臺灣博碩士論文加值系統

成熟雌性哺乳動物其乳腺發育分為四個時期：處女期、懷孕期、泌乳期及離乳期。其中由腦垂腺所分泌之泌乳素 (prolactin, PRL) 於乳腺上皮細胞之生長、分化及泌乳扮演重要的角色。於泌乳時，泌乳素能透過結合於泌乳素接受體 (prolactin receptor, PRLR) 之酪胺酸激脢Jak-2磷酸化泌乳素接受體，然後泌乳素接受體磷酸化主要之訊號傳遞分子 (signal transducer and activator of transcription 5a, STAT5a)，磷酸化之STAT5a會從細胞質轉移至細胞核，進而調控 b-酪蛋白啟動子之活性。此外，在Caveolin-1 (Cav-1) 的研究中亦發現，Cav-1剔除的小鼠其乳腺管及泡狀構造有提早成熟、提前泌乳與高度磷酸化STAT5a的現象，並且Cav-1認定為具有負調控Jak-2/STAT5a訊息傳遞之功能。另外在小鼠乳腺中，亦了解泌乳素能透過Ras-MAP Kinase訊息傳遞之路徑負調控Cav-1蛋白之表現。
經比對山羊與人類、山羊與小鼠之Cav-1啟動子序列，其相似度分別為74%、67%。亦比對山羊與人類、山羊與小鼠 b-酪蛋白基因轉錄起始點上游1683個核酸序列，其相似度分別為46%、49%。由於各物種間序列相似度上的差異，因此本研究之目的想探討山羊Cav-1於不朽化山羊乳腺上皮細胞 (caprine mammary epithelial cells, CMEC) 中之功能是否相似於人類及小鼠。
首先，透過puromycin的篩選，建立穩定表現人類泌乳素接受體 (human prolactin receptor, hPRLR) 之CMEC (CMEC/hPRLR)。為測定山羊 b-酪蛋白啟動子之活性，構築由山羊 b-酪蛋白啟動子 (-1683 - -1) 驅動表現螢火蟲冷光素之質體，並且為矯正轉型效率，亦構築由thymidine kinase (TK) 啟動子驅動表現珊瑚蟲冷光素之質體，並透過將上述二質體共同轉染入CMEC/hPRLR，再經泌乳素刺激，以建立由泌乳素誘導山羊 b-酪蛋白啟動子之系統。經由此系統，山羊 b-酪蛋白啟動子經泌乳素刺激後能提高36.1倍之活性。為了解泌乳素處理濃度對內源性山羊Cav-1表現之影響，以6種不同最終濃度 (濃度從0至15 ug/mL) 之泌乳素處理CMEC/hPRLR，並且在三個時間點 (處理後24、48及72小時) 抽取細胞總蛋白質，經西方吸漬法分析，內源性山羊Cav-1表現量於泌乳素處理後48及72小時，隨處理濃度之增加逐漸減少。接著為探討山羊Cav-1是否會減少泌乳素訊息傳遞誘導之STAT5a磷酸化，將山羊Cav-1質體與山羊STAT5a質體共同轉染入CMEC/hPRLR中，並以泌乳素刺激，經西方吸漬法分析，山羊STAT5a磷酸化程度明顯下降，此外轉染綠螢光蛋白 (EGFP) 之負控制組，並未影響山羊STAT5a的磷酸化。最後為探討山羊Cav-1是否透過降低山羊STAT5a磷酸化，進而降低山羊 b-酪蛋白啟動子之活性，因此於泌乳素誘導山羊 b-酪蛋白啟動子之系統中額外轉染山羊Cav-1，亦觀察到山羊 b-酪蛋白啟動子活性之降低。
由以上結果可知，CMEC/hPRLR經泌乳素之處理能減少內源性山羊Cav-1之表現，若外源性提高表現山羊Cav-1則會降低山羊STAT5a之磷酸化及降低山羊 b-酪蛋白啟動子之活性。因此，泌乳素處理CMEC/hPRLR時，其內源性山羊Cav-1表現降低及STAT5a磷酸化程度提高，對於促進不朽化山羊乳腺上皮細胞活化山羊 b-酪蛋白啟動子為重要之調控。

