Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.

A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein. Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.

A tumor-associated fibronectin isoform generated by alternative splicing of messenger RNA precursors.

Fibronectin (FN) represents the mixture of a number of structurally different molecules (isoforms) whose make-up varies depending on the FN sources. FN from cultured transformed human cells has a very different isoform composition with respect to its normal counterpart. In fact, SV-40-transformed WI-38VAI3 human fibroblasts produce high levels of a FN isoform (B-FN) which is very poorly expressed in their normal, WI-38, counterpart. We have recently demonstrated that the B-FN isoform derives from a differential splicing pattern of the FN primary transcript which leads, in transformed cells, to a high level expression of the exon ED-B (Zardi, L., B. Carnemolla, A. Siri, T. E. Petersen, G. Paolella, G. Sebastio, and F. E. Baralle. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:2337-2342). Here we report on the production and characterization of a monoclonal antibody (BC-1) which recognizes an epitope within the protein sequence coded for by the ED-B exon. This monoclonal antibody makes it possible to carry out immunohistochemical analysis of the distribution of the ED-B-containing FN isoform (B-FN) in human tissues. The results show that while in normal, adult, human tissues total FN has a widespread distribution, the B-FN isoform is restricted only to synovial cells, to some vessels and areas of the interstitium of the ovary, and to the myometrium. On the contrary, the B-FN isoform has a much greater expression in fetal and tumor tissues. These results demonstrate that, in vivo, different FN isoforms have a differential distribution and indicate that the B-FN isoform may play a role in ontogenesis and oncogenetic processes.

Relaxin: a disulfide homolog of insulin.

Relaxin, a peptide hormone responsible for the widening of the birth canal in mammals, has been purified from the ovaries of pregnant hogs. The amino acid sequences of its constituent A and B chains were determined, and the positions of the disulfide cross-links were established. Relaxin was shown to be identical to insulin with respect to its disulfide bond distribution, but significant homology was lacking in other positions. These findings suggest that relaxin and insulin were derived from a common ancestral gene. Since the intrauterine mode of propagation is synonymous with the development of mammals, the genetic distance between insulin and relaxin should therefore permit an estimate of the earliest possible time of commitment of one evolutionary branch to the development of mammals. This event was estimated to have occurred about 5 X 10(8) years ago.

2-Deoxy-alpha-ecdysone from ovaries and eggs of the silkworm, Bombyx mori.

From ovaries dissected from developing and emerged adults of the silkworm, Bombyx mori, two substances having high molting hormone activity were isolated. One of these was identified as 2-deoxy-alpha-ecdysone by means of high-pressure liquid chromatography and mass spectrometry. Although this compound had previously been isolated from the fern Blechnum minus and postulated to be the precursor of alpha-ecdysone, it had not been obtained from insect material. The compound is also contained in the form of a conjugate in ovaries as well as in diapausing silkworm embryos.

5-methylcytidylic acid: absence from mitochondrial DNA of frogs and HeLa cells.

The content of 5-methylcytidylic acid in nuclear DNA and mito chondrial DNA of Xenopus laevis and HeLa cells has been determined. Both nuclear DNA's contain 5-methylcytidylic acid. The 5-methylcytidylic acid content of X. laevis DNA is 1.7 mole percent of total nucleotides, and that of HeLc cell DNA is 0.7 mole percent. In neither mitochondrial DNA could any 5-methylcytidylic acid be detected; the limit of sensitivity was judged at below 0.1 mole percent for X. laevis DNA and below 0.05 mole percent for HeLa cell DNA.

Starfish gonad: action and chemical identification of spawning inhibitor.

The gamete-shedding substance obtained from the radial nerves induces spawning when it is applied to the gonads of mature starfish in vivo and in vitro. A substance that inhibits the action of this spawning factor is present in both ovary and testis; it has been isolated from testis of Asterina pectinifera and chemically identified as L-glutamic acid.

Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family.

