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In this publication we describe a peptide insulin receptor antagonist, S661, which is a single chain peptide of 43 amino acids. The affinity of S661 for the insulin receptor is comparable to that of insulin and the selectivity for the insulin receptor versus the IGF-1 receptor is higher than that of insulin itself. S661 is also an antagonist of the insulin receptor of other species such as pig and rat, and it also has considerable affinity for hybrid insulin/IGF-1 receptors. S661 completely inhibits insulin action, both in cellular assays and in vivo in rats. A biosynthetic version called S961 which is identical to S661 except for being a C-terminal acid seems to have properties indistinguishable from those of S661. These antagonists provide a useful research tool for unraveling biochemical mechanisms involving the insulin receptor and could form the basis for treatment of hypoglycemic conditions.

The type IA topoisomerases have been implicated in the repair of dsDNA breaks by homologous recombination and in the resolution of stalled or damaged DNA replication forks; thus, these proteins play important roles in the maintenance of genomic stability. We studied the functions of one of the two mammalian type IA enzymes, Top3beta, using murine embryonic fibroblasts (MEFs) derived from top3beta(-/-) embryos. top3beta(-/-) MEFs proliferated more slowly than TOP3beta(+/+) control MEFs, demonstrated increased sensitivity to DNA-damaging agents such as ionizing and UV radiation, and had increased DNA double-strand breaks as manifested by increased gamma-H2-AX phosphorylation. However, incomplete enforcement of the G(1)-S cell cycle checkpoint was observed in top3beta(-/-) MEFs. Notably, ataxia-telangiectasia, mutated (ATM)/ATM and Rad3-related (ATR)-dependent substrate phosphorylation after UV-B and ionizing radiation was impaired in top3beta(-/-) versus TOP3beta(+/+) control MEFs, and impaired up-regulation of total and Ser-18-phosphorylated p53 was observed in top3beta(-/-) cells. Taken together, these results suggest an unanticipated role for Top3beta beyond DNA repair in the activation of cellular responses to DNA damage.

To elucidate mechanisms that regulate Vbeta rearrangement, we generated and analyzed mice with a V(D)J recombination reporter cassette of germline Dbeta-Jbeta segments inserted into the endogenous Vbeta14 locus (Vbeta14(Rep)). As a control, we first generated and analyzed mice with the same Dbeta-Jbeta cassette targeted into the generally expressed c-myc locus (c-myc(Rep)). Substantial c-myc(Rep) recombination occurred in both T and B cells and initiated concurrently with endogenous Dbeta to Jbeta rearrangements in thymocytes. In contrast, Vbeta14(Rep) recombination was restricted to T cells and initiated after endogenous Dbeta to Jbeta rearrangements, but concurrently with endogenous Vbeta14 rearrangements. Thus, the local chromatin environment imparts lineage and developmental stage-specific accessibility upon the inserted reporter. Although Vbeta14 rearrangements occur on only 5% of endogenous TCRbeta alleles, the Vbeta14(Rep) cassette underwent rearrangement on 80-90% of alleles, supporting the suggestion that productive coupling of accessible Vbeta14 segments and DJbeta complexes influence the frequency of Vbeta14 rearrangements. Strikingly, Vbeta14(Rep) recombination also occurs on TCRbeta alleles lacking endogenous Vbeta to DJbeta rearrangements, indicating that Vbeta14 accessibility per se is not subject to allelic exclusion.

The mammalian presenilin (PS) proteins mediate the posttranslational cleavage of several protein substrates, including amyloid precursor protein, Notch family members, and CD44, but they have also been suggested to function in diverse cellular processes, including calcium-dependent signaling and apoptosis. We carried out an integrative computational study of multiple genomic datasets, including RNA expression, protein interaction, and pathway analyses, which implicated PS proteins in Toll-like receptor signaling. To test these computational predictions, we analyzed mice carrying a conditional allele of PS1 and a germ line-inactivating allele of PS2, together with Cre site-specific recombinase expression under the influence of CD19 control sequences. Notably, B cells deficient in both PS1 and PS2 function have an unexpected and substantial deficit in both lipopolysaccharide and B cell antigen receptor-induced proliferation and signal transduction events, including a defect in anti-IgM-mediated calcium flux. Taken together, these results demonstrate a fundamental and unanticipated role for PS proteins in B cell function and emphasize the potency of (systems level) integrative analysis of whole-genome datasets in identifying novel biologic signal transduction relationships. Our findings also suggest that pharmacologic inhibition of PS for the treatment of conditions such as Alzheimer's disease may have potential consequences for immune system function.