Development of the adult female mammary gland encounters four distinct stages: virgin, pregnancy, lactation, and involution. During mammary gland development, prolactin (PRL), a pituitary hormone, mediates mammary epithelial cell growth, differentiation and lactation. The act of prolactin in regulating the activity of b-casein promoter via the activation of prolactin tyrosine kinase associated receptor, and its associated protein kinase, Jak-2. Signal transducer and activator of transcription 5a (STAT5a) is the key signaling molecule transfers the prolactin signal from Jak-2 to b-casein promoter. Once STAT5a phosphorylated by Jak-2, STAT5a translocates from the cytoplasm to the nucleus, and activates b-casein promoter during lactation. Besides, based on the study of Caveolin-1 (Cav-1), its null mice accelerated the development of the labuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a. In addition, Cav-1 is identified as a negative regulator of Jak-2/STAT5a signaling pathway. Prolactin down-regulates Cav-1 expression via Ras-MAP Kinase pathway also been identified in mouse mammary epithelial cells.
The Cav-1 promoter sequence identity between goat versus human and goat versus mouse was 74% and 67%, respectively. The comparison of 1683 nucleotides upstream the transcription start site of b-casein gene between goat versus human and goat versus mouse the sequence similarity is 46% and 49%, respectively. Because diversity among those sequences, the goal in present study is to understand whether the function of goat Cav-1 in caprine mammary epithelial cell (CMEC) is similar to that in mouse and human.
First of all, stable expression of PRL receptor CMEC cells (CMEC/PRLR) lines were established via puromycin selection. In order to measure the b-casein promoter activity, a plasmid possessed goat b-casein promoter (-1683 – -1) drove firefly luciferase (Fluc) reporter protein was constructed. For equivalent the transfection efficiency, another plasmid possessed TK (thymidine kinase) promoter drove the renilla luciferase (Rluc) reporter protein also been constructed. A system that prolactin mediated b-casein promoter in CMEC/PRLR cells was established by co-expression of b-casein promoter Fluc plasmid and TK promoter Rluc plasmid in the CMEC/PRLR cells in response to the stimulation of PRL. The activity of goat b-casein promoter activity was enhanced 36.1 fold after PRL treatment. To understand the dosage effect of PRL to the endogenous Cav-1 expression, 6 final PRL concentrations (range from 0 to 15 ug/ml) were used to treat the CMEC/PRLR cells, then the cell proteins were harvested at 3 time courses (24, 48, 72 hr post-treatment). The results show that endogenous Cav-1 down-regulated in response to PRL treatment at 48, and 72 hr and dose dependent. To study whether goat Cav-1 could down-regulate the activity (phosphorylation) of STAT5a via PRL signal cascade, the phsphorylayion state of STAT5a were determined with or without exogenous goat Cav-1 protein expression in CMEC/PRLR cells after PRL stimulation. The phosphorylation of STAT5a was decreased after PRL stimulation, furthermore expression of the control protein EGFP (Enhanced Green Fluorescent Proteins) did not alter STAT5a phosphorylation. Finally, to study whether goat Cav-1 could down-regulate the b-casein promoter activity by down-regulate the phosphorylation of STAT5a. We transient transfect gCav-1 in the PRL induce b-casein promoter system. The gCav-1 also down-regulate the b-casein promoter activity.
In conclusion, these results showed that PRL down-regulated the endogenous Cav-1 expression, and elevated expression of exogenous Cav-1 decreased the phosphorylation of STAT5a proteins and b-casein promoter activity. Therefore, PRL down-regulated endogenous Cav-1 expression and up-regulated STAT5a phosphorylation is important for improving b-casein promoter activity in CMEC.

目錄....................................................II

圖次....................................................VI

壹、前言.................................................1

貳、中文摘要.............................................2

參、英文摘要.............................................4

肆、文獻檢討.............................................6
一、活體乳腺構造與發育...................................6
1. 乳腺構造..............................................6
2. 乳腺發育..............................................6
二、調控乳腺發育之內泌素與乳蛋白表現.....................7
三、影響 b-酪蛋白表現之調控機制..........................9
1. 胞外基質 (extracellular matrix, ECM) 對 b-酪蛋白基因表現之調控...................................................9
2. 生太與類固醇激乳內泌素對 b-酪蛋白基因轉錄之調控......10
3. b-酪蛋白啟動子調控 b-酪蛋白之表現....................11
四、泌乳素..............................................12
1. 概況.................................................12
2. 生理功能.............................................12
3. 泌乳素於乳腺上皮細胞啟動之訊息傳遞...................13
4. 負調控泌乳素誘導之JAK/STAT訊息傳遞...................13
五、Caveolin............................................14
1. 發現與功能...........................................14
2. Caveolin-1於乳腺細胞中對 b-酪蛋白表現之影響..........15

