Eva Szonyi

A prolactin-inhibiting factor within the precursor for human gonadotropin-releasing hormone

The cloned complementary DNA sequence encoding the human gonadotropin-releasing hormone (GnRH) precursor protein was used to construct an expression vector for the bacterial synthesis of the 56-amino acid GnRH-associated peptide (GAP). GAP was found to be a potent inhibitor of prolactin secretion and to stimulate the release of gonadotropins in rat pituitary cell cultures. Active immunization with peptides corresponding to GAP sequences led to greatly increased prolactin secretion in rabbits.

Heregulins and the ErbB-2/3/4 receptors in gliomas

The activation of autocrine loops involving proto-oncogene relatedreceptor tyrosine kinases has led to the analysisof a large number of growth factor systemsin human glioma specimens and cell lines. TheErbB-2 system, also called HER-2 or neu, isanalogous to the epidermal growth factor receptor system(EGF-R, ErbB-1). Neuregulins consist of a large familyof proteins arising from alternative mRNA splicing ofa single gene located at 8p22-p11. Activation ofErbB-2 by neuregulins occurs in heterodimeric complexes withErbB-3 and ErbB-4. A panel of human gliomacell lines, which had previously been analyzed forErbB-2 expression, was examined for ErbB-3 and ErbB-4expression. Coordinate expression of ErbB-2, -3 or -4was not observed in these cell lines. Despitethe presence of a complete system capable ofsignaling in about half the cell lines, noconstitutive activation of ErbB-2, -3 or -4 wasobserved, and autophosphorylation of ErbB-2 in response toheregulin was observed only in one cell linefrom the panel, NCE-G84. Moreover, the addition ofrecombinant heregulin or antibodies capable of disrupting ErbB-2/ErbB-3complexes had no effect on cell proliferation. Weconclude that the role of neuregulins and itsreceptors in the control of glioma cell proliferationmay be limited or may be context dependenton in situ conditions which are lost invitro. Alternatively, neuregulins may be involved in celldifferentiation or survival in the central nervous system.Data supporting these conclusions are described in moredetail herein.

Activity and Biospecificity of Proteolyzed Forms and Dimeric Combinations of Recombinant Human and Murine Nerve Growth Factor

Abstract: Purified recombinant human nerve growth factor (rhNGF) and submaxillary gland‐derived murine NGF (muNGF) were characterized by amino acid composition, polyacrylamide gel electrophoresis (PAGE), reversed‐phase HPLC (RP‐HPLC), and high‐performance ion‐exchange chromatography (HPIEC). Limited tryptic digest of the N and C termini of the 120‐residue form of rhNGF produced a species of 109 residues (10–118). The previously observed natural murine analogue of this variant, muNGF lacking the first eight N‐terminal amino acids, was also isolated as a homodimer. Both species were purified using HPIEC and characterized by amino acid analysis, N‐terminal sequence, PAGE, and RP‐HPLC analysis. Each of the four homodimeric species was evaluated in some or all of the following biological assays for NGF: chick dorsal root and sympathetic ganglion assays and rat pheochromocytoma‐12 cell line neurite extension assay. The 118‐residue homodimeric versions of both rhNGF and muNGF displayed equivalent bioactivity, whereas the N terminal‐modified molecules presented activity reduced by 50‐ to 100‐fold. Utilizing HPIEC, we have examined the ability of the monomeric forms of any two of the homogeneous dimeric species of rhNGF to recombine. We have shown that not only can all of the previously described species form dimers by recombination, but an interspecies dimer can be created between muNGF and rhNGF.

Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone

The secretion of follicle-stimulating hormone (FSH) by pituitary cells is stimulated by activin and gonadotropin-releasing hormone, GnRH. To examine the possible interrelationships between the intracellular actions of these secretagogues, responsiveness to activin was tested following pretreatment with 0, 0.1, or 10 nM GnRH. In cells pretreated with 0 or 0.1 nM GnRH, FSH secretion was increased approximately 2-fold during a subsequent challenge with either activin or GnRH. In contrast, in cells pretreated with 10 nM GnRH, FSH secretion became unresponsive to GnRH but could still be stimulated 2-fold by activin. These results demonstrate that activin is able to stimulate FSH secretion in cells that have undergone desensitization to GnRH.

Gonadal development and gametogenesis in the hypogonadal mouse are restored by gene transfer.

These results describe controlled regulation of a mammalian neural gene in transgenic mice. Analysis of truncated GnRH-GAP genes in transgenic mice will enable us to define the DNA sequences responsible for this control. Furthermore, by separate mutation of the GnRH and GAP coding sequences we will be able to determine the relative importance of these two peptides in the development and maintenance of reproductive function.

The Hypogonadal Mouse: Reproductive Functions Restored by Gene Therapy

The hypogonadal (hpg) mouse lacks a complete gonadotropin-releasing hormone (GnRH) gene and consequently cannot reproduce. Introduction of an intact GnRH gene into the genome of these mutant mice resulted in complete reversal of the hypogonadal phenotype. Transgenic hpg/hpg homozygotes of both sexes were capable of mating and producing offspring. Pituitary and serum concentrations of luteinizing hormone, follicle-stimulating hormone, and prolactin were restored to those of normal animals. Immunocytochemistry and in situ hybridization showed that GnRH expression was restored in the appropriate hypothalamic neurons of the transgenic hpg animals, an indication of neural-specific expression of the introduced gene.