Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile

An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K_m and V_max values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min⁻¹ mg⁻¹, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose.     

N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Growth inhibition of unicellular and colonial Microcystis strains (Cyanophyceae) by compounds isolated from rice (Oryza sativa) hulls

The algicidal effects of crude and pure rice hull extracts on the growth of Microcystis aeruginosa were investigated using cultured unicellular and colonial strains. Upon treatment with rice hull crude extract (RHE), growth inhibition of unicellular M. aeruginosa was much higher than that of colonial M. aeruginosa. However, purified compounds from the crude extract, β-sitosterol-β-d-glucoside and dicyclohexanyl orizane, powerfully inhibited the growth of colonial M. aeruginosa cells. At the same concentrations, the two compounds were almost equipotent (66% and 80% growth inhibition for colonial M. aeruginosa, respectively; P < 0.05). As rice hulls are readily obtainable, and as extracts show high algicidal activity (targeting colonial algae rather than unicellular organisms) at low concentrations, the results suggest that some pure compounds extracted from rice hulls, such as β-sitosterol-β-d-glucoside and dicyclohexanyl orizane, may serve as environmentally friendly agents for controlling the growth of toxic colonial M. aeruginosa in eutrophic waters.     

α2-macroglobulin ‘fast’ forms inhibit superoxide production by activated macrophages

Mouse peritoneal macrophages activated by bacillus Calmette-Guerin (BCG) were incubated with human α₂-macroglobulin converted to its ‘fast’ form with either trypsin or methylamine before being stimulated with phorbol myrystate acetate. Both α₂-macroglobulin-trypsin and α₂-macroglobulin-methylamine inhibited macrophage production of superoxide anion (O₂⁻) while native α₂-macroglobulin had little effect except at high concentration. The α₂-macroglobulin ‘fast’ forms, which bind with a K_d of about 8 nM, inhibited 50% generation of O₂⁻(ID₅₀) at a concentration of 7 nM while α₂-macroglobulin inhibited O₂⁻ production with an ID₅₀ of 141 nM. The ‘fast’ forms of α₂-macroglobulin may play a role in the feedback regulation of inflammatory reactions.     

Lysophosphatidic acid-induced arterial wall remodeling: Requirement of PPARγ but not LPA1 or LPA2 GPCR

Lysophosphatidic acid (LPA) and its ether analog alkyl-glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1 h without injury into the carotid arteries of LPA₁, LPA₂, LPA_1&2 double knockout, and Mx1Cre-inducible conditional PPARγ knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and α-smooth muscle actin markers. In LPA₁, LPA₂, LPA_1&2 GPCR knockout, Mx1Cre transgenic, PPARγ^fl/−, and uninduced Mx1Cre × PPARγ^fl/− mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARγ^−/− knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARγ in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express α-smooth muscle actin.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

Conversion of Major Ginsenoside Rb1 to Ginsenoside F2 by Caulobacter leidyia

Ginsenoside Rb₁ is the most predominant ginsenoside in Panax species (ginseng) and the hydrolysis of this ginsenoside produces pharmaceutically active compounds. Caulobacter leidyia GP45, one of the isolates having strong β-glucosidase-producing activity, converted ginsenoside Rb₁ to the active metabolites by 91%. The structures of the resultant metabolites were identified by NMR. Ginsenoside Rb₁ had been consecutively converted to ginsenoside Rd (1), F₂ (2) and compound K (3) via the hydrolyses of 20-C β-(1→6)-glucoside, 3-C β-(1→2)-glucoside, and 3-C β-glucose of ginsenoside Rb₁.     