Toll-like receptors (TLRs) recognize a limited number of conserved elements in pathogens and, by activating antigen-presenting cells such as dendritic cells and monocytes and macrophages, play a crucial role in the immune response to infection and vaccination. Most data on TLR function in the context of human aging focus on responses to lipopolysaccharide, an integral component of gram-negative bacteria, which signals through TLR4. However, such studies have not led to a consensus conclusion and are limited by differences in epidemiological and laboratory methods. A recent comprehensive evaluation of TLR function in monocytes from older adults was conducted using a multivariable mixed statistical model to account for covariates. It was found that cytokine production after TLR1/2 engagement, which is essential for the recognition of triacylated lipopeptides found in a variety of bacteria, is substantially lower in monocytes from older adults. The upregulation of costimulatory proteins such as CD80, essential for optimal activation of T cells, on monocytes from older adults was less for all TLR ligands tested than for cells from young individuals, and the extent of CD80 upregulation predicted subsequent antibody response to influenza immunization. These and other consequences of aging on human TLR function may impair activation of the immune response and contribute to poorer vaccine responses and greater morbidity and mortality from infectious diseases in older adults. Such age-associated alterations have particular relevance in view of the interest in TLR agonists as therapeutic agents not only for infections, but also for allergic, autoimmune, and malignant disease.

Background: Innate immunity, including Toll-like receptor (TLR)-mediated expression of the B7 costimulatory molecules CD80 and CD86, is critical for vaccine immunity. We examined whether CD80 and CD86 expression vary with aging and predict response to the trivalent inactivated influenza vaccine., Methods: One hundred sixty-two subjects between 21 and 30 years of age (the young group) or > or =65 years of age (the older group) enrolled before vaccination. We determined TLR-induced monocyte CD80/CD86 expression by flow cytometry and vaccine antibody responses by hemagglutination inhibition., Results: The mean increase in TLR-induced CD80(+) monocytes was reduced in older, compared with young, adults by 68% (P=.0002), and each decile increase of CD80(+) cells was associated with an 8.5% increase in mean number of vaccine strains with a > or =4-fold titer increase (P=.01) and a 3.8% increase in mean number of strains with a postvaccine titer > or =1 : 64 (P=.037). Each decile decrease of CD86(+) cells was associated with an 11% increase in the mean number of strains with a 4-fold increase (P=.002) and a 3.9% increase in the mean number of strains with a postvaccine titer > or =1 : 64 (P=.07)., Conclusions: CD80 and CD86 expression on activated monocytes is highly associated with influenza vaccine response. This approach prospectively identifies adults unlikely to respond to immunization who may benefit from alternative vaccines or antiviral prophylaxis during influenza outbreaks.

Mice lacking DNA topoisomerase 3beta are predisposed to a shortened lifespan, infertility, and lesions in multiple organs resulting from inflammatory responses. Examination of the immune system of 6- and 52-week-old top3beta(-/-) mice revealed no significant aberrations in their central and peripheral tolerance or in T lymphocyte activation. However, the older but not the younger cohort shows a high incidence of serum autoantibodies relative to their TOP3beta(+/+) age-mates. The mutant mice also show an increase in numerical aberrations of chromosomes in splenocytes and bone marrow cells, as well as an increase in apoptotic cells in the thymus. Thus, it appears plausible that the inflammatory lesions in top3beta(-/-) mice are caused by the development of autoimmunity as they age: Chromosomal abnormalities in top3beta(-/-) mice might lead to a persistent increase in apoptotic cells, which might in turn lead to the progression of autoimmunity.

The effects of aging on human TLR function remain incompletely understood. We assessed TLR function and expression in peripheral blood monocytes from 159 subjects in 2 age categories, 21-30 and >65 years of age, using a multivariable mixed effect model. Using flow cytometry to assess TLR-induced cytokine production, we observed a substantial, highly significant defect in TLR1/2-induced TNF-alpha (p = 0.0003) and IL-6 (p < 0.0001) production, in older adults compared with young controls. In contrast to findings in aged mice, other TLR (including TLR2/6)-induced cytokine production appeared largely intact. These differences were highly significant even after correcting for covariates including gender, race, medications, and comorbidities. This defect in TLR1/2 signaling may result from alterations in baseline TLR1 surface expression, which was decreased by 36% in older adults (p < 0.0001), whereas TLR2 surface expression was unaffected by aging. Production of IL-6 (p < 0.0001) and TNF-alpha (p = 0.003) after stimulation by N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2R,S)-propyl]-Cys-[S]-Ser1-[S]-Lys(4) trihydrochloride was strongly associated with TLR1 surface expression. Diminished TLR1/2 signaling may contribute to the increased infection-related morbidity and mortality and the impaired vaccine responses observed in aging humans.

