Identification of a novel surface protein on activated CD4+ T cells that induces contact-dependent B cell differentiation (help).

CD4+ T lymphocytes provide contact-dependent stimuli to B cells that are critical for the generation of specific antibody responses in a process termed T helper function. The surface structures on activated CD4+ T cells that mediate this function are not fully known. We previously reported the isolation of a functionally unique subclone of the Jurkat leukemic T cell line (D1.1) that constitutively expressed contact-dependent helper effector function. To identify T cell surface molecules that mediate contact-dependent T helper function, a monoclonal antibody (mAb), designated 5c8, was generated that inhibits D1.1-mediated B cell activation and immunoprecipitates a novel 30-kD protein structure from surface-iodinated D1.1 cells. Normal CD4+ T cells express 5c8 antigen (Ag) transiently 5-6 h after activation by phorbol myristate acetate and phytohemagglutinin with maximal expression 5-6 h after activation and absence of expression by 24 h. In contrast, neither resting nor activated CD8+ T cells express 5c8 Ag. In functional studies, mAb 5c8 inhibits the ability of fixed, activated CD4+ T cells to induce B cell surface CD23 expression. In addition, mAb 5c8 inhibits the ability of CD4+ T cells to direct terminal B cell differentiation driven by pokeweed mitogen. Taken together, these data suggest that 5c8 Ag is a novel, activation-induced surface T cell protein that is involved in mediating a contact-dependent element of the helper effector function of CD4+ T lymphocytes.

Gonadotropin beta subunits determine the rate of assembly and the oligosaccharide processing of hormone dimer in transfected cells.

The glycoprotein hormones lutropin (LH) and chorionic gonadotropin (CG) share a common structure consisting of an identical alpha subunit noncovalently linked to a hormone-specific beta subunit. While LH is produced in the anterior pituitary, CG is synthesized in placenta. To compare the assembly, processing, and secretion of human LH and CG in the same cell type, we have expressed their subunits, individually and together, in mouse C-127 mammary tumor cells. Analysis of transfected clones revealed an unexpected difference in the secretion of individually expressed subunits. Whereas alpha and CG beta subunits were rapidly and quantitatively secreted, only 10% of newly synthesized LH beta subunit reached the medium. The remaining subunit was found in an intracellular, endoglycosidase H (endo H)-sensitive pool that had a turnover rate of approximately 8 h. Coexpression with alpha subunit resulted in "rescue" of LH beta subunit by formation of LH dimer, which was efficiently secreted. However, combination of LH beta with alpha was slow, with an overall efficiency of only 50% despite the presence of excess alpha. In contrast, CG beta was rapidly assembled with the alpha subunit after synthesis. The two beta subunits also differed in their influence on the N-linked oligosaccharide processing of combined alpha. The oligosaccharides of LH dimer were endo H resistant, while those of CG dimer remained partially endo H sensitive. Thus, despite a high degree of homology between LH beta and CG beta, the two subunits differ in their secretion as free subunits, their rate of assembly with alpha subunit, and in their effect on the N-linked oligosaccharide processing of combined alpha.

Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation.

RNA granules are a macromolecular structure observed in neurons, where they serve as motile units that translocate mRNAs. Isolated RNA granules are highly enriched in Staufen protein and ultrastructurally contain densely packed clusters of ribosomes. With depolarization, many mRNAs, including those involved in plasticity, rapidly shift from the RNA granule fraction to polysomes. Depolarization reorganizes granules and induces a less compact organization of their ribosomes. RNA granules are not translationally competent, as indicated by the failure to incorporate radioactive amino acids and the absence of eIF4E, 4G, and tRNAs. We concluded that RNA granules are a local storage compartment for mRNAs under translational arrest but are poised for release to actively translated pools. Local release of mRNAs and ribosomes from granules may serve as a macromolecular mechanism linking RNA localization to translation and synaptic plasticity.

Development of highly sensitive immunoassays to measure human chorionic gonadotropin, its beta-subunit, and beta core fragment in the urine: application to malignancies.

