Expression of functional Anopheles merus alpha-amylase in the baculovirus/Spodoptera frugiperda system.

The Anopheles merus (Diptera, Nematocera, Culicoidea) alpha-amylase gene (AmerAmy, GenBank Accession Number U01210) was amplified with its own or with the Zabrotes subfasciatusalpha-amylase signal peptide (ZsAmerAmy, GenBank Accession Number AY270183) by PCR, using designed primers. The AmerAmy gene was sequenced from its promotor to the TGA codon. As a positive control, the Z. subfasciatusalpha-amylase gene with its own signal peptide (ZsAmy, GenBank Accession Number AF255722) was also amplified by PCR. These three sequences were inserted into the baculovirus genome using the Bac-to-Bac trade mark system. Recombinant baculovirus preparations were used to infect Sf9 Spodoptera frugiperda insect cells. The A. merusalpha-amylase was successfully expressed as an active enzyme detected mainly in cell culture supernatants.

Cell-specific transcriptional regulation of follicle-stimulating hormone-beta by activin and gonadotropin-releasing hormone in the LbetaT2 pituitary gonadotrope cell model.

FSH is secreted by gonadotropes of the anterior pituitary and plays a crucial role in mammalian reproduction. However, little is known about FSH gene regulation due to the lack of a gonadotrope cell line that synthesizes FSH. The LbetaT2 mouse pituitary cell line, isolated by targeted tumorigenesis in transgenic mice, has the characteristics of a mature gonadotrope, including expression of GnRH receptor, steroidogenic factor 1, and both the alpha- and beta-subunits of LH, but was thought not to express FSH. Using RT-PCR, we show that these cells synthesize FSH beta- subunit messenger RNA, which is induced by activin and inhibited by follistatin. Furthermore, in transient transfections an ovine FSHbeta 5'-regulatory region (5.5 kb) confers LbetaT2 cell-specific expression to a reporter gene compared with other pituitary and nonpituitary cell lines. This FSHbeta regulatory region responds to activin specifically in LbetaT2 cells, an effect that is blocked by follistatin. The LHbeta, alpha-subunit, and GnRH receptor regulatory regions are induced by activin and blocked by follistatin. Furthermore, LbetaT2 cells express the components of the activin system, and addition of follistatin alone reduces FSHbeta gene expression, demonstrating that an endogenous activin autocrine loop regulates FSH in these cells. In addition, GnRH stimulates both the FSHbeta and LHbeta regulatory regions, specifically in LbetaT2 cells. Surprisingly, GnRH induction is reduced by follistatin, suggesting its dependence on endogenous activin. As the mouse GnRH receptor promoter is inhibited by follistatin, reduction of GnRH receptor levels might be one mechanism by which follistatin interferes with GnRH induction of gonadotropin genes. In summary, LbetaT2 cells exhibit the characteristics of fully differentiated gonadotropes, including the expression of LH, FSH, GnRH receptor, and components of the activin/follistatin system, as well as display the appropriate responses to activin and GNRH:

Impaired adrenocorticotropin-adrenal axis in combined pituitary hormone deficiency caused by a two-base pair deletion (301-302delAG) in the prophet of Pit-1 gene.

The Prophet of Pit-1 gene (PROP1) encodes a paired-like homeodomain protein, which is expressed early in pituitary gland development. When mutated, it is responsible for combined pituitary hormone deficiency (CPHD) in humans, as well as in Ames dwarf mice (df/df). Several independent mutations in the homeodomain of PROP1 have been identified as causative for the human CPHD phenotype, which has been characterized, thus far, as absence or low levels of GH, PRL, TSH, LH, and FSH. Here, we report 10 CPHD cases, 9 of which were born to consanguineous marriages occurring in a large family living in an isolated area in the Southeast of Brazil. All affected patients present complete absence of puberty and low GH, PRL, TSH, LH, and FSH associated with severe hypoplasia of the pituitary gland, as seen by MRI. All 3 exons of the PROP1 genes of these patients were sequenced. The 301-302delAG frameshift mutation was found in both alleles of each affected case. Surprisingly, we observed ACTH/cortisol insufficiency associated with the PROP1 phenotype. The patients' ages varied between 8 and 67 yr, and cortisol response impairment was identified in 5 of 6 of the older patients and in an 11-yr-old patient. Previous studies have not fully characterized patients at advanced ages, leading us to conclude that the phenotype of this PROP1 mutation includes late-onset adrenal insufficiency. We present an extensive clinical analysis of all of these patients. The presence of ACTH/cortisol deficiency in this family bearing the PROP1 301-302delAG mutation indicates the importance of a complete endocrine characterization and of life-long monitoring of PROP1 patients.

Pro239Ser: a novel recessive mutation of the Pit-1 gene in seven Middle Eastern children with growth hormone, prolactin, and thyrotropin deficiency.

Pit-1, a member of the POU-homeo domain protein family, is one of the transcription factors responsible for anterior pituitary development and pituitary-specific gene expression. Here, we describe seven children with GH, PRL, and TSH deficiency from three, reportedly unrelated, Middle Eastern families, harboring a newly recognized Pro- > Ser recessive mutation in codon 239 of the Pit-1 gene. The mutated residue is located at the beginning of the second alpha-helix of the POU-homeodomain and is strictly conserved among all POU proteins. The Pro239Ser mutant binds DNA normally but is unable to stimulate transcription.

Thyroid hormone inhibits the human prolactin gene promoter by interfering with activating protein-1 and estrogen stimulations.

Transcription of the human PRL (hPRL) gene in the pituitary is subject to tissue-specific and multihormonal regulation involving two main regulatory regions, a proximal promoter and a distal enhancer. In this report we show that thyroid hormone inhibits the expression of the hPRL gene in rat pituitary cells. Transient expression experiments show that thyroid hormone regulation involves a strong inhibitory element, located in the proximal (-164/-35) promoter, which is modulated by a more distal stimulatory response control region. Gel retardation experiments reveal that the thyroid hormone receptor does not bind to the proximal negative element. We show the existence of an activating protein-1 (AP-1) response element located at positions -61 to -54 of the proximal promoter, conferring AP-1 stimulation to the hPRL promoter. This AP-1 induction is abolished when hormone-bound thyroid hormone receptor is present, indicating that there is an interference between the thyroid hormone receptor and AP-1 regulatory pathways. Furthermore, using the complete hPRL upstream region, we show that estrogen induction is abolished by simultaneous thyroid hormone treatment.

The prenatal role of thyroid hormone evidenced by fetomaternal Pit-1 deficiency.

The role of thyroid hormone in the human fetus is uncertain; a significant amount of T4 is transferred from the maternal to the fetal circulation. A mother-infant pair was found to be heterozygotic for a point mutation in codon 271 of the gene encoding Pit-1, a pituitary-specific transcription factor regulating somatotrope, lactotrope, and thyrotrope function. At birth, serum T4 was undetectable in mother and infant. The newborn presented with a striking delay of respiratory, cardiovascular, neurological, and bone maturation. Despite replacement therapy since birth, neurological development of the infant is impaired. Fetomaternal Pit-1 deficiency resulted in unmitigated fetal hypothyroidism that unmasked thyroid hormone as a potent endogenous drive of fetal maturation and revealed placental transfer of maternal T4 as a rescue mechanism for infants with congenital hypothyroidism, preventing fetal and neonatal symptoms of thyroid deficiency and safeguarding developmental potential.