A complementary DNA (cDNA) for the rat luteal lutropin-choriogonadotropin receptor (LH-CG-R) was isolated with the use of a DNA probe generated in a polymerase chain reaction with oligonucleotide primers based on peptide sequences of purified receptor protein. As would be predicted from the cDNA sequence, the LH-CG-R consists of a 26-residue signal peptide, a 341-residue extracellular domain displaying an internal repeat structure characteristic of members of the leucine-rich glycoprotein (LRG) family, and a 333-residue region containing seven transmembrane segments. This membrane-spanning region displays sequence similarity with all members of the G protein-coupled receptor family. Hence, the LH-CG-R gene may have evolved by recombination of LRG and G protein-coupled receptor genes. Cells engineered to express LH-CG-R cDNA bind human choriogonadotropin with high affinity and show an increase in cyclic adenosine monophosphate when exposed to hormone. As revealed by RNA blot analysis and in situ hybridization, the 4.4-kilobase cognate messenger RNA is prominently localized in the rat ovary.

Catechol estrogens: presence in brain and endocrine tissues.

Catechol estrogens have been identified and measured in rat brain and various endocrine tissues with the use of a sensitive radioenzymatic assay. The specificity of this assay was confirmed by thin-layer chromatography and mass spectral analysis of the reaction products. The concentration of catechol estrogens in the hypothalamus and pituitary are at least ten times higher than reported previously for the parent estrogens. Catechol estrogens have potent endocrine effects and, because of their normal occurrence in the hypothalamic-pituitary axis, they have an important role in neuroendocrine regulation.

Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein.

The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.

Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain.

Complementary DNA clones, encoding the LH-hCG (luteinizing hormone-human choriogonadotropic hormone) receptor were isolated by screening a lambda gt11 library with monoclonal antibodies. The primary structure of the protein was deduced from the DNA sequence analysis; the protein contains 696 amino acids with a putative signal peptide of 27 amino acids. Hydropathy analysis suggests the existence of seven transmembrane domains that show homology with the corresponding regions of other G protein-coupled receptors. Three other types of clones corresponding to shorter proteins were observed, in which the putative transmembrane domain was absent. These probably arose through alternative splicing. RNA blot analysis showed similar patterns in testis and ovary with a major RNA of 4700 nucleotides and several minor species. The messenger RNA was expressed in COS-7 cells, yielding a protein that bound hCG with the same affinity as the testicular receptor.

DDT administered to neonatal rats induces persistent estrus syndrome.

The o,p'-isomer of the insecticide DDT when injected into neonatal female rats significantly advanced puberty, induced persistent vaginal estrus after a period of normal estrous cycles, and caused the ovaries to develop follicular cysts and a reduced number of corpora lutea. The uterotropic response to administered estradiol was reduced, and the female pattern of mating behavior was slightly disturbed. Residues of DDT in ovarian, brain, and adipose tissues of the adult animals were the same in both treated and control groups.

Immunoreactive apolipoprotein E is a widely distributed cellular protein. Immunohistochemical localization of apolipoprotein E in baboon tissues.

Apolipoprotein (apo)E is an important protein determinant in cholesterol homeostasis in man. The protein is synthesized by the liver as well as by a number of extrahepatic tissues. In the present study, immunohistochemical techniques were used to identify apoE in specific cells in various baboon organs. In the 11 tissues studied, the following cell types have been found to harbor apoE immunoreactivity: cerebral astrocytes; thyroid follicular cells; alveolar type II pneumocytes; hepatocytes, and Kupffer cells; adrenocortical cells in zona fasciculata and zona reticularis; adrenal medullary cells; some renal tubular epithelia; some pancreatic islet cells; histiocytic macrophages in lymph nodes and the spleen; some gastric mucosal epithelia; and ovarian oocytes. These observations indicate the wide distribution of apoE in many organs and suggest that the protein might perform other important functions such as regulation of local hormonal homeostasis in addition to its role in cholesterol metabolism.

Isolation of alpha 1-protease inhibitor from human normal and malignant ovarian tissue.

