• The Korean Journal of Zoology
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• v.34 no.3
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• pp.394-402
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• 1991

Yolk protein-3 (YP3) was purified from the ovary of Hvpharatria cunea D. and the synthesis and fate during embryogenesis of WP3 were investisated by electrophoresis and fluorography. YP3 purified through gel slice and electrophoretic elution'was determined to have M. W. of 18 Kd and consist of one subunit. Haemolymph and fat body of male and female %were electrophoresed during vifellogenic stages to indentifv the vitellosenin in female. The result showed that there was no distinct difference in electrophoretic patterns betweerl male and female. However, tissue culture of fat body and maturing ovary indicated that YP3 was svuthesized by fat body. Aiso, vP3 in iaid eggs was maintained constant untii naut s artier oviposition and then decreased, indicating that YP3 was drastically used during late embryosenesis. However, a part of YP3 was present even in newly hatched first instar larvae.

• The Korean Journal of Zoology
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• v.33 no.4
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• pp.468-475
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• 1990

An In vitro protein sulfation in the soluble fraction of rat brain was charaderized further by an improved method of alkaline hydrolysis and thin layer ceflulose electrophoresis TLE) The protein sulfation was carried out in a reaction system containing [35 S] 3'-phosphoadenosine-5'-phosphosulfate (PAPS), Tris-maleate buffer (pH 8), MgCI$_2$, and soluble proteins from rat brain. The sulfated proteins were precipitated by acetone and alkaline hydrolysis was performed to obtain sulfated amino acids. The hydrolysate was separated further by TLE and the separated residues were identified by fluorography. The Iluorography of one-dimensional The showed at least nine sulfated residues including tryosine-O-sulfate. The other spots were not identified yet positively. General properties of protein sulfotransferases (PST) using this method were re-examined such as effects of concentrations of PAPS, pH, incubation temperature and $Mg^2$+. These results suggest a possible occurrence of several PST corresponding to each sulfated residue in rat brain and that the sulfation can occur not only in tyrosine but also in other residues as well.

• Journal of Microbiology and Biotechnology
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• v.3 no.4
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• pp.256-260
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• 1993

DNA subfragments, sopA, sopB and sopC which help to maintain the stability of an ori C plasmid, were derived from a mini-F plasmid DNA (EcoRI restriction fragment f5) after digestion with restriction endonuclease, and cloned in the vector plasmid pBR322. The recombinant plasmids obtained were introduced into E. coli KY7231 and E. coli CSR603 strains, and proteins specified by the mini-F fragments were analysed by SDS-PAGE. Two proteins encoded by the F fragments were detected, and their molecular weights were 41,000 and 37,000 daltons. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins had been overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of sopA and sopB proteins were 6.6 and 7.0, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.7
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• pp.962-967
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• 2003

The purpose of the study were to characterize the proteins secreted by elongating caprine conceptus, to identify a group of low molecular weight proteins as retinol-binding protein (RBP), to identify RBP cell-specific localization in conceptus tissue, and to demonstrate that the conceptuses secreted continuously RBP during the time period maternal recognition of pregnancy. Caprine conceptuses were removed from the uterus between days 16 and 22 of pregnancy, the time period maternal recognition of pregnancy. Isolated conceptuses were cultured in a modified minimum essential medium in the presence of radiolabeled amino acids. Proteins synthesized and secreted into medium were analyzed by fluorography of two-dimensional polyacrylamide gel electrophoresis and fluorography. At least five proteins showed consistently a grouping of spots with characteristic location on two-dimensional gels. A major low molecular weight protein consisted of two major isoforms (pI 5.3-6.0) of similar molecular mass (21 kDa) was identified as RBP by using antiserum against RBP. Presence of RBP in conceptus culture medium and uterine flushings between days 16 and 22 of pregnancy were determined by immunoprecipitation and Western blotting using anti-RBP serum. In immunocytochemical study, strong immunostaining for RBP was localized in trophectoderm and endoderm of conceptus. These results clearly demonstrated that the caprine conceptus was active in protein synthesis as early as day 16 of pregnancy. Secretion of RBP by caprine conceptuses (days 16-22) coincident with the rapid transformation of the conceptus from a spherical blastocyst to a filamentous structure. Production of RBP by the elongating conceptuses may be indicative of an important role for conceptus RBP in the transport, availability and metabolism of retinol during maternal recognition of pregnancy.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.12
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• pp.1708-1713
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• 2002

