature into precursor B cells. The pre-BCR consists of a homodimer of microH chains, the covalently associated surrogate L (SL) chain composed of VpreB and lambda5, and the transmembrane signal molecules Ig(alpha) and Igbeta. One way to explain how maturation signals are initiated in late progenitor B cells is that the pre-BCR is transported to the cell surface and interacts from there with a ligand on stroma cells. To address this hypothesis, we first produced soluble Fab-like pre-BCR and BCR fragments, as well as SL chain, in baculovirus-infected insect cells. Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to B and T lymphoid suspension cells. The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion, and not dependent on bivalent cations. The binding of pre-BCR seems to be independent of the H chain of IgM (microH chain), because SL chain alone was able to interact with stroma cells. Finally, soluble pre-BCR specifically precipitated a 135-kDa protein from ST2 cells. These findings not only demonstrate for the first time the capacity of a pre-BCR to specifically bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL chain with a putative ligand on stroma cells.

Abstract: We describe a new population of non-naive B cells in the peripheral blood of quasimonoclonal (QM) mice. Surface Ig of switched isotypes is expressed, but not B220 nor CD19. These cells are larger and denser than naive B cells but smaller than blasts or plasma cells; they do not stain with syndecan, a marker for plasma cells. Telomerase, which is usually expressed in B cell blasts, was not present in this population. We sorted the switched, idiotype-positive, B220(-) B cells from the peripheral blood of QM mice and sequenced Ig H chain and lambda L chain cDNA. There were many point mutations but no V gene replacements, gene conversions or other type of diversifications. As they express switched isotypes and have mutated their Ig genes, cells in the B220(-), CD19(-) population must have been in an immune response and we suggest that it includes the memory B cell subset.

Abstract: The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

Abstract:

Abstract: To study the degradation requirements of unassembled immunoglobulin (Ig) chains, we heterologously expressed a cDNA encoding the secretory form of murine mu in the yeast S. cerevisiae. We found that mu chains were translocated into and retained in the endoplasmic reticulum (ER) as they were N-glycosylated and bound to the yeast homolog of BiP, Kar2p. Similar to mutant yeast carboxypeptidase Y (CPY*), known to undergo cytosolic degradation, mu protein is stabilized in yeast mutants lacking the ubiquitinating enzymes Ubc6p and Ubc7p or in cells overexpressing mutant ubiquitin. Unexpectedly, the translation inhibitor cycloheximide (CHX), but not puromycin, led to the accumulation of polyubiquitinated mu chains that were still glycosylated. By contrast, degradation of CPY* was not impaired by CHX, indicating that the drug affects a substrate-specific degradation step. In contrast to the situation for CPY*, the ER-transmembrane protein Der1p is not essential for mu degradation. Strikingly, however, the CHX-induced accumulation of polyubiquitinated Igmu chains was stronger in deltader1-mutants as compared to wild-type cells, indicating an additive effect of two inhibitory conditions. The results support a previously unknown activity of CHX, i.e. impairing the degradation of transport-incompetent secretory mu chains. Moreover, this activity will allow to dissect substrate-specific steps in ER associated protein degradation.

Abstract: Leflunomide is an immunosuppressive drug capable of inhibiting T and B cell responses in vivo. A number of studies demonstrate that leflunomide functions both as a pyrimidine synthesis inhibitor and as a tyrosine kinase inhibitor. We previously reported that leflunomide inhibits LPS-stimulated B cell proliferation, cell cycle progression, and IgM secretion. This inhibition can be reversed by the addition of exogenous uridine, suggesting that leflunomide functions as a pyrimidine synthesis inhibitor in B cells. We report here that while the addition of uridine restored proliferation and IgM secretion to leflunomide-treated LPS-stimulated B cells, as determined by metabolic labeling and immunoprecipitation, it did not completely restore secretion of IgG Ab. We hypothesized that leflunomide inhibits LPS-induced IgG secretion by inhibiting tyrosine kinase activity required for isotype switch. We tested this hypothesis in a well-defined model of isotype switch, LPS plus IL-4 induction of IgG1. Leflunomide inhibited IgG1 secretion in this model in a dose-dependent manner. The signal transduction pathway utilized by IL-4 to induce IgG1 involves tyrosine phosphorylation of the IL-4 receptor, JAK1, JAK3, and STAT6 proteins induced by IL-4 binding to the IL-4R. Leflunomide diminished the tyrosine phosphorylation of JAK3 and STAT6 in the absence or presence of uridine. In gel mobility shift studies, STAT6 binding to the STAT6 DNA binding site in the IgG1 promoter decreased in the presence of leflunomide or leflunomide plus uridine. Taken together, these data suggest that leflunomide acts as a tyrosine kinase inhibitor to block IgG1 production.

