Purification and characterization of an FSH releasing protein from porcine ovarian follicular fluid

A variety of hypophysiotropic peptides or proteins have been reported to be present in mammalian gonads. Inhibin, a hormone that under most circumstances selectively suppresses the secretion of follicle-stimulating hormone (FSH) but not luteinizing hormone (LH), has been isolated from the gonadal fluids of several species and characterized as a heterodimeric protein consisting of alpha- and beta-polypeptides associated by disulphide bonds. The complete amino-acid sequences of the precursors of porcine and human inhibin alpha-subunits and two distinct porcine inhibin beta-subunits (beta A and beta B) have been deduced from complementary DNA sequences. Gonadotropin releasing peptides have also been found in the gonad and have generally been shown to be active in radioreceptor assays for gonadotropin releasing hormone (GnRH) but to exhibit different chromatographic and immunological characteristics from those of GnRH. During our purification of inhibin from porcine follicular fluid, we noted fractions that could stimulate the secretion of FSH by cultured anterior pituitary cells. We report here the purification of an FSH releasing protein (FRP) and its characterization by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions and by partial sequence analysis. Our results indicate that porcine gonadal FRP is a homodimer consisting of two inhibin beta A-chains linked by disulphide bonds. FRP is highly potent (50% effective concentration (EC50) approximately 25 pM) in stimulating the secretion and biosynthesis of FSH but not of LH or any other pituitary hormone. In contrast to the effects of GnRH and other reported gonadal gonadotropin releasing fractions, the action of FRP is not mediated by GnRH receptors.     

Inhibin beta in central neural pathways involved in the control of oxytocin secretion

Inhibin (I) a gonadal hormone glycoprotein which suppresses follicle-stimulating hormone (FSH) secretion from the anterior pituitary, is a heterodimer consisting of an alpha subunit and one of two distinct beta subunits. S1 nuclease analysis has revealed that RNAs encoding all three subunits (alpha, beta A and beta B) are expressed in rat brain. We report here on the localization, and a potential function, of inhibin beta in the rat brain. A cell group centred in the nucleus of the solitary tract (NTS), a major recipient of visceral sensory information, was stained immunohistochemically with antisera against synthetic fragments of I beta, but not I alpha. The distribution of I beta-stained fibres is consistent with known NTS projections, and includes a prominent projection to oxytocinergic aspects of the magnocellular neurosecretory system.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

K562 human leukaemic cells express fetal type (i) antigen on different glycoproteins from circulating erythrocytes

During the ontogenic change from fetal to adult human erythrocytes, as well as fetal haemoglobin being replaced by adult haemoglobin, the cell-surface antigen i is converted to I (ref. 1). Recently it has been shown that this antigenic change is the conversion of the linear repeating Gal beta 1 leads to 4GlcNac beta 1 leads to 3Gal structure to branched Gal beta 1 leads to 4GlcNac beta 1 leads to 3(Gal beta 1 leads to 4GlcNac beta 1 leads to 6)Gal structure. We have shown that cell-surface labelling followed by endo-beta-galactosidase digestion can distinguish these two forms on the cell surface, and that band 3 and band 4.5 are the major carriers for these antigens on mature erythrocytes. Human leukaemic cell line K562, originally isolated from a patient at blast crisis of chronic myelocytic leukaemia, has recently been shown to synthesize glycophorin A, and to be capable of synthesizing haemoglobin upon induction. I demonstrate here that K562 cells express the fetal type (i) antigen on distinctly different glycoproteins from those of erythrocytes, by the use of cell-surface labelling followed by endo-beta-galactosidase digestion or followed by immunoprecipitation with specific antibodies.     