伍、材料與方法..........................................17
第一部份：建立穩定表現人類泌乳素接受體之山羊乳腺上皮細胞株......................................................17
1-1、細胞培養...........................................17
1-2、細胞繼代...........................................17
1-3、細胞轉型感染.......................................17
1-4、抗生素篩選建立穩定表現外源性蛋白之CMEC細胞株.......18
第二部份：泌乳素誘導山羊 b-酪蛋白基因啟動子之活性.......19
2-1、誘導CMEC細胞株活化 b-casein啟動子..................19
2-2、雙重冷光脢分析系統.................................19
第三部份：細胞內蛋白質表現之偵測........................19
3-1、細胞蛋白質萃取.....................................19
3-2、蛋白質定量.........................................20
3-3、西方吸漬 (Western blotting)........................20
3-4、細胞免疫螢光染色...................................21
第四部份：山羊caveolin-1 cDNA之選殖.....................22
4-1、細胞total RNA萃取..................................22
4-2、反轉錄脢聚合鏈鎖反應...............................22
4-3、聚合脢鏈鎖反應.....................................23
4-4、質體pGEM-T easy gCav-1與pcDNA4 A gCav-1 myc/His建構......................................................24
1. 質體pGEM-T easy gCav-1建構...........................24
2. 質體pcDNA4 A gCav-1 myc/His建構......................25
4-5、小量質體DNA萃取....................................25
4-6、中量質體DNA萃取....................................26
第五部份：腺病毒載體系統之架構..........................27
5-1、細胞培養...........................................27
5-2、重組腺病毒載體pAd/CMV/V5-DEST gCav-1與pAd/CMV/V5-DEST EGFP建構................................................27
5-3、初代重組腺病毒製備.................................29
5-4、重組腺病毒力價擴增.................................29
5-5、重組腺病毒力價測試 (Plaque assay)..................29
5-6、重組腺病毒感染CMEC細胞株MOI (Multipicity of infection) 測試.........................................30
第六部份：統計分析......................................30

陸、結果................................................32
一、建立穩定表現人類泌乳素接受體之山羊乳腺上皮細胞株....32
二、泌乳素處理增加CMEC/hPRLR中外源性山羊 b-酪蛋白基因啟動子活性....................................................34
三、泌乳素負調控CMEC/hPRLR內源性Caveolin-1表現..........37
四、山羊caveolin-1 cDNA之選殖...........................37
五、山羊Caveolin-1蛋白負調控STAT5a磷酸化與 b-酪蛋白啟動子活性......................................................39
六、表現gCav-1與EGFP腺病毒載體系統之構築................40

柒、討論................................................43
一、建立穩定表現人類泌乳素接受體之CMEC..................43
二、泌乳素增加CMEC/hPRLR中外源性山羊 b-酪蛋白基因啟動子活性......................................................44
三、泌乳素負調控CMEC/hPRLR內源性Caveolin-1蛋白表現......45
四、表現goat Caveolin-1蛋白抑制STAT5a磷酸化.............46
五、表現gCav-1與EGFP腺病毒載體系統之構築................47