Abstract: CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor kappaB (NF-kappaB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8alpha) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8alpha chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8alpha revealed the existence of at least two forms of sCD30L/CD8alpha, which exhibited molecular sizes consistent with the existence of monomeric and trimeric forms of the molecule. Binding analyses performed using a soluble CD30 fusion protein (sCD30/gamma1) confirmed the ability of sCD30L/CD8alpha to bind to CD30. Functionally, immobilized sCD30L/CD8alpha-induced cell death in the CD30-expressing lines Karpas-299 and HDLM-2 and reduced proliferative levels in Karpas-299; these effects were inhibitable by the addition of sCD30/gamma1. These studies demonstrate the utility of sCD30L/CD8alpha in characterizing the normal function of CD30L and CD30 and indicate the natural ability of soluble forms of CD30L to trimerize.

Abstract: Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.

Abstract: Our studies examined the expression and DNA binding activity of myocyte enhancer factor 2 (MEF2A-D) transcription factors in lymphopoietic tissues, cell lines, and primary lymphocytes. Our analyses demonstrate that mef2C expression is restricted to B cells within the lymphocyte lineage. Using in situ hybridization, mef2C is detected in foci in fetal liver and postnatal thymic medulla, and both mef2B and mef2C are expressed in areas of the postnatal spleen and lymph node that also express kappa light chain (Ckappa), a B cell-specific marker. Reverse transcriptase-PCR (RT-PCR) analyses demonstrate that all mef2 family members are expressed in B cell lines, and all except mef2C are expressed in T cell lines. Immunoblot analyses of cell lines and primary thymic and splenic lymphocytes show that MEF2C and MEF2D proteins are expressed in B cells and that MEF2D is expressed in T cells; however, MEF2A protein is not detected in lymphocytes. Electrophoretic mobility shift assays (EMSA) demonstrate that B cell lines have MEF2C-containing, MEF2-specific DNA binding complexes whereas T cells do not. Our data is the first to describe mef2C expression in the lymphocyte lineage, and this finding suggests possible roles for MEF2C activity in B cell development and function.

Abstract: OBJECTIVE: To present the concept of nicotine-replacement therapy (NRT) and the pharmacologic approaches, nonprescription and prescription, to smoking cessation. DATA SOURCES: Current clinical literature. DATA SYNTHESIS: NRT can be delivered through a number of different nicotine-containing dosage forms (e.g., gum, patch, nasal spray, oral inhaler). The Agency for Health Care Policy and Research (AHCPR) recommends using the nicotine patches for routine clinical practice and the American Psychiatric Association (APA) recommends the use of the patches and gum as initial pharmacotherapies for smoking cessation. There are no comparative studies indicating the superiority of one form or another at relieving nicotine withdrawal symptoms. Of the other pharmacologic agents used for smoking cessation, bupropion hydrochloride demonstrates the most promise. CONCLUSION: The pharmacist can assist the consumer with the selection of an OTC smoking cessation product and serve as an informational resource to consumers and physicians desiring information on prescription drug products for smoking cessation.