Proteolytic enzymes are associated with normal and neoplastic tissues. Therefore protease inhibitors might also be involved in the control of cell function. alpha 1-protease antigen and antitryptic activity have been found in normal and neoplastic human ovarian homogenate. The inhibitor has been localized to ovarian stromal cells or tumor cells by immunoperoxidase staining. The protein was purified to apparent homogeneity as judged by alkaline gel and sodium dodecyl sulfate (SDS) gel electrophoresis. Immunochemical studies revealed antigenic similarity of plasma alpha 1-protease inhibitor by double immunodiffusion and similar mobility on immunoelectrophoresis and two-dimensional electroimmunodiffusion. The molecular weight was similar to that described for plasma alpha 1-protease inhibitor: 60,000 by gel filtration and 53,500 by SDS electrophoresis. Furthermore, the phenotypic pattern as determined by acid starch gel electrophoresis and immunoprecipitation was PiMM, which is the predominant genetic variant in normal plasma alpha 1-protease inhibitor. An inhibitor ws isolated and purified from an ovarian carcinoma that exhibited functional, immunochemical, and physical similarity to the normal ovarian alpha 1-protease inhibitor. alpha 1-protease inhibitor from normal and malignant ovaries competitively inhibited bovine pancreatic trypsin at incubation times of 5 min at 30 degrees C. Inhibition constant (Ki) values were calculated at 0.67 and 0.51 inhibitory units, respectively. The alpha 1-protease inhibitor in malignant cells may be a factor in the control of proliferation in this tissue. Since ovulation is in part a proteolytic event, the alpha 1-protease inhibitor in ovarian cells may play a role in the control of this specialized tissue. Persistance of this protein in malignant ovarian tissue may be a vestige of its differentiated origin.

Tumor-associated glycoprotein (TAG-72) in ovarian carcinomas defined by monoclonal antibody B72.3.

Murine IgG1 monoclonal antibody (MoAb) B72.3, reactive with a high molecular weight glycoprotein complex [designated tumor-associated glycoprotein-72 (TAG-72)] was shown, with the use of the avidin-biotin-complex-immunoperoxidase technique and surgically resected tissues, to be reactive with a variety of histologic tumor types. TAG-72 is expressed in at least 5% (and up to 100%) of the malignant epithelial cells in 77% (n = 52) of human primary cancers and 71% (n = 31) of metastatic ovarian cancers of the common "epithelia" histologic category. Of these, several histologic types, including serous and mucinous cystadenocarcinomas, undifferentiated carcinomas, and less common types of ovarian carcinoma, were all shown to express TAG-72. In contrast, normal ovarian tissues and 26 of 27 benign ovarian tumors of various histologic types failed to express similar levels of TAG-72. Of interest is the 1 benign tumor that demonstrated unusual glandular complexity, as well as 3 tumors designated as borderline malignancy, that contained elevated TAG-72 expression. MoAb B72.3 also was used successfully to detect ovarian carcinoma cells in 28 cytologic preparations of human serous effusions and peritoneal washings. The reactivity of MoAb B72.3 was shown to be distinct from that of MoAb OC125 and an anti-CEA MoAb B1.1. The potential applications of MoAb B72.3 in the study of human ovarian cancer cell populations, as well as in several aspects of the management of human ovarian cancer, are discussed.

Immunological detection of Chinese hamster ovary cells expressing a multidrug resistance phenotype.

A monoclonal antibody (IgG1) has been prepared that specifically detects Chinese hamster ovary cells expressing a multidrug-resistant phenotype. This antibody recognizes the membrane P-glycoprotein (Mr 170,000) associated with drug resistance as determined by enzyme-linked immunosorbent assay with purified P-glycoprotein and by Western blot analysis of cell extracts from drug-resistant and drug-sensitive cells. By immunofluorescence methods, the antibody also reacts strongly with viable and ether:ethanol-fixed resistant cells but does not react with the parent drug-sensitive cell line. Thus, this antibody can bind with live cells allowing discrimination by immunohistochemistry between drug-resistant and drug-sensitive Chinese hamster ovary cells.

Radioimmunological determination of cellular retinol-binding protein in human tissue extracts.

A sensitive and specific radioimmunoassay was developed for human cellular retinol-binding protein (CRBP) using antibodies raised in rabbits against purified human liver CRBP. The assay could determine concentrations of CRBP in extracts down to 8 micrograms/liter. CRBP could be demonstrated in all tissues investigated except in serum, and the same immunoreactive CRBP seemed to be present in all tissues. The highest concentrations were found in the gonads. In the gastrointestinal tract, the small intestine had a higher concentration than did stomach or colon. Muscle and skin had low concentrations of CRBP.

Solubilization, gel filtration and sedimentation behaviour of prolactin receptors from human ovarian tissue.