A variety of malignancies have been associated with the presence of human chorionic gonadotropin, hCG, its subunits, and fragments of its beta-subunit in blood and urine. The usefulness of these hCG-related tumor markers in nontrophoblastic malignancies has been inhibited by inadequate assay techniques. In order to achieve the required sensitivity and specificity, concentration steps and other procedures to remove cross-reacting human luteinizing hormone were necessary. In addition, the coexistence of a fragment of the hCG-beta or beta human luteinizing hormone subunit contributes to significant errors of measurement in urine. The importance of the hCG-beta fragment as a potential tumor marker has been recognized previously but no method was available to measure this antigen readily. We report here the development of a series of radioimmunometric, two-site assays which will accurately measure hCG, hCG-beta subunit, and the beta-subunit fragment directly in small volumes of unprocessed urine. These assays are highly specific, extremely sensitive, and not labor intensive since they employ microtiter plate procedures. Application of these assays to urine samples from patients with gynecological malignancies indicated that over 50% of all patients tested excreted the hCG-beta fragment in their urine. Also, this fragment comprised more than 50% of the moles of hCG immunoreactive components present in the specimens that were positive for hCG. This cancer marker is also demonstrable in trophoblastic malignant states such as choriocarcinoma in which the low molecular weight fragment can also be visualized directly by immunoblotting procedures. We conclude that a search for hCG immunoreactivity in the urine of patients with malignancies will be improved by the inclusion of accurate measurements of the prominent quantities of the beta fragment excreted by these individuals.

The fd phage and a peptide derived from its p8 coat protein interact with the HIV-1 Tat-NLS and inhibit its biological functions.

Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 - a synthetic peptide derived from it and bearing amino acids 1-20 of the phage p8 protein - interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin beta and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100 microM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.

Developmental regulation of proenkephalin gene expression in osteoblasts.

Proenkephalin (PENK), a classically defined opioid gene, was originally thought to be expressed almost exclusively in the mature nervous and neuroendocrine systems. In the last few years, it was demonstrated, however, that high levels of PENK messenger RNA and PENK-derived peptides are expressed in embryonic mesenchymal tissues during differentiation into mature tissues and organs. Shortly after birth, as development progresses, PENK expression drops in those tissues to undetectable levels. Very little is known about the molecular mechanisms regulating this transient expression. To investigate those mechanisms, we used primary cell cultures of calvaria-derived osteoblasts. These cultures express PENK and exhibit a normal pattern of osteoblastic differentiation. In the present study we demonstrate that 1) a reciprocal interrelationship exists between PENK expression and osteoblastic differentiation in vivo, ex vivo, and in vitro; namely, PENK expression is down-regulated upon cellular differentiation; 2) PENK promoter usage and messenger RNA splicing function similarly in osteoblasts and in neural cells; 3) osteoblastic PENK expression is modulated by bone-targeting hormones; and 4) this down-regulation is inhibited by the serine/threonine kinase inhibitor H-8. The link between osteoblastic differentiation and down-regulation of PENK expression together with our preliminary findings indicating the existence of an osteoblastic opioid receptor suggest that opioids act in an autocrine/paracrine mechanism on undifferentiated osteoblasts and play a significant role in bone development.

Inhibition of beta-adrenergic responsiveness in muscle cell cultures by dexamethasone.

Cultures of two myogenic cell lines, L6E9 and L8, were grown in the absence or presence of dexamethasone. Dexamethasone (1 microM) completely inhibited the formation of myotubes. Partial inhibition (20-40%) was obtained at a concentration as low as 1 nM. Dexamethasone also inhibited beta-adrenergic responsiveness, as noted by decreases in isoproterenol-stimulated adenylate cyclase activity and cAMP accumulation. These effects were both dose and time dependent. In the presence of dexamethasone, the number of beta-adrenergic receptors, as assessed by [125I]iodohydroxybenzylpindolol binding, decreased coordinately with the decrease in cAMP. A high affinity, limited capacity cytosolic binding site for [3H]triamcinolone acetonide observed under control conditions (Kd = 0.7 nM; maximum binding, 2.7 pmol/mg protein) increased in number as a function of developmental state. These data indicate that glucocorticoids inhibit myogenesis and beta-adrenergic responsiveness in vitro.

Immunological detection of membrane-associated human luteinizing hormone correlates with gene expression in cultured human cancer and fetal cells.