Insulin receptor (IR) and insulin-like growth factor I receptor (IGF-IR) are both from the same subgroup of receptor tyrosine kinases that exist as covalently bound receptor dimers at the cell surface. For both IR and IGF-IR, the most described forms are homodimer receptors. However, hybrid receptors consisting of one-half IR and one-half IGF-IR are also present at the cell surface. Two splice variants of IR are expressed that enable formation of two isoforms of the IGF-IR/IR hybrid receptor. In this study, these two splice variants of hybrid receptors were studied with respect to binding affinities of insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II). Unlike previously published data, in which semipurified receptors have been studied, we found that the two hybrid receptor splice variants had similar binding characteristics with respect to insulin, IGF-I, and IGF-II binding. We studied both semipurified and purified hybrid receptors. In all cases we found that IGF-I had at least 50-fold higher affinity than insulin, irrespective of the splice variant. The binding characteristics of insulin and IGF-I to both splice variants of the hybrid receptors were similar to classical homodimer IGF-IR.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors that are an important link between innate and adaptive immunity. Many established, as well as experimental, vaccines incorporate ligands for TLRs, not only to protect against infectious diseases but also in therapeutic immunization against noninfectious diseases, such as cancer. We review the underlying mechanisms by which engagement of TLR signaling pathways might trigger an adaptive immune response after immunization. Although the engagement of TLR signaling pathways is a promising mechanism for boosting vaccine responses, questions of efficacy, feasibility and safety remain the subject of active investigation.

Breast cancer is a leading cause of death for women. The underlying molecular mechanism is still not well understood. In this study, two-dimensional gel electrophoresis combined with mass spectrometry was used to analyze changes in the proteome of infiltrating ductal carcinoma compared to normal breast tissue. Ten sets of two-dimensional gels per experimental condition were analyzed and more than 500 spots each were detected. This revealed 39 spots for which expression in breast cancer cells were reproducibly altered more than twofold compared to normal controls (p < 0.01). These spots represented 25 different proteins after identification using the database search after mass spectrometry, comprising cell defense proteins, enzymes involved in glycolytic energy metabolism and homeostasis, protein folding and structural proteins, proteins involved in cytoskeleton and cell motility, and proteins involved in other functions. In addition, 28 nondifferentially expressed proteins with different functions were also mapped and identified, which might help to establish a two-dimensional gel electrophoresis reference map of human breast cancer. Our study shows that proteomics offers a powerful methodology to detect the proteins that show different expression patterns in breast cancer tissue and may provide an accurate molecular classification. The differentially expressed proteins may be used as potential candidate markers for diagnostic purposes or for determination of tumor sensitivity to therapy. The functional implications of the identified proteins are discussed.

LEOPARD syndrome (lentigines, electrocardiographic conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) is an autosomal dominant condition. The main clinical features include multiple lentigines, cardiovascular defects, and facial anomalies, some of which are shared with Noonan syndrome (NS). Recent reports have shown that LEOPARD syndrome can be caused by mutations in PTPN11, the gene in which mutations can produce NS. Here we report the findings of mutation screening and linkage analysis of PTPN11 in three families with LEOPARD syndrome. We identified a novel mutation in one family. The mutation (1529A>C) substitutes proline for glutamine at amino acid 510 (Gln510Pro). No variations in sequence were observed in the other two families, and negative LOD scores excluded linkage to the PTPN11 locus, showing that LEOPARD syndrome is genetically heterogeneous.

T cell antigen recognition involves the formation of a structured interface between antigen-presenting and T cells that facilitates the specific transmission of activating and desensitizing stimuli. The molecular machinery that organizes the signaling molecules and controls their disposition in response to activation remains poorly understood. We show here that in T cells Discs large (Dlg1), a PDZ domain-containing protein, is recruited upon activation to cortical actin and forms complexes with early participants in T cell activation. Transient overexpression of Dlg1 attenuates basal and Vav1-induced NFAT reporter activation. Reduction of Dlg1 expression by RNA interference enhances both CD3- and superantigen-mediated NFAT activation. Attenuation of antigen receptor signaling appears to be a complex, highly orchestrated event that involves the mutual segregation of important elements of the early signaling complex., (Copyright The Rockerfeller University Press)