捌、結論................................................70

玖、參考文獻............................................71

附錄....................................................78

Biswas, R. and B. K. Vonderhaar. 1987. Role of serum in the prolactin responsiveness of MCF-7 human breast cancer cells in long-term tissue culture. Cancer Res. 47: 3509-3514.
Chilton, B. S. and A. Hewetson. 2005. Prolactin and growth hormone signaling. Curr. Top. Dev. Biol. 68: 1-23.
Chomczynski, P., P. Qasba, and Y. J. Topper. 1984. Essential role of insulin in transcription of the rat 25,000 molecular weight casein gene. Science 226: 1326-1328.
Cohen, A. W., R. Hnasko, W. Schubert, and M. P. Lisanti. 2004. Role of caveolae and caveolins in health and disease. Physiol. Rev. 84: 1341-1379.
Couet, J., S. Li, T. Okamoto, T. Ikezu, and M. P. Lisanti. 1997. Identification of peptide and protein ligands for the caveolin-scaffolding domain. Implications for the interaction of caveolin with caveolae-associated proteins. J. Biol. Chem. 272: 6525-6533.
Couet, J., M. M. Belanger, E. Roussel, and M. C. Drolet. 2001. Cell biology of caveolae and caveolin. Adv. Drug Deliv. Rev. 49: 223-235.
Doppler, W., B. Groner, and R. K. Ball. 1989. Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat b-casein gene promoter constructs in a mammary epithelial cell line. Proc. Natl. Acad. Sci. U.S.A. 86: 104-108.
Ebert, K. M., P. DiTullio, C. A. Barry, J. E. Schindler, S. L. Ayres, T. E. Smith, L. J. Pellerin, H. M. Meade, J. Denman, and B. Roberts. 1994. Induction of human tissue plasminogen activator in the mammary gland of transgenic goats. Biotechnology (N. Y.) 12: 699-702.
Eisenstein, R. S. and J. M. Rosen. 1988. Both cell substratum regulation and hormonal regulation of milk protein gene expression are exerted primarily at the posttranscriptional level. Mol. Cell. Biol. 8: 3183-3190.
Engelman, J. A., C. Chu, A. Lin, H. Jo, T. Ikezu, T. Okamoto, D. S. Kohtz, and M. P. Lisanti. 1998. Caveolin-mediated regulation of signaling along the p42/44 MAP kinase cascade in vivo. A role for the caveolin-scaffolding domain. FEBS Lett. 428: 205-211.
Fantozzi, A. and G. Christofori. 2006. Mouse models of breast cancer metastasis. Breast Cancer Res. 8: 212-222.
Farrelly, N., Y. J. Lee, J. Oliver, C. Dive, and C. H. Streuli. 1999. Extracellular matrix regulates apoptosis in mammary epithelium through a control on insulin signaling. J. Cell Biol. 144: 1337-1348.
Ferrag, F., J. J. Lebrun, P. Touraine, M. Nagano, M. Dardenne, and P. A. Kelly. 1994. Prolactin and the immune system. Immunomethods 5: 21-30.
Fox, P. F. and P. L. H. McSweeney. 1998. Dairy Chemistry and Biochemistry. Blackie Academic and Professional, London.
Freeman, M. E., B. Kanyicska, A. Lerant, and G. Nagy. 2000. Prolactin: structure, function, and regulation of secretion. Physiol. Rev. 80: 1523-1631.
Gilbert, S. F. 2000. Cell-cell communication in development. In Development Biology P. 143-154. Sinauer Associates, Inc., Publishers, Massachusetts.
Glasow, A., M. Breidert, A. Haidan, U. Anderegg, P. A. Kelly, and S. R. Bornstein. 1996. Functional aspects of the effect of prolactin (PRL) on adrenal steroidogenesis and distribution of the PRL receptor in the human adrenal gland. J. Clin. Endocrinol. Metab. 81: 3103-3111.
Glenney, J. R. 1989. Tyrosine phosphorylation of a 22-kD protein is correlated with transformation with Rous sarcoma virus. J. Biol. Chem. 264: 20163-20166.
Guyette, W. A., R. J. Matusic, and J. M. Rosen. 1979. Prolactin-mediated transcriptional and post-transcriptional control of casein gene expression. Cell 17: 1013-1023.
Hennighausen, L. and G. W. Robinson. 2001. Signaling pathways in mammary gland development. Dev. Cell 1: 467-475.
Hennighausen, L. and G. W. Robinson. 2005. Information networks in the mammary gland. Nat. Rev. Mol. Cell. Biol. 6: 715-725.