Endometrial explants obtained from does between days 13 and 21 of pregnancy were cultured in a modified minimum essential medium in the presence of [$^35S$]methionine and [$^3H$]-leucine. Proteins synthesized and secreted into medium were analyzed by fluorography of two-dimensional polyacrylamide gel electrophoresis and fluorography. No marked qualitative changes in patterns of protein production by caprine endometrium between days 13-21 of pregnancy. At least 11 proteins showed consistently a clear spot or a grouping of spots with characteristic location on two-dimensional gels. A major low molecular weight protein consisted of two major isoforms (pI 5.3-6.0) of similar molecular mass (21 kDa). Limited N-terminal sequence analysis of these two isoforms showed that the protein had complete homology with bovine placental and plasma retinol-binding protein (RBP) over the first 20 amino acids. Through use of the antiserum raised against bovine placental RBP, immunoreactive RBP was detected in cultures conditioned by uterine explants prepared at days 13, 15 and 21 of pregnancy. In the present study, proteins synthesized and secreted by caprine endometrium during periattachment period of early pregnancy were characterized. The pregnant endometrium secreted a number of neutral-to-acidic proteins which constituted, in part, the histotroph. A vitamin A-transport protein, RBP, was identified in cultures conditioned by endometrium of days 13-21 of pregnancy. The uterine endometrium is the only source of retinol for embryonic tissues. The uterine RBP appears to transport retinol locally toward embryonic tissues. Secretion of RBP by caprine endometrium of days 13, 15 and 21 of pregnancy suggested that retinol played an important role in conceptus development during periattachment period of early pregnancy.

• Journal of Microbiology and Biotechnology
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• v.1 no.4
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• pp.232-239
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• 1991

Caboxymethyl cellulase (CMCase) I was purified from the induced culture filtrate of Penicllium verruculosum F-3 by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and Bio-gel P-150 filtration. The purified enzyme was assumed to be a glycoprotein consisting of 8.5% carbohydrate and having a molecular weight of 70.000 in SDS-polycrylamide gel electrophoresis (SDS-PAGE). The purified enzyme-specific anti-CMCase I IgG was obtained by rabbit immunization and protein A-sepharose CL-4B chromatography. The fungal poly($A^+$) RNA was isolated from the total RNA of the mycelium grown under cellulase induction conditions by oligo(dT)-cellulosse chromatography. The translation products in vitro were prepared by translating the isolated poly ($A^+$) RNA in rabbit reticulocyte lysate and analyzed by SDS-PAGE and fluorography. Of the translation products, CMCase I was identified by the immunoprecipitation against anti-CMCase I IgG.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1986.12a
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• pp.516.2-516
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• 1986

DNA subfragments, sopA, sopB, and sopC supporting stable maintenance of an oriC plasmid, were derived from mini-F plasmid DNA (EcoRI restriction fragment, f5) after digestion with restriction endonucleases, and cloned in vector plasmid pBR322. The recombinant plasmid obtained were introduced into E. coli KY7231 and E. coli CSR603, and proteins specified by the mini-F fragments were analysed by SDS-polyacrylamide gel electrophoresis. Two proteins encoded by the F fragments were detected, having molecular weights of 41,000 and 37.000. The sopA protein (41K) encoded by a plasmid pXX288 was observed in the cytoplasm, whereas the sopB protein (37K) encoded by a plasmid pXX157 was in the membrane fraction. There was no novel protein band detected in the cell with a plasmid pXX300, which contained sopC fragment. Gene products of a plasmid pXX167, which is comprised of sopA, sopB, and sopC, were not detectable. Fluorography after one and two dimensional gel electrophoresis of the lysates showed that these two proteins were overproduced in the cells which were allowed to incorporate radioactive amino acid after plasmid amplification by chloramphenicol treatment. The isoelectric points of the sopA and sepB proteins were 6.6 and 7.0, respectively.

