Diss. (sammanfattning) Göteborg : Univ. , 2007. / Härtill 4 uppsatser.

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 70-85).

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 56-69).

Growth hormone-releasing hormone (GHRH) is a neuropeptide which stimulates the synthesis and release of growth hormone (GH) from the pituitary gland. The primary structure of this peptide has been identified in 7 mammalian species while the gene has been isolated from only rat and human. GHRH is a member of the glucagon superfamily which includes vasoactive intestinal peptide (VIP), glucagon, secretin, peptide histidine methionine (PHM), gastric inhibitory peptide (GIP) and a recently identified peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The evolutionary relationships of this superfamily are not well understood because the gene structure of these molecules has only been identified in mammals. This thesis presents immunological evidence of a GHRH-like molecule, and identifies a GHRH/PACAP precursor and gene that encode two peptides, a GHRH-like molecule structurally related to PACAP-related peptide (PRP) and PACAP, from sockeye salmon brain. An antiserum directed against a topologically assembled epitope of human GHRH 1-44 (NH2) was produced and used to develop a radioimmunoassay for detection of immunoreactive GHRH in brain extracts of salmon, guinea pig, mouse and alligator. An immunoreactive GHRH from salmon brain extracts with a retention time on reverse phase high-performance liquid chromatography (HPLC) distinct from human GHRH was present. In alligator, the same antiserum also detected a GHRH-like molecule. During attempts to purify alligator GHRH, alligator brain neuropeptide Y (NPY) was identified. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The sequence identity between alligator and human NPY suggests that this sequence is the same as the ancestral amniote NPY. Molecular biological techniques were used for the structural identification of the salmon GHRH-like molecule and another peptide. The salmon GHRH/PACAP precursor contains 173 amino acids and has dibasic and monobasic processing sites for cleavage of a 45 amino acid GHRH-like peptide with a free acid C-terminus and a 38 amino acid PACAP with an amidated C-terminus. The salmon GHRH-like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP-related peptide (PRP). Salmon PACAP-38 is highly conserved (89%) with only 4 amino acid substitutions compared with the human, ovine and rat PACAP-38 peptides. Nucleotide sequencing and use of the polymerase chain reaction show the exon/intron organization of the salmon GHRH/PACAP gene to be similar to the human PACAP gene. Unlike the mammalian PACAP genes, the salmon gene produces two precursor forms by post-transcriptional processing. One form is similar to the mammalian PACAP precursors, while the second form is shorter due to the excision of exon 4. This deletion results in the loss of the first 32 amino acids of the GHRH-like peptide from the precursor. The high sequence identity and structural organization between the GHRH(PRP)/PACAP and PHM(PHI)/VIP genes suggest a duplication event occurred in an ancestral gene after the divergence from the other glucagon superfamily members. GHRH in mammals may have arisen by gene duplication after the divergence of the tetrapods from the other vertebrate lines. Thus, GH in fish may be controlled by the two molecules, GHRH-like peptide and PACAP, located on a single GHRH/PACAP gene. / Graduate

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13362号 / 論医博第2204号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 稲垣 暢也, 教授 渡邉 大, 教授 影山 龍一郎 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abnormal regulation of growth hormone (GH) secretion has been reported in some patients with insulin-dependent diabetes (IDD). We compared the GH responses in 32 healthy subjects (age 25 ± 2 SE years) and in 23 IDD patients (28 ± 1.9 years old, diabetes duration 10.4 ± 2 years, and glycohemoglobin levels 9.3 ± 2.0%). During acute, severe hypoglycemia (glucose < 40 mg/dl), the mean GH levels were similar. When prolonged mild hypoglycemia was induced (58.0 ± 2.0 mg/dl in the controls and 54.0 ± 2.0 mg/dl in the IDD patients), the mean GH levels were similar, although the increase in GH was delayed in the latter group. During brief (30 min) exercise at 40-50% of VO2 max, GH rose comparably in both groups (IDD patients maintained euglycemia with basal insulin infusion). However, with more prolonged and intense exercise using a glucose clamp to maintain euglycemia, GH rose to 5.4 ± 2.2 ng/ml in controls and 26.4 ± 12.6 ng/ml in the diabetics (P < 0.05). When the combination of intense exercise and hypoglycemia (~ 55 mg/dl) was used, GH rose to a peak of 21.7 ± 2.7 ng/ml in the controls and to 33 ± 3.0 ng/ml in the diabetics (P = NS). Our data show that in insulin-infused IDD patients made euglycemic for these experiments: a) The GH response to acute, severe hypoglycemia was identical to that in the controls and the response to mild, prolonged hypoglycemia was delayed, but of similar magnitude compared with controls; b) Exercise-induced GH responses were observed in both groups, but exaggerated in the diabetics at a higher exercise intensity; c) Hypoglycemia during exercise produced an additive effect on GH secretion in the controls but not in the IDD patients. We conclude that the wide range of abnormal GH secretory responses in type I diabetes reflects a central, possibly hypothalamic, defect in GH regulation.

No description available.

Growth Hormone (GH), also known as somatotropin, is a protein hormone secreted from the anterior pituitary gland. GH action results in longitudinal bone growth in children and adolescents and continues later to control a variety of metabolic reactions in adults. DNA polymorphisms leading to a decrease in GH expression result in persons of short stature, whereas those leading to overexpression of GH produce either acromegaly or gigantism. In order to diagnose and treat patients with altered GH production, understanding the control of GH expression is thus clinically relevant. In order to understand how transcription of GH is determined by selective regulatory modulators, two rat somatotroph cell lines were studied that differed in their relative differentiation: MtT/S cells are considered as fully differentiated somatotrophs that express GH exclusively; MtT/Se cells are related somatotrophs with GH expression that is responsive to estrogen treatment. These cell lines were obtained from the Riken Cell Bank (Japan) and then grown and cultured in accordance with established protocols. After harvesting of cells from each line, total RNA was extracted using a rapid affinity method (Qiagen). After validation of the RNA integrity index of each RNA isolate (Affymetrix), they were sent to an off-site laboratory (NovoGene) for RNA-Seq analysis. Samples were examined in triplicate for comparison using standard procedures for adapter ligation, library construction, amplification and sequencing. In our previous work with single gene quantitative RT-PCR, we measured expression of 9 separate targets, primarily transcriptional controllers, in these cell lines. Now, the use of RNA-Seq quantifies the levels of all mRNAs in each sample without the need for specific primers or probes. Therefore, it is possible to find nearly all of the mRNAs that demonstrate changing abundance without requiring prior evidence of their role in the terminal differentiation of the MtT/S from MtT/Se cells. After extensive QC for validation, initial analysis of the data shows more than 36 million validated sequenced reads from each replicate sample with >96.39% mapping to the reference rat genome sequence. Basic analysis shows that 13,012 expressed genes were 87% similar, with 1,175 (9.0%) unique to MtT/Se cells and 484 (3.7%) genes selectively present in MtT/S cells. Of these, 329 genes were upregulated in expression while only 57 were reduced. For comparison with our previous study, we are now confirming differences in GH transcripts and the transcription factors/regulators previously measured, particularly the GH regulator Zn16, a protein with 16 zinc fingers known to bind to the GH promoter DNA. Further analysis of the RNA-Seq profile is focused on the unique and unidentified changes in expression that differentiate these cell lines. Analysis of the differentiation of MtT/S and MtT/Se cells will further understanding of GH regulation in the body.

No description available.

No description available.