Houdebine, L. M. 2000. Transgenic animal bioreactors. Transgenic Res. 9: 305-320.
Jenness, R. 1974. The composition of milk. In: Lactation. B. L. Larson and V. R. Smith (Ed.) Vol. III, pp. 3-107. Academic Press, New York.
Jensen, R. G. 1995. Hankbook of milk composition. Academic Press, New York.
Kolb, A. F. 2002. Structure and regulation of the murine a-casein gene. Biochim. Biophys. Acta 1579: 101-116.
Krasnow, J. S., G. J. Hickey, and J. S. Richards. 1990. Regulation of aromatase mRNA and estradiol biosynthesis in rat ovarian granulose and luteal cells by prolactin. Mol. Endocrinol. 4: 13-21.
Kulaeva, O. I., S. Draghici, L. Tang, J. M. Kraniak, S. J. Land, and M. A. Tainsky. 2003. Epigenetic silencing of multiple interferon pathway genes after cellular immortalization. Oncogene 22: 4118-4127.
Lechner, J., T. Welte, J. K. Tomasi, P. Bruno, C. Cairns, J. Gustafsson, and W. Doppler. 1997. Promoter-dependent synergy between glucocorticoid receptor and stat5 in the activation of b-casein gene transcription. J. Biol. Chem. 272: 20954-20960.
Li, S., J. Couet and M. P. Lisanti. 1996. Src tyrosine kinases, Galpha subunits, and H-Ras share a common membrane-anchored scaffolding protein, caveolin. Caveolin binding negatively regulates the auto-activation of Src tyrosine kinases. J. Biol. Chem. 271: 29182-29190.
Lindeman, G. J., S. Wittlin, H. Lada, M. J. Naylor, M. Santamaria, J. Zhang, R. Starr, D. J. Hilton, W. S. Alexander, C. J. Ormandy, and J. Visvader. 2001. SOCS1 deficiency results in accelerated mammary gland development and rescues lactation in prolactin receptor-deficient mice. Genes Dev. 15: 1631-1636.
Maaskant, R. A., L. V. Bogic, S. Gilger, P. A. Kelly, and G. D. Bryant-Greenwood. 1996. The human prolactin receptor in the fetal membranes, decidua, and placenta. J. Clin. Endocrinol. Metab. 81: 396-405.
Merez, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Cambell, K. Carver Moore, R. N. DuBois, R. Clark, M. Aguet, and R. D. Schreiber. 1996. Targeted disruption of the stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84: 431-442.
Murtagh, J., E. McArdle, E. Gilligan, L. Thornton, F. Furlong, and F. Martin. 2004. Organization of mammary epithelial cells into 3D acinar structures requires glucocorticoid and JNK signaling. J. Cell Biol. 166: 133-143.
Park, D. S., H. Lee, C. Riedel, J. Hulit, P. E. Scherer, R. G. Pestell, and M. P. Lisanti. 2001. Prolactin negatively regulates caveolin-1 gene expression in the mammary gland during lactation, via a Ras-dependent mechanism. J. Biol. Chem. 276: 48389-48397.
Park, D. S., H. Lee, P. G. Frank, B. Razani, A. V. Nguyen, A. F. Parlow, R. G. Russell, J. Hulit, R. G. Pestell, and M. P. Lisanti. 2002. Caveolin-1-deficient mice show accelerated mammary gland development during pregnancy, premature lactation, and hyperactivation of the Jak-2/STAT5a signaling cascade. Mol. Biol. Cell 13: 3416-3430.
Pauloin, A., G. C. Rogel, F. Piumi, H. Hayes, M. L. Fontaine, E. Chanat, P. Chardon, and E. Devinoy. 2002. Structure of the rabbit a1- and b-casein gene cluster, assignment to chromosome 15 and expression of the a1-casein gene in HC11 cells. Gene 283: 155-162.
Provot, C., M. A. Persuy, and J. C. Mercier. 1995. Complete sequence of the ovine beta-casein-encoding gene and interspecies comparison. Gene 154: 259-263.
Purvis, K., O. P. Clausen, A. Olsen, E. Haug, and V. Hansson. 1979. Prolactin and Leydig cell responsiveness to LH/hCG in the rat. Arch. Androl. 3: 219-230.
Riddle, O., R. W. Bates, and S. W. Dykshorn. 1933. The preparation, identification and assay of prolactin – a hormone of anterior pituitary. Am. J. Physiol. 105: 191-216.
Roberts, B., P. DiTullio, J. Vitale, K. Hehir, and K. Gordon. 1992. Cloning of the goat b-casein-encoding gene and expression in transgenic mice. Gene 121: 255-262.
