Alternately binding probe competitive PCR as a simple, cost-effective, and accurate quantification method for JAK2V617F allele burden in myeloproliferative neoplasms.

We developed a simple, cost-effective, and accurate JAK2 allele burden quantification method named alternately binding probe competitive PCR (ABC-PCR). ABC-PCR can be performed to quantify target JAK2 allele burdens in a single reaction. The throughput and running cost of ABC-PCR are markedly improved compared with those of allele-specific quantitative PCR (AS-qPCR). The quantification of samples with known JAK2 allele burdens revealed that ABC-PCR had a small assay-to-assay variation. The JAK2 allele burdens in the patients with myeloproliferative neoplasms measured by ABC-PCR and AS-qPCR showed a good fitting. ABC-PCR would be a powerful tool for quantifying target JAK2 allele burdens.

Structures and diverse functions of frog growth hormone-releasing peptide (fGRP) and its related peptides (fGRP-RPs): a review.

A new Arg-Phe-NH(2) (RFamide) peptide has been discovered in the amphibian hypothalamus. The cell bodies and terminals containing this peptide were localized in the suprachiasmatic nucleus and median eminence, respectively. This peptide was further revealed to have a considerable growth hormone (GH)-releasing activity in vitro and in vivo and hence designated as frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the fGRP precursor polypeptide revealed that it encodes fGRP and its putative gene-related peptides (fGRP-RP-1, -RP-2, and -RP-3). Subsequently, we identified these putative fGRP-RPs as mature peptides and analyzed their hypophysiotropic activities. Only fGRP-RP-2 stimulated the release of GH and prolactin (PRL) in vitro and in vivo. Thus, in addition to fGRP, fGRP-RP-2 acts as a hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

Identification of immunoreactive plasma and stomach ghrelin, and expression of stomach ghrelin mRNA in the bullfrog, Rana catesbeiana.

In this study, we established a radioimmunoassay (RIA) specific for ghrelin from the bullfrog Rana catesbeiana using a novel antibody raised against the C-terminal amino acid sequence of bullfrog ghrelin [13-28]. We also examined the distribution of ghrelin-producing cells in the stomachs of bullfrogs using this antibody and a cRNA probe specific for the bullfrog ghrelin gene. Ghrelin levels in plasma and stomach extracts were approximately 150 fmol/ml and 83-135 fmol/mg wet tissue, respectively. Reverse-phase high performance liquid chromatographic analysis, combined with bullfrog ghrelin RIA, revealed that ghrelin immunoreactivity in the stomach was composed of non-acylated ghrelin (des-acyl ghrelin) and several acylated forms of ghrelin bearing different fatty acid modifications, which could induce increases in intracellular Ca2+ in cells expressing the rat GH secretagogue receptor. In the stomach, the major storage form was acylated ghrelin. In bullfrog plasma, however, the majority of ghrelin immunoreactivity was des-acyl ghrelin and C-terminal fragments of frog ghrelin. Acylated ghrelin forms comprised only minor peaks. Ghrelin-immunopositive and ghrelin mRNA-expressing cells were observed within the mucosal layer of the stomach. Following starvation, significant increases in plasma ghrelin levels and stomach ghrelin mRNA levels were observed as early as 10 days after starvation. These results indicate that ghrelin is present in the stomach and plasma of the bullfrog, which can be detected with our novel antibody. Interestingly, the primary storage form of ghrelin in the stomach differed from the circulating form dominating in the plasma. Furthermore, increases in ghrelin levels in plasma and mRNA levels in the stomach after starvation suggest the possible involvement of ghrelin in energy homeostasis in the bullfrog.

Development of radioimmunoassay for bullfrog thyroid-stimulating hormone (TSH): effects of hypothalamic releasing hormones on the release of TSH from the pituitary in vitro.