CFC (cardiofaciocutaneous) syndrome (MIM 115150) has been considered by several authors to be a more severe expression of Noonan syndrome. Affected patients present with congenital heart defects, cutaneous abnormalities, Noonan-like facial features and severe psychomotor developmental delay. We have recently demonstrated that Noonan syndrome can be caused by missense mutations in PTPN11(MIM 176876), a gene that encodes the non-receptor protein tyrosine phosphatase SHP-2. In this report, we have evaluated the possible involvement of mutations in PTPN11 in CFC syndrome. A cohort of 28 CFC subjects rigorously assessed as having CFC based on OMIM diagnostic criteria was examined for mutations in the PTPN11 coding sequence by using DHPLC analysis. The results showed no abnormalities in the coding region of the PTPN11 gene in any CFC patient, nor any evidence of major deletions within the gene suggesting that mutations in other gene(s) are responsible for this syndrome.

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.

Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D-PAGE profiles of [35S]-labelled Chlamydia proteins from whole lysates of infected cells to 2D-PAGE profiles of proteins from purified Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the 'chlamydial protease- or proteasome-like activity factor' (CPAF) and were clearly present in 2D-PAGE profiles of whole lysates of infected cells but absent from purified Chlamydia. CPAF was recently identified by Zhong and colleagues as a secreted protease which cleaves host cell transcription factors essential for MHC class I and II antigen presentation. The identification of CPAF in this paper verifies the applicability of the described method for the identification of secreted proteins. We extend the findings by Zhong et al. by proteome studies of expression and turnover of C. trachomatis CPAF showing that the degradation of C. trachomatis D CPAF in the host cell is very limited. Furthermore, we show that two fragments of CPAF exist in C. pneumoniae as well as in C. trachomatis.

Chlamydia trachomatis represents a group of human pathogenic obligate intracellular and gram-negative bacteria. The genome of C. trachomatis D comprises 894 open reading frames (ORFs). In this study the global expression of genes in C. trachomatis A, D and L2, which are responsible for different chlamydial diseases, was investigated using a proteomics approach. Based on silver stained two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), gels with purified elementary bodies (EB) and auto-radiography of gels with 35S-labeled C. trachomatis proteins up to 700 protein spots were detectable within the range of the immobilized pH gradient (IPG) system used. Using mass spectrometry and N-terminal sequencing followed by database searching we identified 250 C. trachomatis proteins from purified EB of which 144 were derived from different genes representing 16% of the ORFs predicted from the C. trachomatis D genome and the 7.5 kb C. trachomatis plasmid. Important findings include identification of proteins from the type III secretion apparatus, enzymes from the central metabolism and confirmation of expression of 25 hypothetical ORFs and five polymorphic membrane proteins. Comparison of serovars generated novel data on genetic variability as indicated by electrophoretic variation and potentially important examples of serovar specific differences in protein abundance. The availability of the complete genome made it feasible to map and to identify proteins of C. trachomatis on a large scale and the integration of our data in a 2-D PAGE database will create a basis for post genomic research, important for the understanding of chlamydial development and pathogenesis.

Early stages of B cell development are dependent on the expression of a pre-B cell receptor (BCR), composed of a mu heavy chain (HC) in association with surrogate light chain (SLC) proteins and the signaling molecules, Igalpha and Igbeta. During the formation of the variable region of the mu chain by somatic gene rearrangement, a truncated form of the mu protein (called Dmu) is sometimes produced by the rearrangement of a D(H) segment to a J(H) segment using one of three reading frames (designated rf2). When a Dmu protein is formed, subsequent B cell development is blocked by down-regulation of further HC rearrangements, so that a full-length muHC cannot be formed. In this study, we demonstrate that in recombinase activating gene (RAG)-2-deficient B220(+) CD43(+) pro-B cells in which B lymphopoiesis has been arrested at fraction C, transgenic expression of Dmu promoted partial developmental progression to fraction C', but was unable to mediate the pro-B to pre-B cell transition to fraction D effected by full-length muHC protein. These data suggest that the intracellular signaling pathways engaged by the Dmu pre-BCR are insufficient to facilitate the expansion and/or survival of pre-B cells, and are distinct from those engaged by the pre-BCR-containing full-length muHC.

