Call number: LD2668 .T4 1949 C71 / Master of Science

Call number: LD2668 .T4 1956 S74 / Master of Science

Call number: LD2668 .T4 1958 P35

Avian intestinal cytosoluble receptors for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) were subjected to limited trypsin digestion, endogenous proteolytic action, as well as carboxypeptidase treatment, and the physical and functional properties of the resulting discrete polypeptide fragments were identified and contrasted with the native 1,25(OH)₂D₃ receptor. Resultant fragments were followed by tracing either radioactive 1,25(OH)₂D₃ or by probing with anti-receptor monoclonal antibodies. Two differentially trypsin-sensitive effects on the 1,25(OH)₂D₃ receptor were noted when fragments were detected by their ability to bind 1,25(OH)₂[³H]D₃. Two hormone-bound fragments of 40 and 30 kDa were formed; neither bound to DNA-cellulose nor anti-receptor monoclonal antibodies. Immunoblot technology was used to show the disappearance of the 60 kDa receptor with increasing trypsin concentrations, paralleling the appearance of an immunoreactive 20 kDa fragment. The 20 kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60 kDa receptor. This fragment is likely the complementary fragment to the hormone-bound fragment of 40 kDa that is described above. In contrast to the exogeneous effect of trypsin, incubation of chick intestinal cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. This species retained the hormone-binding site and the antibody determinant, but was devoid of DNA-binding activity. Moreover, it did not generate the trypsin-dependent 20 kDa fragment and therefore was derived from the opposite end of the receptor molecule. Carboxypeptidase treatment of the 1,25(OH)₂D₃ receptor produces a 56 kDa fragment which does not retain hormone, but which does bind to DNA-cellulose and monoclonal antibody. These combined data from various limited enzymatic cleavage studies of the receptor have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the N-terminal DNA-binding domain and the C-terminal hormone-binding domain. This map for the 1,25(OH)₂D₃ receptor protein is consistent with the general structure of steroid and thyroid hormone receptors and places the vitamin D hormone receptor in a class of macromolecules that are postulated to bind enhancer regions of responsive DNA and thereby control target gene transcription.

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Doctor of Medicine

published_or_final_version / Zoology / Master / Master of Philosophy

The active form of vitamin D is the steroid hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Central to the mechanism of action of 1,25(OH)₂D₃ is its specific, high affinity intracellular receptor. This research focused on the participation of this receptor in the biology, biochemistry, and molecular biology of the vitamin D regulatory system. The effects of 1,25(OH)₂D₃ on the differentiation of hematopoietic cells were investigated using the cultured human promyelocytic leukemia cell line, HL-60, as a model. It was observed that 1,25(OH)₂D₃ induced macrophage differentiation in HL-60 cells and that a direct biochemical correlation existed between 1,25(OH)₂D₃ receptor saturation and a 1,25(OH)₂D₃-stimulated bioresponse. These data implicate 1,25(OH)₂D₃ as a natural cell differentiating agent and the 1,25(OH)₂D₃ receptor as the mediator of this hormone's action. Since the most fundamental level of control occurs by the regulation of gene expression, studies were undertaken to define the transcriptional control by 1,25(OH)₂D₃ over a known vitamin D-regulated endpoint protein. This work resulted in the molecular cloning of cDNAs to two avian intestinal calcium binding proteins, vitamin D-dependent calcium binding protein and a novel calmodulin-like protein. To gain further insight into the role of the 1,25(OH)₂D₃ receptor as a transcriptional regulator, avian and mammalian 1,25(OH)₂D₃ receptor mRNAs were characterized extensively by the techniques of in vitro translation and immunoprecipitation. These mRNAs were then utilized to construct cDNA libraries from which avian and human intestinal 1,25(OH)₂D₃ receptor cDNAs were isolated and their identity verified by hybrid-selected translation, sequencing, and Northern analysis. It was concluded that demonstrated 1,25(OH)₂D₃ receptors are polypeptides of 52-60 kDa whose activity is regulated by 1,25(OH)₂D₃ at both an mRNA and posttranslational level. Furthermore, the deduced amino acid sequence of receptor mRNA included a highly conserved cysteine, lysine, and arginine rich region that is homologous to other steroid receptors and the oncogene product v- erbA. Thus, the vitamin D receptor to be a specific trans -acting factor, modulating the pleiotropic effects of vitamin D including calcium homeostasis, and cellular differentiation.

The preparation of 11 new oxytocin analogs is described. The synthesis of the protected peptides were performed using solid phase peptides synthetic methodology. The protected peptides were deprotected and cyclized using sodium in liquid ammonia followed by aqueous potassium ferricyanide treatment. The purification of each peptide was accomplished using partition chromatography followed by gel filtration. Final purity was checked using high-performance liquid chromatography. Several amino-acid derivatives were prepared and incorporated as racemates into the synthetic peptides. The synthetic diastereomeric peptides were separated and purified by high pressure liquid chromatography using aqueous trifluoroacetic acid:acetonitrile mixtures. The oxytocin analogs prepared in this dissertation were divided into 2 classes: oxytocin agonists and oxytocin antagonists. The oxytocin agonist analogs prepared are [2-cycloleucine]oxytocin and [8-cycloleucine]oxytocin. The oxytocin antagonists described in this dissertation are [Pen¹,Cle²]oxytocin, [Pen¹,Cle⁸]oxytocin, [Pen¹,L-TyrMe²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-TyrEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,D-TyrEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-PheMe²,Thr⁴,Orn⁸]oxytocin, [Pen¹,L-PheEt²,Thr⁴,Orn⁸]oxytocin, [Pen¹,D-PheMe²,Thr⁴,Orn⁸]oxytocin and [Pen¹,D-PheEt²,Thr⁴,Orn⁸]oxytocin. A conformational study of the synthetic peptides was also undertaken in order to determine possible solution conformations for the various peptides. Two biophysical methods were used in the conformational study of these peptides; they include nuclear magnetic resonance spectroscopy (H-1 and C-13) and circular dichroism spectroscopy. Two somewhat different solution conformations were discovered for peptides containing all L-amino acids and for peptides containing a D-amino acid residue in position 2. A possible correlation between biological potency and observed solution conformation is suggested; the proposed models may aid in the design of more potent peptide inhibitor analogs.