• Molecules and Cells
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• 제28권1호
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• pp.37-42
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• 2009

Iron binding lactoferrin (Lf) is involved in the control of cell cycle progression. However, the molecular basis underlying the effects of Lf on cell cycle control, as well as its target genes, remains incompletely understood. In this study, we have demonstrated that a relatively low level of ironsaturated Lf, Lf($Fe^{3+}$), can stimulate S phase cell cycle entry, and requires Akt activation in MCF-7 cells. Lf($Fe^{3+}$) immediately induced Akt phosphorylation at Ser473, which subsequently induced the phosphorylation of two G1-checkpoint Cdk inhibitors, $p21^{Cip/WAF1}$ and $p27^{kip1}$. The Lf($Fe^{3+}$)-induced phosphorylation of Cdk inhibitors impaired their nuclear import behavior, thereby inducing cell cycle progression. However, the treatment of cells with a PI3K inhibitor, LY294002, almost completely blocked Lf($Fe^{3+}$)-stimulated cell cycle progression. LY294002 treatment abrogated Lf($Fe^{3+}$)-induced Akt activation, and prevented the cytoplasmic localization of $p27^{kip1}$. Higher levels of $p21^{Cip/WAF1}$ were also detected in the cytoplasmic sub-cellular compartment as a measure of cellular response to Lf($Fe^{3+}$). Consequently, the degree of phosphorylation of retinoblastoma protein was enhanced in response to Lf($Fe^{3+}$). Therefore, we conclude that Lf($Fe^{3+}$), as a potential antagonist of Cdk inhibitors, can facilitate the functions of E2F during progression to S phase via the Akt signaling pathway.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.235-239
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• 2005

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

• Clinical and Experimental Pediatrics
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• 제63권6호
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• pp.226-231
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• 2020

Background: Probiotics and prebiotics have strain-specific effects on the host. Synbiotics, a mixture of probiotics and prebiotics, are proposed to have more beneficial effects on the host than either agent has alone. Purpose: We performed a randomized controlled trial to investigate the effect of Lactobacillus and Bifidobacterium together with oligosaccharides and lactoferrin on the development of necrotizing enterocolitis (NEC) or sepsis in very low birth weight neonates. Methods: Neonates with a gestational age ≤32 weeks and birth weight ≤1,500 g were enrolled. The study group received a combination of synbiotics and lactoferrin, whereas the control group received 1 mL of distilled water as placebo starting with the first feed until discharge. The outcome measures were the incidence of NEC stage ≥2 or late-onset cultureproven sepsis and NEC stage ≥2 or death. Results: Mean birth weight and gestational age of the study (n=104) and the control (n=104) groups were 1,197±235 g vs. 1,151±269 g and 29±1.9 vs. 28±2.2 weeks, respectively (P>0.05). Neither the incidence of NEC stage ≥2 or death, nor the incidence of NEC stage ≥2 or late-onset culture-proven sepsis differed between the study and control groups (5.8% vs. 5.9%, P=1; 26% vs. 21.2%, P=0.51). The only significant difference was the incidence of all stages of NEC (1.9% vs. 10.6%, P=0.019). Conclusion: The combination of synbiotics and lactoferrin did not reduce NEC severity, sepsis, or mortality.

• IMMUNE NETWORK
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• 제15권1호
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• pp.37-43
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• 2015

It is well established that TGF-${\beta}1$ and retinoic acid (RA) cause IgA isotype switching in mice. We recently found that lactoferrin (LF) also has an activity of IgA isotype switching in spleen B cells. The present study explored the effect of LF on the Ig production by mouse peritoneal B cells. LF, like TGF-${\beta}1$, substantially increased IgA production in peritoneal B1 cells but little in peritoneal B2 cells. In contrast, LF increased IgG2b production in peritoneal B2 cells much more strongly than in peritoneal B1 cells. LF in combination with RA further enhanced the IgA production and, interestingly, this enhancement was restricted to IgA isotype and B1 cells. Similarly, the combination of the two molecules also led to expression of gut homing molecules ${\alpha}4{\beta}7$ and CCR9 on peritoneal B1 cells, but not on peritoneal B2 cells. Thus, these results indicate that LF and RA can contribute to gut IgA response through stimulating IgA isotype switching and expression of gut-homing molecules in peritoneal B1 cells.