Structural analogues of the phytohormone (+)-abscisic acid (ABA) have been synthesized and tested as inhibitors of the catabolic enzyme (+)-ABA 8'-hydroxylase. Assays employed microsomes from suspension-cultured corn cells. Four of the analogues [(+)-8'-acetylene-ABA, (+)-9'-propargyl-ABA, (-)-9'-propargyl-ABA, and (+)-9'-allyl-ABA] proved to be suicide substrates of ABA 8'-hydroxylase. For each suicide substrate, inactivation required NADPH, increased with time, and was blocked by addition of the natural substrate, (+)-ABA. The most effective suicide substrate was (+)-9'-propargyl-ABA (K(I) = 0.27 microM). Several analogues were competitive inhibitors of ABA 8'-hydroxylase, of which the most effective was (+)-8'-propargyl-ABA (K(i) = 1.1 microM). Enzymes in the microsomal extracts also hydroxylated (-)-ABA at the 7'-position at a low rate. This activity was not inhibited by the suicide substrates, showing that the 7'-hydroxylation of (-)-ABA was catalyzed by a different enzyme from that which catalyzed 8'-hydroxylation of (+)-ABA. Based on the results described, a simple model for the positioning of substrates in the active site of ABA 8'-hydroxylase is proposed. In a representative physiological assay, inhibition of Arabidopsis thaliana seed germination, (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA exhibited substantially stronger hormonal activity than (+)-ABA itself.

The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.

The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web.

Interferon gamma (IFN-gamma) is a potent immunomodulatory lymphokine, secreted by activated T-lymphocytes and NK-cells during the cellular immune response. Actions of IFN-gamma are mediated through binding to the IFN-gamma-receptor, present on most cells, and the subsequent activation of a great magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting.

Chlamydia trachomatis is an obligate intracellular bacterium causing human ocular and genital disease. The lymphokine interferon gamma (IFN-gamma) is an important immune effector exerting antimicrobial effects towards several intracellular parasites, the chlamydia included. IFN-gamma has been reported to inhibit the chlamydial replication in vitro in part by depleting intracellular levels of tryptophan in a dose-dependent manner. In addition, down-regulation of important immunogens has been described. These findings are extended in this paper, in which we are combining pulse labeling with [35S]methionine and two-dimensional gel electrophoresis with immobilized pH gradients in order to investigate changes in the protein expression of C. trachomatis serovar A and L2 caused by treatment with IFN-gamma. In contrast to what was observed in C. trachomatis L2, our results showed that, in C. trachomatis A, down-regulations of the chlamydia major outer membrane protein and of several other proteins were detectable upon IFN-gamma treatment. In addition, we report the up-regulations of C. trachomatis A and L2 proteins with molecular masses of approximately 30 kDa and 40 kDa which may be part of an, as yet, uncharacterized chlamydial response to IFN-gamma treatment.

During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promote development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, we have tested the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression. For this purpose, we introduced an activated Ras expression construct into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes and assayed differentiation potential by recombination activating gene (RAG) 2-deficient blastocyst complementation. We found that activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. We discuss these findings in the context of potential mechanisms that may regulate Ig LC gene rearrangement.

To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras-RAG). Similar to the effects of an immunoglobulin (Ig) mu heavy chain (HC) transgene, activated Ras caused progression of RAG1-deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of lambda5, RAG2, and germline kappa locus transcripts. However, these Ras-RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig mu HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cells induces developmental progression by activating both differentiation and survival signals.

Wheat (Triticum aestivum L.) embryo germination is inhibited by natural (S)-(+)-abscisic acid (ABA). In this report we have determined critical structural features of the ABA molecule, particularly the methyl and ketone groups of the ABA ring, required for inhibitory activity. To examine the ring residues a series of new optically active ABA analogs have been synthesized in which the 4[prime]-keto, 7[prime]-, 8[prime]-, or 8[prime]- and 9[prime]-carbons have been replaced with hydrogen atoms. Each of the analogs was tested over a range of concentrations as a germination inhibitor. Enantiomers of the analogs altered at the 4[prime]-keto or 8[prime]- and 9[prime]-methyl groups were active, but less so than ABA. Both enantiomers of 7[prime]-demethylABA were inactive as germination inhibitors. The results show that the 7[prime]-methyl group is absolutely required for activity, but that the other residues are less critical for hormone recognition.

Serum C-reactive protein and neopterin concentrations were measured in samples taken at an early stage in different types of infection to see whether the combination of markers could contribute to the diagnosis of infection and help distinguish between bacterial and viral infections, tuberculosis, and infections due to "other" pathogens. Both markers were significantly raised in all categories of infection compared with controls, and there were significant differences between the means of both markers when comparing several of the categories of infection. Only C-reactive protein concentrations in bacteraemic patients, however, were both sensitive and specific at distinguishing the type of infection. The additional use of neopterin estimation played only a minor part in increasing the specificity of diagnosis in tuberculosis and in viral infections. On the basis of this study it was not considered worth the time and expense of performing neopterin assays in addition to C-reactive protein estimations to differentiate viral from bacterial infection.