We have demonstrated the expression of membrane-associated hCG and its subunits and fragments by cells from 78 human cancer cell lines of different types and origins, indicating that such expression is a common phenotypic characteristic of cultured human malignant cells. Because human (h) LH beta has 80% homology with hCG beta and is coded by one of the seven genes in the gene cluster located in chromosome 19, it was important to determine whether hLH and its beta-subunit are also expressed as membrane-associated proteins by cells from human cancer cell lines. Thus, 11 cancer cell lines of different types and origins were adapted to grow in serumless medium, with Nutridoma-HU or SP as serum substitute, and analyzed by flow cytometry using two monoclonal antibodies directed to different conformational epitopes of intact hLH and a monoclonal antibody reacting with an epitope of hLH beta-free. The cells were also analyzed simultaneously for the expression of hCG and its subunits and fragments. Determination of translatable levels of hLH beta and hCG beta messenger RNAs (mRNAs) was performed in cells from some of the cancer cell lines, including the JEG-3 choriocarcinoma cell line, and in cells from a human fetal lung cell line. The analytical flow cytometry studies showed that in addition to the expression of membrane-associated hCG in all of its forms, expression of membrane-associated intact (holo) hLH and its free beta-subunit occurred in every case. These findings were corroborated by the presence of translatable levels of hLH beta and hCG beta mRNAs in all of the cancer cell lines analyzed, indicating that the expression of these membrane-associated glycoproteins is a phenotypic characteristic of human cancer cells and that the activation of the hCG beta-hLH beta gene cluster is nonselective. The presence of translatable levels of hCG beta-hLH beta mRNAs in the cultured human fetal lung cells punctuates once more the in vivo and in vitro biochemical similarities between fetal and cancer cells.

Development and characterization of a new, highly specific antibody to the human chorionic gonadotropin-beta fragment.

In addition to high concentrations of hCG, pregnancy urine contains even higher concentrations of a fragment of the hCG beta-subunit. This biologically inactive material complicates immunological measurement of hCG, since it cross-reacts with many polyclonal and monoclonal antibodies to the hCG beta-subunit that are employed for assays of hCG in urine. Although we and others have developed antibodies to this fragment, specific measurement of the fragment in the presence of free hCG beta has remained difficult due to intrinsic cross-reactivity of these antibodies with the intact hCG beta. Rather than attempt to increase specificity by assay optimization, we developed a new, highly specific monoclonal antibody, designated B210, which cross-reacts less than 0.1% with the free hCG beta-subunit in both liquid and solid phase immunoassay formats. We have used this new monoclonal antibody in immunoradiometric assays to measure specifically the hCG beta fragment in urine throughout pregnancy as well as in the sera of two individuals with cancers producing the hCG beta-subunit. We discovered that the hCG beta fragment can bind three monoclonal antibodies simultaneously, indicating that although the epitope for antibody B210 is a new determinant exposed on the hCG beta fragment and not on intact hCG or on free hCG beta-subunit, the hCG beta fragment retains at least two other hCG beta-related epitopes intact, i.e. those that bind monoclonal antibodies B108 and B201.

Development, characterization, and application of monoclonal antibodies to the native and synthetic beta COOH-terminal portion of human chorionic gonadotropin (hCG) that distinguish between the native and desialylated forms of hCG.

Although the pregnancy hormone hCG has been extensively mapped immunochemically, few monoclonal antibodies have been produced to the unique COOH-terminal region of its beta-subunit (beta CTP). We now report the development and characterization of five such monoclonal antibodies. Three of these antibodies were developed to the synthetic peptide analog of the hCG beta-(109-145) region coupled to diphtheria toxoid, and two antibodies to a conjugate of bovine thyroglobulin and the peptide hCG beta-(115-145) prepared from hCG with its carbohydrate moieties intact. The monoclonal antibodies raised against the synthetic peptide bound hCG, desialylated hCG, and synthetic peptide to a similar extent, whereas antibodies generated to the natural hCG peptide did not bind to the synthetic peptide analog of the COOH-terminal peptide (beta CTP) region or to desialyated hCG. These new monoclonal antibodies could distinguish between native and desialyated hCG in liquid phase immunoassays as well as by Western blots. They are highly specific reagents for such Western blotting and were used for studies of a crude human pituitary gonadotropin preparation to demonstrate that it contained intact hCG beta without the internal peptide bond cleavages found in the subunit present in human blood and urine. Competition experiments using combinations of monoclonal antibodies and rabbit anti-beta CTP antiserum demonstrated that two epitopes exist within the beta-(115-145) region of hCG, one of which depends on the presence of carbohydrate. In summary, the new monoclonal hCG beta CTP antibodies reported here can 1) discriminate between native and desialylated hCG, 2) identify hCG and nicked hCG on Western blots, 3) provide an immunoaffinity purification tool for hCG, and 4) bind to two distinct epitopes on the beta CTP.