Identification of a potent Xenopus mesoderm-inducing factor as a homologue of activin A

The first inductive interaction in amphibian development is mesoderm induction, when a signal from the vegetal hemisphere of the blastula induces mesoderm from overlying equatorial cells. Recently, several 'mesoderm-inducing factors' (MIFs) have been discovered. These cause isolated Xenopus animal caps to form mesodermal cell types such as muscle, instead of their normal fate of epidermis. The MIFs fall into two classes. One comprises members of the fibroblast growth factor (FGF) family, and the other members of the transforming growth factor type beta (TGF-beta) family. Of the latter group, the most potent is XTC-MIF, a protein produced by Xenopus XTC cells. Here we show that XTC-MIF is the homologue of mammalian activin A. Activins modulate the release of follicle-stimulating hormone from cultured anterior pituitary cells and cause the differentiation of two erythroleukaemia cell lines. Our results indicate that these molecules may also act in early development during formation of the mesoderm.     

Pituitary FSH is released by a heterodimer of the beta-subunits from the two forms of inhibin

Inhibin is a gonadal protein that specifically inhibits the secretion of pituitary follicle-stimulating hormone (FSH). Two forms of inhibin (A and B) have been purified from porcine follicular fluid and characterized as heterodimers of relative molecular mass (Mr) 32,000 (ref. 2). Each inhibin is comprised of an identical alpha-subunit of Mr 18,000 and a distinct but related beta-subunit of Mr 13,800-14,700 linked by interchain disulphide bond(s). Throughout the purification of inhibins, we consistently observed two fractions which stimulated the secretion of pituitary FSH. We report here the isolation of one of the FSH-releasing proteins; it has a Mr of 24,000 and its N-terminal sequences up to residue 32 are identical to those of each beta-subunit of inhibins A and B. In the presence of reducing agents, SDS-polyacrylamide gel electrophoresis resolves the FSH-releasing substance into two subunits which are identical in their migration behaviour to the reduced beta-subunits of inhibins A and B. Based on the N-terminal sequence data and Mr of the intact and reduced molecules, we propose that the FSH-releasing substance, which is active in picomolar concentrations, is a heterodimeric protein composed of the two beta-subunits of inhibins A and B linked by interchain disulphide bond(s). The structural organization of the FSH-releasing substance is homologous to that of transforming growth factor-beta (TGF-beta), which also possesses FSH-releasing activity in the same bioassay. We suggest that the substance be called activin to signify the fact that it has opposite biological effects to inhibin.     

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.     

Nucleotide sequence of cloned cDNA of human c-myc oncogene

Like other transforming genes of retroviruses, the v-myc gene of the avian virus, MC29, has a homologue in the genome of normal eukaryotic cells. The human cellular homologue, c-myc, located on human chromosome 8, region q24 leads to qter (refs 1, 2), is translocated into the immunoglobulin heavy-chain locus on human chromosome 14 (ref. 3) in Burkitt's lymphoma, suggesting that c-myc has a primary role in transformation of some human haematopoietic cells. In addition, c-myc is amplified in the human promyelocytic leukaemia cell line, HL60 (refs 6, 7) which also contains high levels of c-myc mRNA. Recently, Colby et al. reported the nucleotide sequence of the human c-myc DNA isolated from a genomic recombinant DNA library derived from human fetal liver. This 4,053-base pair (bp) sequence includes two exons and one intron of the myc gene, and the authors have suggested the existence of a human c-myc mRNA of 2,291 nucleotides that has a coding capacity for a protein of molecular weight (Mr) 48,812. We have approached the problem of accurately defining the characteristics of the human c-myc mRNA and c-myc protein by determining the sequence of the c-myc cDNA isolated from a cDNA library prepared from mRNA of a clone of the K562 human leukaemic cell line. K562 cells are known to contain c-myc mRNA which is similar in size to the c-myc mRNA of other human cell types. We report here the sequence of 2,121 nucleotides of a human c-myc mRNA and demonstrate that its 5' noncoding sequence does not correspond to the sequence of the reported genomic human sequence. However, our data confirm that the intact human c-myc mRNA can encode a 48,812-Mr protein with a sequence identical to that reported by Colby et al.     