Structural elucidation of an exopolysaccharide from mycelial fermentation of a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis

A novel polysaccharide designated EPS-1A with an average molecular weight around 40 kDa was fractionated and purified by anion-exchange and gel-filtration chromatography from the crude exopolysaccharide (EPS) isolated from fermentation broth of Cs-HK1, a Tolypocladium sp. fungus isolated from wild Cordyceps sinensis. The structural characteristics of EPS-1A were determined with various methods (e.g. GC, GC–MS, FT-IR, ¹H NMR and ¹³C NMR) and through acid hydrolysis, methylation, periodate-oxidation and Smith degradation. The results suggested that EPS-1A was composed of glucose, mannose and galactose at 15.2:3.6:1.0 M ratio. EPS-1A was a slightly branched polysaccharide and its backbone was composed of (1 → 6)-α-d-glucose residues (77%) and (1 → 6)-α-d-mannose residues (23%). Branching occurred at O-3 position of (1 → 6)-α-d-mannose residues of the backbone with (1 → 6)-α-d-mannose residues and (1 → 6)-α-d-glucose residues, and terminated with β-d-galactose residues.     

Unique transglycosylation potential of extracellular α-d-galactosidase from Talaromyces flavus

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.     

Expression, purification and characterization of the chitinolytic β-N-acetyl-d-hexosaminidase from the insect Ostrinia furnacalis

Insect β-N-acetyl-d-hexosaminidases are of particular interest due to their multiple physiological roles in many life processes. Chitinolytic β-N-acetyl-d-hexosaminidases, which function only in chitin degradation in insects, have long been regarded as species-specific target potentials in developing environmental friendly pesticides. Here the chitinolytic β-N-acetyl-d-hexosaminidase from the insect Ostrinia furnacalis was cloned and expressed in the yeast strain, Pichia pastoris, to meet the demands of biochemical studies and drug development. Enzymatic assay as well as Western blot confirmed that the high-level expression could be achieved after the induction of methanol for 120 h. Through the sequential combination of ammonium sulfate precipitation, metal chelating chromatography as well as anion exchange chromatography, 7.7 mg of the recombinant OfHex1 with high purity was obtained from 1 liter of culture supernatant. The recombinant OfHex1, characterized as a homodimer with molecular weight of 130 kDa, exhibited the same enzymatic activities as its native form, which could efficiently degrade the chitooligosaccharide substrate (GlcNAc)₂ and release 4-methylumbelliferone (4MU) from substrates, 4MU-β-GlcNAc and 4MU-β-GalNAc. This work provides a low-costing and high-efficient purification procedure for the preparation of insect β-N-acetyl-d-hexosaminidases.     

Gas exchange of Ginkgo biloba leaves at different CO2 concentration levels

One and a half year-old Ginkgo saplings were grown for 2 years in 7 litre pots with medium fertile soil at ambient air CO₂ concentration and at 700 μmol mol⁻¹ CO₂ in temperature and humidity-controlled cabinets standing in the field. In the middle of the 2nd season of CO₂ enrichment, CO₂ exchange and transpiration in response to CO₂ concentration was measured with a mini-cuvette system. In addition, the same measurements were conducted in the crown of one 60-year-old tree in the field. Number of leaves/tree was enhanced by elevated CO₂ and specific leaf area decreased significantly.CO₂ compensation points were reached at 75–84 μmol mol⁻¹ CO₂. Gas exchange of Ginkgo saplings reacted more intensively upon CO₂ than those of the adult Ginkgo. On an average, stomatal conductance decreased by 30% as CO₂ concentration increased from 30 to 1000 μmol mol⁻¹ CO₂. Water use efficiency of net photosynthesis was positively correlated with CO₂ concentration levels. Saturation of net photosynthesis and lowest level of stomatal conductance was reached by the leaves of Ginkgo saplings at >1000 μmol mol⁻¹ CO₂. Acclimation of leaf net CO₂ assimilation to the elevated CO₂ concentration at growth occurred after 2 years of exposure. Maximum of net CO₂ assimilation was 56% higher at ambient air CO₂ concentration than at 700 μmol mol⁻¹ CO₂.     