Abstract: This study, we present evidence for a negatively acting control mechanism that coordinately suppresses the synthesis of the Th2 lymphokines IL-4, IL-5 and IL-6. This control mechanism operates in the murine thymoma cell line BW 5147. When cells of this line were fused to four independently established, well-defined Th2 cell clones, all resulting 74 lymphokine-secreting hybridomas secreted IL-2 which was not secreted by any of the parental Th2 cell clones. Most interestingly, however, none of the 74 hybridomas retained the capacity of the parental Th2 cells to express IL-4. Likewise, the secretion of IL-5 and IL-6 was also suppressed. Obviously, BW 5147 cells dominated the pattern of lymphokines produced, although the lymphokine pattern of Th2 cells was previously considered to be irreversibly fixed due to terminal differentiation of these cells. Suppression of IL-4 production was also observed at the mRNA level, as tested in Northern blot assays. Putative DNA target sequences for suppression of IL-4 gene transcription were not part of the proximal IL-4 promotor regions. Remote DNA control sequences may exist which coordinately regulate the proper, stage-specific expression of the Th2 lymphokines IL-4, IL-5 and IL-6.

Abstract: Levels of most nonsense mRNAs are normally reduced in prokaryotes and eukaryotes when compared with that of corresponding functional mRNAs. Genes encoding polypeptides that selectively reduce levels of nonsense mRNA have so far only been identified in simple eukaryotes. We have now cloned a human cDNA whose deduced amino acid sequence shows the highest degree of homology to that of UPF1, a bona fide Saccharomyces cerevisiae group I RNA helicase required for accelerated degradation of nonsense mRNA. Based on the total sequence of the shorter yeast UPF1 protein, the overall identity between the human protein and UPF1 is 51%. Besides NTPase and other RNA helicase consensus motifs, UPF1 and its human homolog also share similar putative zinc finger motifs that are absent in other group I RNA helicases. Northern blot analysis with the human cDNA probe revealed two transcripts in several human cell lines. Further, antibodies raised against a synthetic peptide of the human polypeptide detected a single 130 kDa polypeptide on Western blots from human and mouse cells. Finally, immunofluorescence and Western blot analyses revealed that the human and mouse polypeptides, like yeast UPF1, are expressed in the cytoplasm, but not in the nucleus. We have thus identified the first mammalian homolog of yeast UPF1, a protein that regulates levels of nonsense mRNA, and we tentatively name this protein human HUPF1 (for human homolog of UPF1).

Abstract: The etiology and pathogenesis of Kawasaki syndrome (KS) remain unknown. Clinical and epidemiologic features of KS are consistent with an infectious cause. To search for an etiologic agent of KS, a phage cDNA expression library was constructed from the aorto-iliac junction of a patient with fatal acute KS and screened with convalescent KS serum followed by anti-human Ig. Unexpectedly, 0.1% of the clones in the library react with anti-human Ig, indicating the presence of many Ig-producing B lymphocytes in the vasculitic tissue. To confirm this finding and to determine the isotypes produced, frozen vascular tissue sections from the patient and paraffin sections from coronary arteries from six additional patients with fatal acute or subacute KS were incubated with Abs to Ig isotypes. Histopathology of the tissues revealed the presence of many plasma cells in the inflammatory infiltrate. IgA was the predominant isotype produced in vascular tissue in all seven KS patients. IgM- and IgG-producing cells were less often detected. We conclude that there is a marked plasma cell response within the vasculitic tissue in KS, with unusual IgA production locally in this nonlymphoid, nonmucosal tissue. We suggest that the prominence of IgA plasma cells in the vascular infiltrate in the early, acute, and subacute stages of KS indicates an Ag-driven immune response to an etiologic agent with a respiratory or gastrointestinal portal of entry and speculate that this unusual immune response is integral to the pathogenesis of the illness.