• Korean Journal of Animal Reproduction
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• v.21 no.4
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• pp.325-333
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• 1997

Recent evidence indicates that growth factors modulate response of mammary epithelial cells to environmental stress. The objective of this study was to examine the cellular and biochemical responses of mammary tissue to environmental stress caused by artificial mastitis. For experimental a, pp.oach, toxins of most mastitis causing organisms(Staph. aureus or Strep. agalactiae) and heat stress(42$^{\circ}C$) were artificially exposed to mammary tissue. Effects of these environmental stresses on cell growth, cell death and heat shock protein synthesis were examined. Lactating mammary tissure were cultured under basal medium(DMEM) su, pp.emented with insulin(10$\mu\textrm{g}$/ml) and aldosterone(1$\mu\textrm{g}$/ml). All treatment groups in heat stress at 42$^{\circ}C$ incubation significantly decreased DNA synthesis rates in comparison with those at 39$^{\circ}C$(P<0.05), however, these decreased DNAa synthesis rates were recovered by addition of adenosine(10$\mu$M) and IGFI(10ng/ml). Similar results were obtained when tissue growth rates were measured by DNA content/tissue. Strep. agalactiae toxin did not significantly decreased DNA content/tissue in comparison with no treatment of bacterial toxin with or without heat stress, however, tended to decrease DNA contents/tissue without heat stress. In the fluorography analysis, heat stress(42$^{\circ}C$ incubation) slightly increased 35S-methoionine labelled 70kd protein synthesis. These results indicate that environmental stress caused by artificial mastitis slightly decreased mammary growth or mammary size, however, these results could be recovered by addition of adenosine and IGF-I.

• Korean Journal of Animal Reproduction
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• v.19 no.2
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• pp.95-104
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• 1995

Mouse mammary epithelial cells(NMuMG) were maintained onto 6-well plates (3$\times$105 cells/well) or chambered slide (1$\times$104 cells/well), in DMEM supplemented with 10% fetal calf serum. After serum starvation for 24 hours, DMNB (1$\mu$M) was added and exposed to UV light (300nm, 3 second pulse) after 2 hours from DMNB addition in order to activate DMNB which induces a rapid transient increase in intracellular cAMP upon UV irradiation. EGF (100ng/ml) and/or IGF-I (10ng/ml) were treated at the time of UV irradiation. Nuclear labeling index was estimated as percent of nuclear labeled cells(percent of S phase of cells) by incorporation of 3H-thymidine into DNA(1 hour pulse with 1$\mu$Ci/ml). DMNB(1$\mu$M), EGF (100ng/ml) and/or IGF-I (10ng/ml) signifciantly increased nuclear labeling index than those of control (P<0.05). Addition of DMNB＋EGF or DMNB＋EGF＋IGF-I showed the interaction effect in nuclear labeling index (P<0.05). Protein kinase A activities by addition of EGF, IGF-I or EGF＋IGF-I were 10.5, 9.8 or 9.4 unit/mg protein, respectively, and no statistical difference was found in comparison with control (P>0.05). Additon of DMNB＋EGF showed the moderate interaction effect on tyrosyl kinase activity (P<0.1). In the fluorography analysis, there were no specific protein phosphorylation patterns were found at 1 or 15 minute by addition of DMNB. EGF and/or IGF-I. These results suggest that the interaction effect in nuclear labeling index by addition DMNB and EGF could be mediated through the modulation of tyrosyl kinase activity by cAMP.

• The Korean Journal of Zoology
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• v.23 no.3
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• pp.149-160
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• 1980

The effects of mutagens, MMS and captan, on the patterns of proteins synthesized during the early developmental stages in mice were analyzed using two-dimensional electrophoresis. Three classes of proteins were observed in terms of synthetic pattern during the preimplantation stages. The first class is synthesized from the m-RNA, which was made and preserved throughout oogenesis and activated at the fertilization. The synthesis of these proteins did not seem to be influenced by MMS. The second class, which may be stagespecific proteins synthesized by newly transcribed m-RNA, was selectively inhibited by MMS. The third class, the synthesis of which is also suppressed by MMS, is the proteins synthesized by the m-RNA transcribed in augmented fashion. While MMS inhibits protein synthesis dependent on thenew transcription, this mutagen enhances a synthesis of a few proteins which were not observed in the untreated embryos. Captan did not affect protein synthesis at morula stage.