A bullfrog (Rana catesbeiana) thyroid-stimulating hormone (TSH) beta-subunit (TSHbeta) antiserum was produced by employing a C-terminal peptide synthesized on the basis of the amino acid sequence deduced from bullfrog TSHbeta cDNA. Immunohistochemical studies revealed that the bullfrog adenohypophyseal cells that immunologically reacted with the anti-bullfrog TSHbeta corresponded to those positively stained with an antiserum against human (h) TSHbeta. The antiserum was used for the development of a specific and sensitive radioimmunoassay (RIA) for the measurement of bullfrog TSH. The sensitivity of the RIA was 0.75+/-0.07ng TSH/100microl assay buffer. The interassay and intraassay coefficients of variation were 7.6 and 5.3%, respectively. Several dilutions of pituitary homogenates of larval and adult bullfrogs, or medium in which bullfrog pituitary cells were cultured, yielded dose-response curves that were parallel to the standard curve. Bullfrog prolactin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and alpha-subunit derived from glycoprotein hormones did not react in this assay. Immunoassayable TSH in the pituitary culture medium was confirmed to exist in the form of TSHbeta coupled with the alpha-subunit by an immunoprecipitation experiment using the TSHbeta antiserum and an alpha-subunit antiserum. TSH released from pituitary cells into the medium was also confirmed to possess a considerable activity in stimulating the release of thyroxine from the thyroid glands of larval bullfrogs in vitro. The effects of hypothalamic hormones such as mammalian gonadotropin-releasing hormone (mGnRH), ovine corticotropin-releasing hormone (oCRH), and thyrotropin-releasing hormone (TRH) on the release of TSH by dispersed anterior pituitary cells of the bullfrog larvae and adults were also studied. CRH markedly stimulated the release of TSH from both adult and larval pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from the larval cells. This is the first report on the development of an RIA for amphibian TSH, which has provided the direct evidence that the release of TSH from the amphibian pituitary is enhanced by the hypothalamic releasing hormones such as CRH, TRH, and GnRH.

Novel neuropeptides related to frog growth hormone-releasing peptide: isolation, sequence, and functional analysis.

We previously identified in the bullfrog a novel hypothalamic RFamide peptide (SLKPAANLPLRF-NH(2)) that stimulated GH release in vitro and in vivo and therefore was designated frog GH-releasing peptide (fGRP). Molecular cloning of cDNA encoding the deduced fGRP precursor polypeptide further revealed that it encodes fGRP and its related peptides (fGRP-RP-1, -RP-2, and -RP-3). In this study immunoaffinity purification using the antibody against fGRP was therefore conducted to determine whether these three putative fGRP-RPs exist as mature endogenous ligands in the frog brain. The mass peaks of the isolated immunoreactive substances were detected at 535.78, 1034.14, and 1079.71 m/z ([M+2H](2+)), and their sequences, SIPNLPQRF-NH(2), YLSGKTKVQSMANLPQRF-NH(2), and AQYTNHFVHSLDTLPLRF-NH(2), were revealed by the fragmentation, showing mature forms encoded in the cDNA sequences of fGRP-RP-1, -RP-2, and -RP-3, respectively. All of these fGRP-RPs contained a C-terminal -LPXRF-NH(2) (X = L or Q) sequence, such as fGRP. This study further analyzed hypophysiotropic activities of the identified endogenous fGRP-RPs. Only fGRP-RP-2 stimulated, in a dose-related way, the release of PRL from cultured frog pituitary cells; its threshold concentration ranged from less than 10(-7) M. A similar stimulatory action of fGRP-RP-2 on GH release was evident. It was ascertained that fGRP-RP-2 was also effective in elevating the circulating GH and PRL levels when administered systemically. In contrast, fGRP-RPs did not have any appreciable effect on the release of gonadotropins. Thus, fGRP-RP-2 may act as a novel hypothalamic factor on the frog pituitary to stimulate the release of GH and PRL.

A novel amphibian hypothalamic neuropeptide: isolation, localization, and biological activity.

Neuropeptides similar to the molluscan cardioexcitatory Phe-Met-Arg-Phe-NH2 have been identified in several vertebrates and characterized by the RFa motif at their C terminus (RFa peptides). In this study, we sought to identify an amphibian hypothalamic RFa peptide that may regulate secretion of hormones by the anterior pituitary gland. An acid extract of bullfrog hypothalami was passed through C-18 reversed-phase cartridges, and then the retained material was subjected to HPLC, initially using a C-18 reversed-phase column. RFa immunoreactivity was measured in the eluted fractions by a dot immunoblot assay employing an antiserum raised against RFa. Immunoreactive fractions were subjected to further cation exchange and reversed-phase HPLC purification. The isolated peptide was a novel RFa peptide and shown to have the sequence Ser-Leu-Lys-Pro-Ala-Ala-Asn-Leu-Pro-Leu-Arg-Phe-NH2. The cell bodies and terminals containing this peptide were localized immunohistochemically in the suprachiasmatic nucleus and median eminence, respectively. This RFa peptide stimulated, in a dose-related way, the release of GH from cultured pituitary cells, its threshold concentration ranging between 10(-9) and 10(-8) M. This peptide did not have any appreciable effect on the secretion of PRL and gonadotropins. It was ascertained that the peptide was also effective in elevating the circulating GH level when administered systemically. Thus, the amphibian hypothalamus was revealed to contain a novel functional RFa peptide that stimulates GH release. This peptide was designated frog GH-releasing peptide.