Inhibin is absent from azoospermic semen of infertile men

There is ample evidence that the secretion of follicle stimulating hormone (FSH) by the pituitary gland is regulated by a nonsteroidal hormone of gonadal origin, termed inhibin. In several species, testicular extracts, gonadal lymph and seminal plasma have been shown to contain proteins which inhibit FSH release; lack of suitable assays for inhibin has, however, prevented clear definition of its physiological significance. Men whose tests show the histological changes of germinal cell failure usually have raised FSH levels in the blood, but may have normal levels of luteinizing hormone (LH) and testosterone. Such patients would thus be predicted to show reduced inhibin production. To test this, we have measured the FSH inhibitory activity of seminal plasma from azoospermic subjects with raised plasma FSH levels. We report here our confirmation of diminished inhibin levels in seminal plasma of these patients, thus providing convincing support for its physiological role as a modulator of FSH production in man.     

Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-beta

Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.     

Isolation of a cDNA clone encoding rat insulin-like growth factor-II precursor

Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic polypeptides of relative molecular mass (Mr) approximately 7,500 isolated from human plasma each containing four peptide domains in a single chain and identical at more than 60% of their amino acid loci. The B- and A-domains of the IGFs are approximately 40% identical to the B- and A-chains of human insulin. IGF-I and IGF-II have similar in vitro biological activities and receptor reactivity, but are immunologically distinct. IGF-I appears to mediate the effects of growth hormone on cartilage to promote skeletal growth whereas IGF-II may have a special role in fetal development and in the central nervous system. To investigate the in vivo role of IGF-II, we have studied IGF-II biosynthesis in the BRL-3A rat liver cell line. BRL-3A cells synthesize and secrete a 7,484 Mr protein 93% identical to human IGF-II and representing rat IGF-II (rIGF-II). Rat IGF-II is synthesized as a approximately 22,000 Mr prepro-rIGF-II (ref. 12) from 12 S poly(A)+mRNA. In addition, approximately 20,000 Mr pro-rIGF-II has been identified in lysates of biosynthetically labelled intact BRL-3A cells. We report here the isolation of an almost complete cDNA clone for rIGF-II. Our results indicate that pro-rIGF-II is synthesized as a 156 amino acid peptide precursor (17,619 Mr) containing mature rIGF-II 1-67 at its amino-terminus and an 89-residue carboxy-terminal peptide extension.     

Beta 2-microglobulin from serum associates with MHC class I antigens on the surface of cultured cells

Beta 2-microglobulin (beta 2-m) is a highly conserved polypeptide (12,000 molecular weight; 12K) noncovalently associated with the heavy chain (45-48K) of the major histocompatibility complex (MHC) class I antigens. Its synthesis is required for expression of the HLA-A/B and H-2K/D heavy chains at the cell surface; beta 2-m is also associated with the human cell-surface antigens T6 and M241 isolated from thymocytes. However, on the T leukaemic cell line MOLT-4 some of the T6 antigens contain a different 12K subunit, termed beta t (refs 3, 7, 8). Purified human beta 2-m can exchange partially both with human beta 2-m associated with HLA-antigens, and with mouse beta 2-m associated with murine alloantigens. As MOLT-4 cells were grown in the presence of fetal calf serum (FCS) and as serum is known to contain some free beta 2-m, we examined whether beta t was bovine beta 2-m which had replaced endogenous beta 2-m on the surface of the cell. Here we show both that beta 2-m from FCS or human serum (HuS) used in cell culture can exchange with beta 2-m on the cell surface, and that beta t is in fact bovine beta 2-m.     

Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1

The product of the gene RCC1 (regulator of chromosome condensation) in a BHK cell line is involved in the control of mitotic events. Homologous genes have been found in Xenopus, Drosophila and yeast. A human genomic DNA fragment and complementary DNA that complement a temperature-sensitive mutation of RCC1 in BHK21 cells encode a protein of relative molecular mass 45,000 (Mr 45K) which is located in the nucleus and binds to chromatin. We have recently isolated a protein from HeLa cells that strongly binds an anti-RCC1 antibody and has the same molecular mass, DNA-binding properties, and amino-acid sequence as the 205 residues already identified. HeLa cell RCC1 is complexed to a protein of Mr 25K. We have shown that this 25K protein has a sequence homologous to the translated reading frame of TC4, a cDNA found by screening a human teratocarcinoma cDNA library with oligonucleotides coding for a ras consensus sequence, and that the protein binds GDP and GTP. We have referred to this protein as the Ran protein (ras-related nuclear protein). In addition to the fraction of Ran protein complexed to RCC1, a 25-fold molar excess of the protein over RCC1 was found in the nucleoplasm of HeLa cells. Here we show that RCC1 specifically catalyses the exchange of guanine nucleotides on the Ran protein but not on the protein c-Ha-ras p21 (p21ras).     

Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluids.

Cerebral deposition of the beta-amyloid peptide (A beta) is an invariant feature of Alzheimer's disease. Since the original isolation and characterization of A beta (ref. 1) and the subsequent cloning of its precursor protein, no direct evidence for the actual production of discrete A beta has been reported. Here we investigate whether A beta is present in human biological fluids using antibodies specific for an epitope within A beta that spans the site of normal constitutive cleavage. These antibodies were used to construct a sandwich-type enzyme-linked immunosorbent assay that detects A beta in cerebrospinal fluid, plasma and conditioned medium of human mixed-brain cells grown in vitro (see also ref. 14). By affinity chromatography, we have purified and sequenced A beta and a novel A beta fragment from human cerebrospinal fluid and conditioned medium of human mixed-brain cell cultures. These findings demonstrate that A beta is produced and released both in vivo and in vitro. These observations offer new opportunities for developing diagnostic tests for Alzheimer's disease and therapeutic strategies aimed at reducing the cerebral deposition of A beta.     

Serum beta 2-microglobulin binds to a T-cell differentiation antigen and increases its expression

The human T-cell leukaemia and differentiation antigen HTA 1 is defined by the monoclonal antibody NA1/34 (ref. 1) and also recognized by the monoclonal antibody OKT6. Like class I products of the human major histocompatibility complex, it has a glycosylated heavy (alpha) chain of approximately 45-50,000 molecular weight (MW) in non-covalent association with beta 2-microglobulin (beta 2m) (MW 11,900). A particular feature of HTA 1 is the presence in significant amounts of an additional beta 2m-like subunit, called beta t (refs 3, 4). Top facilitate biochemical studies we have prepared a high HTA 1 expressor variant (NH17) of the human thymoma line MOLT-4. The N-terminal amino acid sequence of the beta t purified from this cell line was shown to be indistinguishable from that of bovine beta 2m. Further, beta t was present when the cells were grown in medium containing fetal calf serum (FCS), but absent from cells grown with human serum (HuS). We show here that addition of human and bovine beta 2m to MOLT-4 and NH17 cells grown in serum-free medium produces a significant elevation of HTA 1 antigen expression, providing evidence for a regulatory or stabilizing function for the exchange of extracellular beta 2m with a cell-surface antigen.     

Cloning and identification of a novel human glioma differentiation related gene GDR1

In order to identify the genes associated with glioblastoma differentiation, some ESTs, expressed differentially in the control cell and the differentiated human glioblastoma cell line BT-325 induced by the all-trans retinoid acid, have been isolated by the method of DDRT-PCR. Of the 46 ESTs sequenced, 19 are from new genes. A full-length 1 535-bp cDNA, termed gene GDR1, has been isolated from the human cDNA library using the probe designed according to one of the novel ESTs, HGBB098. The open reading frame of GDR1 gene encodes a putative protein containing 334 amino acid residues. Blast against the current GenBank DNA and protein sequence database did not reveal significant homology with any known proteins. RT-PCR shows that GDR1 mRNA level increased in the differentiated BT-325 cells after being treated with RA. The different expression patterns of GDR1 mRNA in human tissues have been detected through the multiple tissue Northern blot hybridization.     

Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity

Alzheimer's disease is characterized by cerebral deposits of amyloid beta-protein (AP) as senile plaque core and vascular amyloid, and a complementary DNA encoding a precursor of this protein (APP) has been cloned from human brain. From a cDNA library of a human glioblastoma cell line, we have isolated a cDNA identical to that previously reported, together with a new cDNA which contains a 225-nucleotide insert. The sequence of the 56 amino acids at the N-terminal of the protein deduced from this insert is highly homologous to the basic trypsin inhibitor family, and the lysate from COS-1 cells transfected with the longer APP cDNA showed an increased inhibition of trypsin activity. Partial sequencing of the genomic DNA encoding APP showed that the 225 nucleotides are located in two exons. At least three messenger RNA species, apparently transcribed from a single APP gene by alternative splicing, were found in human brain. We suggest that protease inhibition by the longer APP(s) could be related to aberrant APP catabolism.     

Cyclin/PCNA is the auxiliary protein of DNA polymerase-delta

Identification of the cellular proteins whose expression is regulated during the cell cycle in normal cells is essential for understanding the mechanisms involved in the control of cell proliferation. A nuclear protein called cyclin of relative molecular mass 36,000 (Mr 36K), whose synthesis correlates with the proliferative state of the cell, has been identified in several cell types of human, mouse, hamster and avian origin. The rate of cyclin synthesis is very low in quiescent cells and increases several fold after serum stimulation shortly before DNA synthesis. Immunofluorescence and autoradiography studies have shown that the nuclear staining patterns of cyclin during S phase have a sequential order of appearance and a clear correlation can be found between DNA synthesis and cyclin positive nuclei. The proliferating cell nuclear antigen (PCNA) and cyclin have many common properties and it has been shown that these two are identical. Recently a protein which is required by DNA polymerase-delta for its catalytic activity with templates having low primer/template ratios has been isolated from calf thymus. We report here that cyclin and the auxiliary protein of DNA polymerase-delta are identical.     

Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells

Major histocompatibility complex (MHC) class I molecules are integral membrane proteins present on virtually all vertebrate cells and consist of a heterodimer between the highly polymorphic alpha-chain and the beta 2-microglobulin (beta 2-m) protein of relative molecular mass 12,000 (ref. 1). These cell-surface molecules play a pivotal part in the recognition of antigens, the cytotoxic response of T cells, and the induction of self tolerance. It is possible, however, that the function of MHC class I molecules is not restricted to the immune system, but extends to a wide variety of biological reactions including cell-cell interactions. For example, MHC class I molecules seem to be associated with various cell-surface proteins, including the receptors for insulin, epidermal growth factor, luteinizing hormone and the beta-adrenergic receptor. In mice, class I molecules are secreted in the urine and act as highly specific olfactory cues which influence mating preference. The beta 2-m protein has also been identified as the smaller component of the Fc receptor in neonatal intestinal cells, and it has been suggested that the protein induces collagenase in fibroblasts. Cells lacking beta 2-m are deficient in the expression of MHC class I molecules, indicating that the association with beta 2-m is crucial for the transport of MHC class I molecules to the cell surface. The most direct means of unravelling the many biological functions of beta 2-m is to create a mutant mouse with a defective beta 2-m gene. We have now used the technique of homologous recombination to disrupt the beta 2-m gene. We report here that introduction of a targeting vector into embryonic stem cells resulted in beta 2-m gene disruption with high frequency. Chimaeric mice derived from blastocysts injected with mutant embryonic stem cell clones transmit the mutant allele to their offspring.