Structure of the human chorionic gonadotropin beta-subunit fragment from pregnancy urine.

A major portion of the hCG immunoreactivity detectable in pregnancy urine is derived from a fragment of hCG beta. This lacks the COOH-terminal portion of hCG beta, but retains immunoreactivity with most antibodies raised against the beta-subunit of hCG. To improve clinical measurements of hCG and assess the importance of such fragments in human urine, we have isolated and determined the structure of this molecule. The hCG beta fragment was isolated from a partially purified commercial preparation of hCG (Organon) by gel filtration and immunoaffinity chromatography using monoclonal antibodies. It was found to consist of two polypeptide chains composed of residues beta-(6-40) disulfide-bridged to residues beta-(55-92). It also differs from the beta-subunit of hCG in its carbohydrate structure, lacking sialic acid and having a low but variable amount of galactose. A beta-fragment containing the same two NH2-terminal sequences was also isolated from a single pregnant woman's urine. The two major polypeptides comprising the beta-fragment contain a total of nine half-cystine residues, raising the possibility that a free thiol may exist or that a third undetected disulfide-bridged peptide is present in the intact fragment. However, tests for the presence of a free thiol have been negative. Another intrinsic characteristic of the beta-fragment is the formation of a variable amount of dimer in solutions of neutral pH. beta-fragment will not combine with intact alpha-subunit. Despite the absence of regions beta-(1-5), beta-(41-54), and beta-(93-145), the beta fragment is recognized by the SB-6 antibody and most monoclonal antibodies elicited to the beta-subunit, thus excluding half of the amino acids of the beta-subunit from the epitope(s) where these antibodies bind.

Preparation and characterization of antibodies to the urinary fragment of the human chorionic gonadotropin beta-subunit.

hCG is a glycoprotein hormone composed of two dissimilar subunits (alpha and beta) and is normally synthesized by trophoblastic tissue. Its measurement by immunoassay is widely employed as a test for pregnancy, but can be complicated by cross-reactivity with human (h) LH. Immunoassays based on the beta-subunit of hCG have been employed to decrease this cross-reactivity with hLH, but when these assays are used with urine specimens, the antibodies employed also detect a fragment of hCG beta, which can lead to significant differences in measurement. To overcome these problems, we have developed a series of monoclonal antibodies to the beta fragment of hCG recovered from pregnancy urine. Some of the antibodies that bind to this beta fragment are directed to a region of hCG beta that is different from the epitopes recognized by antibodies raised against the intact beta-subunit. The new epitopes available in the hCG beta fragment form the basis for novel immunoassays. These beta fragment antibodies are used in conjunction with other antibodies, directed to different epitopes of the hormone, to produce a series of immunoradiometric assays that can discriminate among intact hormone, free hCG beta, and hCG beta fragment. The hCG beta fragment antibodies described herein have affinities between 10(9) and 10(11) M-1 for the beta fragment and exhibit varying degrees of discrimination between the hCG beta fragment, the beta-subunits of hCG and hLH, and intact hCG and hLH.