Receptors for 125I-labelled human prolactin have been identified in the crude membrane fraction isolated from human ovarian tissue. The non-ionic detergent Triton X-100, has been used to solubilize the membrane fraction. The presence of the receptor in the detergent extract was demonstrated by gel filtration and sucrose density gradient centrifugation. The binding was time-temperature dependent, being maximal at 23 degrees C after 15 h of incubation. Large amounts of other peptide hormones did not inhibit the binding of 125I-labelled human prolactin. The binding Scatchard analysis demonstrated that the affinity of the soluble receptor (Ka 1.13 +/- 0.15 X 10(10) M-1) for the labelled hormone was slightly greater than that of the crude membrane fraction (Ka 0.91 +/- 0.12 X 10(10) M-1). The binding capacity of the solubilized receptor was also significantly greater than that seen in the particulate before solubilization. The apparent Stokes radius of the solubilized receptor was estimated to be 57 A and that the hormone-receptor complex 60 A. The sedimentation coefficient of the solubilized receptor was 7.0 +/- 0.1 s, whereas that of the hormone-receptor complex was 7.5 +/- 0.2 s.

The development of a radioimmunoassay for the detection of photoproducts in mammalian cell DNA.

Antiserum was prepared in rabbits against ultraviolet-irradiated DNA. Using a 125I-labeled protein A binding assay it was shown to be specific for ultraviolet-irradiated DNA, binding increasing as a function of logarithmic increase in dose. This antiserum was then used to develop a radioimmunoassay in which the competition between labeled UV-DNA and unlabeled sample DNA for antibody binding sites is monitored. Using this system the specificity of the assay could be changed depending on the nature of the labeled probe. The inability of poly(dA-dT) . poly-(dA-dT), as compared with poly(dA) . poly(dT), to act as a competitive inhibitor established that the primary lesion recognized by the antiserum is the thymine dimer. This antigenic response did, however, depend on the presence of at least one nucleotide adjacent to the dimer. The sensitivity of the assay was optimized by using 32P-labeled plasmid DNA as competitive probe and is capable of detecting photodamage in cellular DNA at doses as low as 2.5 J . m-2.

DNA binding protein from ovaries of the frog, Xenopus laevis which promotes concatenation of linear DNA.

A soluble extract of Xenopus laevis ovaries catalyzed ATP-dependent concatenation of linear duplex DNA molecules. DNA ligase and a unique X. laevis DNA binding protein were required for the formation of concatemers. A linear DNA concatenation system was reconstituted using T4 DNA ligase and homogeneous X. laevis DNA binding protein. This system catalyzed intermolecular ligation of DNA molecules into linear concatemers of up to ten or more times monomer length.

Characterization and regulation of testicular inhibin beta-subunit mRNA.

To understand the possible structures of testicular inhibin, we have isolated cDNAs coding for inhibin subunits from human testicular cDNA libraries. In this study we report that the nucleotide and predicted amino acid sequences for human testicular inhibin beta-B-subunit are similar to those of human ovary. In rat testis two species of beta-B-subunit mRNA [4.4 and 3.3 kilobases (kb)] appeared to be present in equal concentration, as opposed to rat ovary where a predominant band of 4.4 kb and a minor band of 3.3 kb were observed. One major species of beta-A-subunit mRNA (6.5 kb) was identified in both testis and ovary. The concentration of beta-A-subunit mRNA in the testis was very low, representing only 0.5% of that in rat ovary. The accumulation of beta-B-subunit mRNA peaked at 20 days of age and declined thereafter in a pattern similar to that of the alpha-subunit gene. Hypophysectomy caused a marked increase in the concentration as well as the total content of beta-B-subunit but no change in beta-A-subunit mRNA in rat testis. We have previously reported that FSH markedly increased alpha-subunit mRNA levels both in vivo and in vitro. By contrast, neither FSH nor testosterone has any significant effect on the accumulation of beta-A- or beta-B-subunit mRNAs in hypophysectomized animals or Sertoli cell primary cultures. We conclude that 1) the mRNAs for both beta-subunits are not regulated by FSH; and 2) hypophysectomy does not change and increases, respectively, the mRNAs for the beta-A- and beta-B-subunits. We conclude that the inhibin subunit mRNAs are differentially regulated in rat testis.