Abstract: BACKGROUND: The immunoglobulin framework has been mutagenized to engineer recombinant libraries of proteins as potential diagnostics and novel catalysts, although the often shallow binding cleft may limit the utility of this framework for binding diverse small organic molecules. By contrast, the glutathione S-transferase (GST) family of enzymes contains a deep binding cleft, which has evolved to accommodate a broad range of hydrophobic xenobiotics. We set out to determine whether GST molecules with novel ligand-binding characteristics could be produced by random mutagenesis of segments of the binding cleft. RESULTS: We have identified two ligand-recognition segments (LRSs) in human GST P1, which are near the active site in the folded protein, but have characteristics indicating that the integrity of their sequence is not essential for the overall structure or activity of the protein. Libraries of GST P1-derived proteins were produced by substituting randomized sequences for an LRS or inserting random sequences into an LRS. The recombinant proteins in the libraries, collectively designated as 'glubodies,' generally retain enzymatic activity but differ markedly both from each other and from the parent enzyme in sensitivity to inhibition by diverse small organic compounds. In some instances, a glubody is inhibited by completely novel structures. CONCLUSIONS: We have shown that a non-antibody framework can be used to create large libraries of proteins with a wide range of binding specificities for small organic molecules. The glubodies provide a rich source of data for correlating the structural and functional features of proteins relevant to ligand binding. The criteria applied for identifying an LRS in GST P1 are generally applicable to other protein frameworks.

Abstract:

Abstract: CD30 is normally expressed by a subset (15 to 20%) of CD45RO+ T cells after activation by a variety of T cell stimuli and defines the principal IFN-gamma-producing T cells subset. Inasmuch as the production of IFN-gamma is regulated by IL-2 and IL-12, we have examined the effects of these cytokines on the proliferation, induction, and function of CD30+ and CD30- T cell subsets. Upon isolation, CD30+CD25+ T cells exhibit high levels of baseline proliferation compared with CD30-CD25+ T cells. Neutralizing Abs specific for IL-2, IL-4, IL-6, or IL-12 had no effect on basal levels of proliferation in CD30+CD25+ T cells, whereas anti-IL-2 inhibited the basal proliferative activity of CD30-CD25+ T cells. The addition of exogenous rIL-12 to purified CD30+CD25+ and CD30-CD25+ T cells subsets induced significantly higher maximal levels of proliferation in the CD30+ subset, whereas rIL-2 supported comparable levels of maximal proliferation in each subset. The addition of rIL-2 to anti-CD3-activated PBMC increased the relative numbers of CD30+ T cells by expansion of CD30+ T cells, as opposed to recruitment of CD30+ T cells from the CD30- population. Furthermore, the development of the CD30+ T cell subset was inhibited by either anti-IL-2, anti-IL-12, or rIL-10. Inhibition by anti-IL-2 and rIL-10 was overcome by the addition of rIL-12, but not IL-2, to the cultures. Finally, rIL-12 increased IFN-gamma production by CD30+ T cells, yet had little effect on IFN-gamma production by CD30- T cells. Thus, CD30+ T cells are preferentially regulated by IL-12, and the effects of IL-12 on T cell IFN-gamma production are mediated largely through its effects on the CD30+ subset.

Abstract: IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence.

Abstract: Bone marrow B cell precursors frequently rearrange the Ig heavy chain variable (VH) gene segment VH81X. It is puzzling, therefore, that mature B cells in adult mice rarely express mu-heavy chains bearing this VH gene segment. We show in this work in transformed pre-B cell lines that two VH81X/mu-chains that differ in their VH-D-JH joining sequences are not assembled covalently with the B cell precursor-specific surrogate light (SL) chain and are not expressed on the cell surface. From these findings, we propose that a B cell precursor clonally expands and proceeds to the next developmental stage only if it expresses a mu-chain with a VH domain that, together with the SL chain, directs the formation of a signal-transducing mu/SL chain membrane complex. Therefore, a checkpoint exists early in B cell development, at which SL chain not only screens B cell precursors for the presence of a full-length mu-chain, but also for a VH domain that promotes the assembly of a mu/SL chain complex.