Membrane-bound immunoglobulin (mIg) is the antigen receptor on B lymphocytes mediating early events in antigen presentation and signal transduction. Wild-type human mIgM constructs transfected into the murine B-cell lymphoma A20 are expressed as transmembrane proteins with antigen presentation and signaling functions comparable to the endogenous mIgG2A; the transfected wild-type mIgM is internalized rapidly after anti-Ig cross-linking. Transfected constructs lacking the normal three-amino acid cytoplasmic tail are expressed exclusively as phosphatidylinositol-linked proteins, lack both antigen presentation and signal transduction functions, and are internalized slowly following anti-Ig binding. The molecular mass of the cytoplasmic tail-deleted phosphatidylinositol-linked Ig molecule is consistent with cleavage of the transmembrane residues during processing. Cytoplasmic domains may therefore regulate the mode of expression of membrane proteins and thereby influence their functional capabilities.

The membrane-bound form of immunoglobulin serves as an antigen-specific receptor for B cells mediating signal transduction and antigen presentation. We have developed an assay that reconstitutes both these physiologic responses with respect to the antigen phosphorylcholine. By introducing specific mutations in the human Ig mu chain gene, we have shown that certain transmembrane residues and the short cytoplasmic domain are crucial for these two activities. Moreover, elimination of a single transmembrane hydroxyl group severely inhibits antigen presentation without affecting signal transduction, suggesting that these two functions are mediated by different protein interactions.

A general method for synthesis of insect pheromones having alkyl branched carbon skeletons is demonstrated with the preparation of a diastereomeric mixture of 8-methyl-2-decanol, whose propionate is an attractant of someDiabrotica species. The procedure involves reaction of a ketone with lithium acetylide ethylenediamine complex to afford a propargylic alcohol containing the branch of the target molecule. Copper (1) mediated alkylation of the derived propargylic acetate with a primary alkyl halide yields a trisubstituted allene having the desired chain length, and isomerization with an alkali metal amide of either ethylenediamine or 1,3-diaminopropane, affords the alkyl branched terminal acetylene. The triple bond is converted to the methyl ketone and reduced to the methyl carbinol. The reactions proceed in good yield, and can be conveniently carried out on large scale. The method should prove useful for production of pheromone components in cases where diastereomeric mixtures can be employed.

Transgenic mice carrying an immunoglobulin enhancer-driven c-myc oncogene develop rapid-onset pre-B cell lymphomas. The incidence of these malignancies is greatly reduced when an additional transgene encoding the membrane-bound form (but not the secreted form) of human Ig mu is bred into the susceptible strain. This suppressive effect correlates with a subtle alteration in B-cell development induced by the immunoglobulin transgene.

Expression of the membrane-bound version of the human mu chain in transgenic mice results in the allelic exclusion of endogenous mouse Ig heavy chain genes (6). The secreted version of the human Ig transgene has no such effect. F1 hybrid animals that carry transgenes for both secreted and membrane-bound human mu chains produce both forms of the human heavy chain while strongly suppressing endogenous mouse mu expression. The simultaneous expression of the two rearranged transgenes in primary B cells suggests that allelic exclusion operates before the formation of a second functionally rearranged heavy chain gene in vivo.

Antibody-producing cells display a special form of regulation whereby each cell produces immunoglobulin from only one of its two sets of antibody genes. This phenomenon, called allelic exclusion, is thought to be mediated by the product of one heavy chain allele restricting the expression of the other. Heavy chains are synthesized in two molecular forms, secreted and membrane bound. In order to determine whether it is specifically the membrane-bound form of the immunoglobulin M (IgM) heavy chain (mu) that mediates this regulation, transgenic mice were created that carry a human mu chain gene altered so that it can only direct the synthesis of the membrane-bound protein. The membrane-bound form of the human mu chain was made by most of the B cells in these animals as measured by assays of messenger RNA and surface immunoglobulins. Further, the many B cells that express the human gene do not express endogenous mouse IgM, and the few B cells that express endogenous mouse mu do not express the transgene. Thus, the membrane-bound form of the mu chain is sufficient to mediate allelic exclusion. In addition, the molecular structures recognized for this purpose are conserved between human and mouse systems.