Preparation of a biologically active apo-cytochrome b5 via heterologous expression in Escherichia coli

Cytochrome b₅ (b₅) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b₅ on CYP-catalyzed reactions, but also that of the apo-cytochrome b₅ (apo-b₅). Therefore, the apo-b₅ protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b₅ was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5α cells. The gene sequence was verified by DNA sequencing. The sequence coding b₅ was cleaved from pUC19 by NdeI and XhoI restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b₅ was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The b₅ protein, produced predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepharose. Using such procedures, the homogenous preparation of apo-b₅ protein was obtained. Oxidized and reduced forms of the apo-b₅ reconstituted with heme exhibit the same absorbance spectra as native b₅. The prepared recombinant apo-b₅ reconstituted with heme can be reduced by NADPH:CYP reductase. The reconstituted apo-b₅ is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b₅.     

Influence of chelators and iron ions on the production and degradation of H2O2 by β-amyloid–copper complexes

β-Amyloid peptide (Aβ) 1–42, involved in the pathogenesis of Alzheimer’s disease, binds copper ions to form Aβ · Cu_n complexes that are able to generate H₂O₂ in the presence of a reductant and O₂. The production of H₂O₂ can be stopped with chelators. More reactive than H₂O₂ itself, hydroxyl radicals HO (generated when a reduced redox active metal complex interacts with H₂O₂) are also probably involved in the oxidative stress that creates brain damage during the disease. We report in the present work a method to monitor the effect of chelating agents on the production of hydrogen peroxide by metallo-amyloid peptides. The addition of H₂O₂ associated to a pre-incubation step between ascorbate and Aβ · Cu_n allows to study the formation of H₂O₂ but also, at the same time, its transformation by the copper complexes. Aβ · Cu_n peptides produce but do not efficiently degrade H₂O₂. The reported analytic method, associated to precipitation experiments of copper-containing amyloid peptides, allows to study the inhibition of H₂O₂ production by chelators. The action of a ligand such as EDTA is probably due to the removal of the copper ions from Aβ · Cu_n, whereas bidentate ligands such as 8-hydroxyquinolines probably act via the formation of ternary complexes with Aβ · Cu_n. The redox activity of these bidentate ligands can be modulated by the incorporation or the modification of substituents on the quinoline heterocycle.     

Growth inhibitory activity of extracts and compounds from Cimicifuga species on human breast cancer cells

The purpose of this report is to explore the growth inhibitory effect of extracts and compounds from black cohosh and related Cimicifuga species on human breast cancer cells and to determine the nature of the active components. Black cohosh fractions enriched for triterpene glycosides and purified components from black cohosh and related Asian species were tested for growth inhibition of the ER⁻ Her2 overexpressing human breast cancer cell line MDA-MB-453. Growth inhibitory activity was assayed using the Coulter Counter, MTT and colony formation assays.Results suggested that the growth inhibitory activity of black cohosh extracts appears to be related to their triterpene glycoside composition. The most potent Cimicifuga component tested was 25-acetyl-7,8-didehydrocimigenol 3-O-β-d-xylopyranoside, which has an acetyl group at position C-25. It had an IC₅₀ of 3.2 μg/ml (5 μM) compared to 7.2 μg/ml (12.1 μM) for the parent compound 7,8-didehydrocimigenol 3-O-β-d-xylopyranoside. Thus, the acetyl group at position C-25 enhances growth inhibitory activity.The purified triterpene glycoside actein (β-d-xylopyranoside), with an IC₅₀ equal to 5.7 μg/ml (8.4 μM), exhibited activity comparable to cimigenol 3-O-β-d-xyloside. MCF7 (ER⁺Her2 low) cells transfected for Her2 are more sensitive than the parental MCF7 cells to the growth inhibitory effects of actein from black cohosh, indicating that Her2 plays a role in the action of actein. The effect of actein on Her2 overexpressing MDA-MB-453 and MCF7 (ER⁺Her2 low) human breast cancer cells was examined by fluorescent microscopy. Treatment with actein altered the distribution of actin filaments and induced apoptosis in these cells.These findings, coupled with our previous evidence that treatment with the triterpene glycoside actein induced a stress response and apoptosis in human breast cancer cells, suggest that compounds from Cimicifuga species may be useful in the prevention and treatment of human breast cancer.     