Abstract: Two full-length cDNA clones encoding rabbit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were isolated from a lambda gt10 rabbit spleen cDNA library and sequenced. As predicted from the open reading frame (ORF) in vitro translation of a sense orientation GAPDH cDNA clone yielded a protein product with a molecular mass of 37 kDa. Rabbit GAPDH exhibits a high degree of homology to the mouse, rat, hamster, chicken and human GAPDH on both the nucleotide (nt) and amino acid (aa) levels.

Abstract:

Abstract: CD30 is a lymphoid activation Ag expressed by activated B and T lymphocytes, as well as selected lymphoid malignancies. In T cells, CD30 expression is limited to a minority (15 to 25%) of activated CD45RO+ T cells. Studies were undertaken to define unique functional properties of CD30+ T cells by identifying the profile of cytokine production by CD30+ T cells, and by assessing their ability to provide help for B cell Ig production. Assessment of cytokine mRNA transcripts by reverse transcription-PCR (RT-PCR) revealed the presence of IFN-gamma mRNA transcripts only in CD30+ T cells derived from FACS purified from activated CD45RO+ T cells. IFN-gamma mRNA was present neither in the CD30- subset of activated CD45RO+ T cells, nor in activated CD45RA+ T cells. Both CD30+ and CD30- subsets expressed IL-10 and IL-2R alpha mRNAs after 3 days of activation, but neither population expressed IL-4 or IL-6 transcripts at this time. Furthermore, production of secreted IFN-gamma was significantly greater in activated CD30+CD45RO+ cells (564 +/- 250 pg/ml) than in CD30-CD45RO+ (50 +/- 33 pg/ml) or CD45RA+ (0 +/- 0) T cells. CD30+ T cells also expressed IL-5 mRNA and secreted significantly higher levels of IL-5 (320 +/- 73) than activated CD30-CD45RO+ (17 +/- 5) T cells. In contrast, CD30- T cells produced significantly higher levels of IL-2 than CD30+ T cells (1270 +/- 380 vs 450 +/- 128). Induction of IFN-gamma in CD30+ T cells was not a result of CD30 engagement by Abs used during the isolation process, although an anti-CD30 Ab (M44) known to exert agonist activity was capable of inducing IFN-gamma production by T cells when immobilized to plastic. Finally, CD30+CD4+ T cells exhibit significantly greater helper activity for B cell Ig production than CD30-CD4+ T cells. These results demonstrate that CD30 identifies a unique subset of T cells that comprise the major IFN-gamma- and IL-5-producing cells in the T cell compartment, and exhibit potent helper activity for B cell Ig production.

Abstract: CD30 has been extensively studied as a cell surface marker expressed by Reed-Sternberg cells of Hodgkin's disease and other hematologic malignancies, although little is known about its expression by normal lymphoid cells. We therefore characterized the requirements for the induction of CD30 expression and identified the subsets of T cells that express CD30. CD30 is inducible on approximately 15% of normal PBMC stimulated with any of a variety of nonspecific T cell activators, including PHA, Con A, anti-T11(2) + T11(3), and anti-CD3; ionomycin alone induced lower percentages of CD30+ T cells (3 +/- 2%) compared to other stimuli. Maximal numbers of CD30+ cells were observed at 48 to 72 h of activation and the addition of rIL-2 did not affect these kinetics. However, CD30 expression was enhanced by the addition of exogenous rIL-2 to any of the stimuli tested, although rIL-2 alone did not lead to CD30 expression. The induction of CD30 during anti-CD3 mitogenesis was completely inhibitable by anti-IL-2 antibodies and partially inhibitable by rIL-4, indicating a requirement for both TCR triggering and IL-2 for its expression. Dual immunofluorescence analysis revealed that CD30+ cells were confined to CD3+ T cells that coexpressed higher levels of the p55 IL-2 receptor (CD25) than the CD30- population. Furthermore, CD30 expression was restricted to a subset of cells derived from CD45RO+ T cell precursors. Cell cycle analysis showed that CD30+ expression was not cell cycle dependent. Cross-linking of membrane CD30 induced Ca2+ in TCR+, but not TCR- Jurkat T cells. These results demonstrate that CD30 can serve as a T cell signal-transducing molecule and expressed by a unique subset of activated CD45RO+ T cells.