Development of an assay for a biomarker of pregnancy and early fetal loss.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone, secreted by the syncytiotrophoblast cells of the fertilized ovum, that enters the maternal circulation at the time of endometrial implantation. It is composed of two nonidentical subunits; alpha and beta, with molecular weights of 14 kD and 23 kD, respectively. Its alpha subunit is identical in primary structure to its glycoprotein homologs, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). Human chorionic gonadotropin binds to the same receptor as hLH and displays the same biological response, namely, to stimulate the declining function of the corpus luteum to produce progestins and estrogen late in the menstrual cycle. The differences in the structures of hCG and hLH have been exploited to develop antibodies that can measure hCG specifically in the presence of hLH. Two-site antibody binding assays have been developed, based on a surface immunological concept of hCG epitopes, that involve four distinct regions to which antibodies against hCG can bind simultaneously. Antibody cooperative effects, in conjunction with kinetic advantages derived from the concentration factors by use of the sandwich assay technique (immunoradiometric assay, IRMA), have enabled development of extremely sensitive and specific measurement protocols for urinary hCG. The assay described herein permits the detection of pregnancy on an average 25.4 days after the first day of the preceding menses, as opposed to 29.5 days for conventional radioimmunoassay techniques. In addition, the greater sensitivity and specificity of this assay method has permitted the detection of episodes of fetal loss not detected by radioimmunoassay of urine specimens. A large scale epidemiological study is in progress using this assay technique as a way to identify pregnancies that are lost before becoming clinically apparent. This methodology provides a valuable tool for the determination of the rate of early fetal loss.

Characterization and relative orientation of epitopes for monoclonal antibodies and antisera to human chorionic gonadotropin.

We analyzed ten monoclonal antibodies and three polyclonal antisera directed against human chorionic gonadotropin by immunoassay in order to determine which pairs of antibodies are capable of binding simultaneously to hCG. A relative orientation of epitopes on the surface of hCG could be inferred from a matrix of data describing the abilities of particular antibody pairs to inhibit competitively or enhance the binding of each other. These results indicated that the binding site of each of the antibodies could be assigned to one of three different regions of the hCG molecule, and at least one antibody binding within each region exhibited a substantial preference for hCG relative to human luteinizing hormone. One of these regions is the COOH-terminal portion of the hCG beta subunit. A second region contains the binding site for the SB6 rabbit antiserum (which has been widely employed in clinical studies) as well as the binding sites of several monoclonal antibodies. Antibodies to this second region characteristically bind both the whole hormone and the free beta subunit. A third region contains the binding sites of a previously reported human antiserum and also several monoclonal antibodies. These antibodies characteristically react preferentially with the intact hormone relative to the free subunits. The information contained in the type of epitope map described here can be used as the rational basis for the design of two-site immunoradiometric assays for hCG and/or its subunits.

Immunochemical mapping of hCG and hCG-related molecules.

The general observations and experiences of three laboratory groups that have studied the immunochemistry of hCG are summarized. A basic model of the types of antibodies which are most frequently observed has been constructed using the information gathered. Although the immunochemical mapping of the surface of any globular protein is very complex, it was possible to segregate the immune response of mice to hCG into five distinct classes of antibodies based on their binding properties. Each of these types or classes of antibodies can be further subdivided and are obviously binding to different combinations of amino acids on the surface of the hormone. We have attempted to coordinate the nomenclature of the various investigators into a simple scheme for better understanding. The recognition of these different types of antibodies has permitted the development of sensitive and specific immunological measurement systems.

Translational control of specific genes during differentiation of HL-60 cells.

Eukaryotic gene expression can be regulated through selective translation of specific mRNA species. Nevertheless, the limited number of known examples hampers the identification of common mechanisms that regulate translation of specific groups of genes in mammalian cells. We developed a method to identify translationally regulated genes. This method was used to examine the regulation of protein synthesis in HL-60 cells undergoing monocytic differentiation. A partial screening of cellular mRNAs identified five mRNAs whose translation was specifically inhibited and five others that were activated as was indicated by their mobilization onto polysomes. The specifically inhibited mRNAs encoded ribosomal proteins, identified as members of the 5'-terminal oligopyrimidine tract mRNA family. Most of the activated transcripts represented uncharacterized genes. The most actively mobilized transcript (termed TA-40) was an untranslated 1.3-kilobase polyadenylated RNA with unusual structural features, including two Alu-like elements. Following differentiation, a significant change in the cytoplasmic distribution of Alu-containing mRNAs was observed, namely, the enhancement of Alu-containing mRNAs in the polysomes. Our findings support the notion that protein synthesis is regulated during differentiation of HL-60 cells by both global and gene-specific mechanisms and that Alu-like sequences within cytoplasmic mRNAs are involved in such specific regulation.