Screening strains for directed biosynthesis of β-d-mono-glucuronide-glycyrrhizin and kinetics of enzyme production

One fungus, tentatively named Penicillium sp. Li-3, was screened to biosynthesize β-d-mono-glucuronide-glycyrrhizin (GAMG), directly. Using glycyrrhizin as elicitor and the sole carbon source, this strain was capable of expressing β-d-glucuronidase, one intracellular enzyme with high substrate specificity. And glycyrrhizin was hydrolyzed directly into GAMG by enzyme from Penicillium sp. Li-3 with high production. It was found that the mol conversion ratio of this reaction was up to 88.45%. Research about kinetics of β-d-glucuronidase production showed that the cell growth and enzyme production of this strain was partial coupled. During the expressing of target enzyme, carbon catabolite repression existed, so only glycyrrhizin was the best carbon source as well as the elicitor. It was found that the surfactant (Tween 80 0.12%) could improve the ability of enzyme production markedly. Under the condition of initial pH 4.8 of the medium and 32 °C of the culture temperature, the maximum enzyme activity of 181.53 U ml⁻¹ was obtained.     

Structural elucidation, modification and characterization of seed gum from Cassia javahikai seeds: A non-traditional source of industrial gums

A water-soluble seed gum was isolated from seed endosperm of Cassia javahikai. The acid-catalyzed fragmentation, methylation, selective enzymatic degradation and periodate oxidation suggested a heteropolymeric structure for the polysaccharide. The polysaccharide was shown to have a linear chain of β(1 → 4) linked d-mannopyranosyls units with side chains of α(1 → 6) d-galactopyranosyl units. Grafting of polyacrylamide onto the gum was performed using K₂S₂O₈/ascorbic acid redox system in presence of Ag⁺ as catalyst at 35 ± 2 °C. The viscosity of the gum solution increased on grafting and the grafted gum was observed to resist biodegradation for more than 256 h. Thermogravimetric analysis revealed that grafted gum was more thermally stable than native gum.     

Synthesis of monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside for epitope mapping of anti-Candida albicans antibodies

A panel of six complementary monodeoxy and mono-O-methyl congeners of methyl β-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (1) were synthesized by stereoselective glycosylation of monodeoxy and mono-O-methyl monosaccharide acceptors with a 2-O-acetyl-glucosyl trichloroacetimidate donor, followed by a two-step oxidation–reduction sequence at C-2′. The β-manno configurations of the final deprotected congeners 2–7 were confirmed by measurement of ¹J_C1,H1 heteronuclear and ³J_1′,2′ homonuclear coupling constants. These disaccharide derivatives will be used to map the protective epitope recognized by a protective anti-Candida albicans monoclonal antibody C3.1 (IgG3) and to determine its key polar contacts with the binding site.     

Delayed parturition in cloned calves associated with persistently elevated placentomal TGF-β1 expression

The objective of this study was to investigate hormonal and TGF-β₁ characterizations of delayed parturition in the SCNT recipients (Korean native beef cattle: Hanwoo). The SCNT blastocysts produced by Hanwoo fetal fibroblast cells were transferred into the synchronized Hanwoo recipients. The artificially inseminated Hanwoo recipients (AI-R) were used as control. All AI-R were labored by natural delivery. The SCNT recipients (SCNT-R) with no signs of delivery were operated by Caesarean section. The blood and placentomes were collected during parturition. The weight of placentomes in SCNT-R (n = 12, 301 ± 41.22 g) was significantly higher than that of AI-R (n = 10, 204.8 ± 24.89 g) (p < 0.05). There were significantly lower E2 (p < 0.05) or higher P4 (p < 0.01) and TGF-β₁ (p < 0.01) levels in the SCNT-R compared to that of AI-R, respectively. The SCNT-R showed a higher placentomal TGF-β₁ protein level compared to that of AI-R (p < 0.01). Interestingly, the TGF-β₁ protein level in SCNT-R with normal delivery was dramatically decreased as same as AI-R, but it was highly maintained in C-sec at days 250 of pregnancy in AI-R. These results suggest that delayed parturition in clone calving may be associated with persistence of elevated TGF-beta-1 expression in late pregnancy.