Abstract: The Ig kappa L chain synthesized by the mouse hybridoma line F10 forms large fibrils in the lumen of the endoplasmic reticulum. Despite the formation of these inclusion bodies, free kappa-chain is secreted. Both intracellular and secreted kappa-chains in this line migrate faster on SDS gels; thus, the F10 kappa-chain seems to be more compact than normal Ig L chain. In normal kappa-chain, four cysteine residues form intrachain disulfide bonds and the fifth connects the L chain to the H chain. Although the five cysteine residues of the aberrant kappa-chain are in the normal positions, they display an unusual gel pattern when the intrachain disulfide bonds are opened with 2-ME; that is, the intrachain disulfide bonding pattern of F10 kappa-chain seems to be unusual. It is suggested that the abnormal folding pattern favors fibril formation.

Abstract: The heavy chain variable region of the immunoglobulin receptors on cells of the B lymphoma NYC are almost identical to that of other independent B-cell tumors of B/W mice. NYC IgM binds a viral antigen produced by the tumor cells; and despite extensive screening, immunoglobulin-negative variants were never found in the NYC cells, suggesting that NYC loses the capacity to grow in culture when it does not synthesize surface immunoglobulin. These findings indicate that the interaction of endogenous antigen with surface IgM continuously stimulates growth and, thus, that the tumorigenesis of B lymphomas in B/W mice is mediated by antigen receptors.

Abstract: NYC is a B lymphoma cell line derived from B/W mice. Upon fusion of NYC cells with a plasmacytoma, which itself produces no immunoglobulin, the resulting NYCH hybridoma cells are Mott cells; i.e., they contain large intracellular vesicles filled with immunoglobulin, the so-called Russell bodies. When NYCH.kappa, a variant of NYCH that had lost the ability to produce heavy chain, was transfected with a heavy-chain construct, this concentration of immunoglobulin in the intracellular vesicles occurred only when the transfected immunoglobulin heavy chain had the same variable region as NYC. Moreover, unlike conventional Mott cells, the hybrid cells secrete immunoglobulin at a normal rate.

Abstract: Although AIDS patients lose human immunodeficiency virus (HIV)-specific cytotoxic T cells, their remaining CD8-positive T lymphocytes maintain cytotoxic function. To exploit this fact we have constructed bispecific antibodies that direct cytotoxic T lymphocytes of any specificity to cells that express gp120 of HIV. These bispecific antibodies comprise one heavy/light chain pair from an antibody to CD3, linked to a heavy chain whose variable region has been replaced with sequences from CD4 plus a second light chain. CD3 is part of the antigen receptor on T cells and is responsible for signal transduction. In the presence of these bispecific antibodies, T cells of irrelevant specificity effectively lyse HIV-infected cells in vitro.

Abstract: The class of immunoglobulin is defined by the constant region of its heavy chain. When a B lymphocyte switches the class of heavy chain it produces, the constant region of mu-type heavy chain is replaced; this occurs through a DNA rearrangement that brings the gene segment encoding the new constant region close to the VDJ segment encoding the variable region. The pre-B-cell line 18-81, which switches from heavy chain mu to gamma 2b production in culture, occasionally abnormally rearranges the heavy chain locus so that DNA sequences between the switch regions of mu and gamma 2b are inverted. Because looping-out is an intermediate step in generating an inversion, the switch rearrangement could occur by looping-out and deletion. Provided that recombination is reciprocal, this would produce a circle of DNA. Indeed, circular DNA molecules have been isolated as products of rearrangement among gene segments encoding the variable regions of the T-cell receptor and of the immunoglobulin heavy chain and light chain. But whereas the breakpoints for the variable region rearrangement are precisely defined, the breakpoints for any given heavy chain class switch are scattered over a length of greater than 6 kilobases, including both switch regions. We have now isolated circular DNA containing the sequences deleted by class-switching, thereby showing that the immunoglobulin heavy chain class switch occurs through looping-out and deletion.