Rodig, S. J., M. A. Meraz, and J. M. White. 1998. Disruption of the Jak1 gene demonstrates obligatory and nonredundant roles of the Jaks in cytokine-induced biologic responses. Cell 93:373-383.
Rose, M. T., H. Aso, S. Yonekura, T. Komatsu, A. Hagino, K. Ozutsumi, and Y. Obara. 2002. In vitro differentiation of a cloned bovine mammary epithelial cell. J. Dairy Res. 69: 345-355.
Rosen, J. M., S. Li, B. Raught, and D. Hadsell. 1996. The mammary gland as a bioreactor: factors regulating the efficient expression of milk protein-based transgenes. Am. J. Clin. Nutr. 63: 627S-32S.
Rosen, J. M., S. L. Wyszomierski, and D. Hadsell. 1999. Regulation of milk protein gene expression. Annu. Rev. Nutr. 19: 407-436.
Schedin, P., T. Mitrenga, S. McDaniel, and M. Kaeck. 2004. Mammary ECM composition and function are altered by reproductive state. Mol. Carcinog. 41: 207-220.
Scherer, P. E., T. Okamoto, M. Chun, I. Nishimoto, H. F. Lodish, and M. P. Lisanti. 1996. Identification, sequence, and expression of caveolin-2 defines a caveolin gene family. Proc. Natl. Acad. Sci. U.S.A. 93: 131-135.
Scherer, P. E., R. Y. Lewis, D. Volonte, J. A. Engelman, F. Galbiati, J. Couet, D. S. Kohtz, E. van Donselaar, P. Peters, and M. P. Lisanti. 1997. Cell-type and tissue-specific expression of caveolin-2. Caveolins 1 and 2 co-localize and form a stable hetero-oligomeric complex in vivo. J. Biol. Chem. 272: 29337-29346.
Schmidhauser, C., M. J. Bissell, C. A. Myers, and G. F. Casperson. 1990. Extracellular matrix and hormones transcriptionally regulate bovine beta-casein 5’ sequences in stably transfected mouse mammary cells. Proc. Natl. Acad. Sci. U.S.A. 87: 9118-9122.
Schmidhauser, C., G. F. Casperson, C. A. Myers, K. T. Sanzo, S. Bolten, and M. J. Bissell. 1992. A novel transcriptional enhancer is involved in the prolactin- and extracellular matrix-dependent regulation of b-casein gene expression. Mol. Biol. Cell 3: 699-709.
Shome, B. and A. F. Parlow. 1977. Human pituitary prolactin (hPRL): the entire linear amino acid sequence. J. Clin. Endocrinol. Metab. 45: 1112-1115.
Sotgia, F., W. Schubert, R. G. Pestell, and M. P. Lisanti. 2006. Genetic ablation of caveolin-1 in mammary epithelial cells increases milk production and hyper-activates stat5a signaling. Cancer Biol. Ther. 5: 292-297.
Starr, R. and D. J. Hilton. 1999. Negative regulation of the JAK/STAT pathway. Bioessays 21: 47-52.
Stocklin, E., M. Wissler, F. Gouilleux, and B. Groner. 1996. Functional interactions between Stat5 and the glucocorticoid receptor. Nature 383: 726-728.
Streuli, C. H., C. Schmidhauser, N. Bailey, P. Yurchenco, A. P. N. Skubitz, C. Roskelley, and M. J. Bissell. 1995. Laminin mediates tissue-specific gene expression in mammary epithelia. J. Cell Biol. 129: 591-603.
Stricker, P. and R. Grueter. 1928. Action du lobe anterieur de l’hypophyse sur la montee laiteuse. C. R. Seances Soc. Biol. Fil. 99: 1978-1980.
Vorburger, S. A. and K. K. Hunt. 2002. Adenoviral gene therapy. Oncologist 7: 46-59.
Winklehner-Jennewein, P., S. Geymayer, J. Lechner, T. Welte, L. Hansson, S. Geley, and W. Doppler. 1998. A distal enhancer region in the human b-casein gene mediates the response to prolatin and glucocorticoid hormones. Gene 217: 127-139.
Yamazaki, T., T. Kimoto, K. Higuchi, Y. Ohta, S. Kawato, and S. Kominami. 1998. Calcium ion as a second messenger for o-nitrophenylsulfenyl-adrenocorticotropin (NPS-ACTH) and ACTH in bovine adrenal steroidogenesis. Endocrinology 139: 4765-4771.
Ziomek, C. A. 1998. Commercialization of proteins produced in the mammary gland. Theriogenology 49: 139-144.
Zucchi, I., L. Bini, D. Albani, R. Valaperta, S. Liberatori, R. Raggiaschi, C. Montagna, L. Susani, O. Barbieri, V. Pallini, P. Vezzoni, and R. Dulbecco. 2002. Dome formation in cell cultures as expression of an early stage of lactogenic differentiation of the mammary gland. Proc. Natl. Acad. Sci. U.S.A. 99: 8660-8665.