Abstract: Immunoglobulin genes are generated during differentiation of B lymphocytes by joining gene segments. A mouse pre-B cell contains a functional immunoglobulin heavy-chain gene, but no light-chain gene. Although there is only one heavy-chain locus, there are two light-chain loci: kappa and lambda. It has been reported that kappa loci in the germ-line configuration are never (in man) or very rarely (in the mouse) present in cells with functionally rearranged lambda-chain genes. Two explanations have been proposed to explain this: (a) the ordered rearrangement theory, which postulates that light-chain gene rearrangement in the pre-B cell is first attempted at the kappa locus, and that only upon failure to produce a functional kappa chain is there an attempt to rearrange the lambda locus; and (b) the stochastic theory, which postulates that rearrangement at the lambda locus proceeds at a rate that is intrinsically much slower than that at the kappa locus. We show here that lambda-chain genes are generated whether or not the kappa locus has lost its germ-line arrangement, a result that is compatible only with the stochastic theory.

Abstract: During the switch in expression of an immunoglobulin class, the gene segment encoding the constant region of the heavy chain is replaced in a way that leads to a deletion. Three different models of how this deletion is generated have been proposed: recombination between homologs, unequal sister chromatid exchange, and looping out and deletion. While none of the predicted recombination products of the first two models have been found, the products of the looping out--inversions and circular DNA--have been isolated. Thus looping out and deletion appears to be the appropriate model to explain the genetic events leading to the immunoglobulin heavy chain class switch.

Abstract: The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.

Abstract: When termination codons were introduced into exons of the gene for Ig mu chain, steady-state levels of mu mRNA were reduced, both at the pre-B cell stage and at the plasma cell stage. A termination codon in the variable region gene segment and a termination codon in the second exon of the constant region gene segment had effects of similar magnitude. When the termination codon was deleted, the original level of mRNA was restored. The rate of mu gene transcription was the same whether or not a termination codon was present. Therefore, the termination codons must reduce the amount of the mRNA by reducing its stability. Since the introduced termination codons prematurely terminate translation and, in so doing, change the ribosome load on the mRNA, we conclude that mu mRNA stability is conferred in part by ribosomal protection from enzymatic degradation. We propose that the differences in mu mRNA stability during B lymphocyte differentiation are due to different amounts of ribosomes available for translation.

Abstract: In the mouse pre-B-cell line 18-81, cells can switch production in vitro from immunoglobulin mu chain to gamma 2b chain. The gene encoding the gamma 2b chain is created by a rearrangement of the mu gene. This rearrangement always takes place within a homolog. In cells with a gamma 2b gene, most of the time the gene segment encoding the constant region of the mu chain is deleted, but often the rearrangement leads to cells that produce no immunoglobulin, and all DNA sequences are retained. The latter result is due to an inversion. Inversions exclude the unequal sister chromatid exchange model of the heavy-chain class switch. Looping out is an intermediate step in the process of generating an inversion. Our findings demonstrate that the switch rearrangement occurs by looping out and deletion.

Abstract: During differentiation of B lymphocytes, the change in the amount of immunoglobulin heavy chain produced is reflected by a change in the steady state level of heavy chain mRNA. At the pre-B cell stage, the earliest stage at which immunoglobulin chain is produced, and later at the small resting B cell stage, there is a low steady state level of heavy chain mRNA. After the small B cell has differentiated to become a plasma cell, the steady state level of heavy chain mRNA is much higher. We confirm that the transcription rate at the immunoglobulin mu heavy chain gene does not change during differentiation from the pre-B cell to the plasma cell stage. In contrast, we show here that differences in the stability of mu mRNA are sufficient to account for the differences in the steady state level at the various differentiation stages.

Abstract: Spontaneous deletions at the immunoglobulin heavy-chain locus are frequently found in myelomas, hybridomas and pre-B-cell lines. We have measured the rates for large and small deletions within the constant-region gene segment for mu chain in a pre-B-cell lines. The large deletions, which include the entire first and second exons, occurred at a rate of 1.7 X 10(-5) per cell generation. The small deletions, which span a few base pairs, occurred at a rate of 1.4 X 10(-7) per cell generation. The rate for the reversion of a termination codon in the second exon is even less than that for the small deletions and is 1000 times lower than the reversion rate that had been determined for the variable-region gene segment. Therefore, the variable-region gene segment is likely to be the preferred target for hypermutation.

Abstract: In a pre-B-cell line that rearranges its heavy chain gene segments in vitro, we found that the rate of productive rearrangement on one allele was not influenced by the presence of heavy chain protein encoded by the other allele. This shows that allelic exclusion of heavy chain genes is not regulated at the genetic level.

Abstract: It is established that somatic mutation is an important source of antibody diversity in vivo. It is also established that Igh-V gene segments are hypermutable in vitro. This is not a completely satisfactory situation. While there is no reason to believe that Igh-V genes are not hypermutable in vivo as well, direct experimental evidence is lacking. Perhaps experiments with transgenic mice will soon fill this gap. It is not so clear how much higher than normal the rate of hypermutation is. As far as we are aware, there are no direct measurements of mutation rates per base pair per cell generation in mammals, certainly not for lymphocyte cell lines. For a variety of reasons, it is difficult to measure very low mutation rates. The general consensus is that the normal rate should be somewhere between 10(-10) and 10(-12) mutations per base pair per cell generation. Therefore, an experiment designed to directly determine a rate using the compartmentalization test would involve hundreds of cultures, each containing at least 10(9) cells. It is not a trivial problem to find one or a few mutants among so many cells. It is simple to study mutation to resistance to a drug, for example, ouabain or azaguanine, but, as we discussed, there are technical and conceptual pitfalls. The vast excess of dead cells influences the growth of a few mutant cells, particularly in lymphocyte cell lines. Even if this problem could be solved, the mutation rate so obtained would be "per gene(s)" and not "per base pair". The problems associated with cytotoxic agents can be avoided by immunofluorescence methods in conjunction with selective cloning or cell sorting. Using these techniques, we have carried out extensive experiments to determine whether the immunoglobulin mutator system acts, at least partially, on genetic elements other than those in or near the heavy chain variable region gene segment. For an opal termination codon in a heavy chain constant region gene segment, the rate of reversion was less than 10(-7) per base pair per cell generation. This upper limit was fixed by the high rate of small deletions at the heavy chain locus. For an allotype mutation at B2m, the gene encoding beta 2 microglobulin, the rate of mutation was less than 10(-8). This upper limit could be lowered by at least two orders of magnitude by using a high-speed cell sorter.

Abstract: Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.

Abstract: By fusing interphase cells to cells undergoing mitosis, the interphase chromosomes can be visualized. When analyzed in this way, chromosomes of normal mouse cells show characteristic undercondensed centromeric regions. We have found that the centromeric regions of chromosomes from Abelson virus-transformed cells are fully condensed. Abelson virus transforms mouse cells by introducing into them a virally encoded phosphokinase that is expressed constitutively. Thus, we propose that the condensation of centromeric chromatin is a result of overphosphorylation by the Abelson virus phosphokinase, and that the centromeric region is the relevant target of overphosphorylation in transformed cell growth.

Abstract: The "silent" allele at the immunoglobulin heavy-chain locus in the pre-B-lymphocyte line 18-81 contains a correctly assembled gene. However, an amber termination codon within the variable-region gene segment prematurely terminates translation into complete heavy chain. Revertants that do produce heavy chain are generated at a high rate, which is termed hypermutation. By DNA sequencing of subclones, we have confirmed that whenever mu chain is produced by the usually silent allele, a true reversion is found in the DNA. Mutations are not confined to the position of the amber termination codon but are also found at other sites in and near the variable-region gene segment.