• Bulletin of the Korean Chemical Society
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• 제33권11호
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• pp.3671-3675
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• 2012

${\beta}$-Amyloid accumulation in the brain is a pathological hallmark of Alzheimer's disease (AD). Since early detection of ${\beta}$-amyloid may facilitate more successful and timely therapeutic interventions, many investigators have focused on developing AD diagnostic reagents that can penetrate the blood-brain barrier (BBB). Oleanolic acid (OA) is a substance found in a variety of plants that has been reported to prevent the progression of AD in mice. In this study, we synthesized and evaluated a new radioligand in which OA was conjugated to lactoferrin (Lf, an iron-binding glycoprotein that crosses the BBB) for the diagnosis of AD. In an in vitro study in which OA-Lf was incubated with ${\beta}$-amyloid (1-42) aggregates for 24 h, we found that OA-Lf effectively inhibited ${\beta}$-amyloid aggregation and fibril formation. In vivo studies demonstrated that $^{123}I$-OA-Lf brain uptake was higher than$^{123}I$-Lf uptake. Therefore, radiolabeled OA-Lf may have diagnostic potential for ${\beta}$-amyloid imaging.

• Animal Bioscience
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• 제35권9호
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• pp.1351-1359
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• 2022

Objective: The objective of this study was to analyse the differentially abundant proteins caused by freeze-thawing of ram sperm and explore candidate proteins of interest for their ability to improve ram sperm cryopreservation outcomes in vitro. Methods: Sperm were from three mature Dorper. Fresh and frozen sperm proteins were extracted, and the differentially abundant proteins were analysed by mass spectrometry. Among these proteins, lactoferrin (LTF) was selected to be added before cryopreservation. Next, sperm samples were diluted in Tris extender, with the addition of 0, 10, 100, 500, and 1,000 ㎍/mL of LTF. After thawing, sperm quality was evaluated by motility, plasma membrane integrity, mitochondrial activity and reactive oxygen species (ROS). Results: Cryopreservation significantly altered the abundance of 40 proteins; the abundance of 16 proteins was increased, while that of 24 proteins was decreased. Next, LTF was added to Tris extender applied to ram sperm. The results showed that sperm motility and plasma membrane integrity were significantly improved (p<0.05) by supplementation with 10 ㎍/mL LTF compared to those in the control group. There was no significant difference in mitochondrial activity between the 0 ㎍/mL group and other groups (p>0.05). Supplementation of the cryoprotective extender with 10 ㎍/mL LTF led to decreased ROS levels compared with those in the control and other groups (p<0.05). Conclusion: The LTF is an important protein during cryopreservation, and the addition of 10 ㎍/mL LTF to a cryoprotective extender can significantly improve the function of frozen ram sperm.

• IMMUNE NETWORK
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• 제23권5호
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• pp.37.1-37.11
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• 2023

Forkhead box P3-positive (Foxp3⁺)-inducible Tregs (iTregs) are readily generated by TGF-β1 at low TCR signaling intensity. TGF-β1-mediated Foxp3 expression is further enhanced by retinoic acid (RA) and lactoferrin (LF). However, the intensity of TCR signaling required for induction of Foxp3 expression by TGF-β1 in combination with RA and LF is unknown. Here, we found that either RA or LF alone decreased TGF-β1-mediated Foxp3 expression at low TCR signaling intensity. In contrast, at high TCR signaling intensity, the addition of either RA or LF strongly increased TGF-β1-mediated Foxp3 expression. Moreover, decreased CD28 stimulation was more favorable for TGF-β1/LF-mediated Foxp3 expression. Lastly, we found that at high signaling intensities of both TCR and CD28, combined treatment with TGF-β1, RA, and LF induced robust expression of Foxp3, in parallel with powerful suppressive activity against responder T cell proliferation. Our findings that TGFβ/RA/LF strongly generate high affinity Ag-specific iTreg population would be useful for the control of unwanted hypersensitive immune reactions such as various autoimmune diseases.

• IMMUNE NETWORK
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• 제21권3호
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• pp.22.1-22.17
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• 2021

Chitinase-3-like-1 (CHI3L1) is known to induce inflammation in the progression of allergic diseases. Previous our studies revealed that 2-({3-[2-(1-cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284-6111; K284), the CHI3L1 inhibiting compound, has the anti-inflammatory effect on neuroinflammation. In this study, we investigated that K284 treatment could inhibit the development of atopic dermatitis (AD). To identify the effect of K284, we used phthalic anhydride (5% PA)-induced AD animal model and in vitro reconstructed human skin model. We analyzed the expression of AD-related cytokine mediators and NF-κB signaling by Western blotting, ELISA and quantitative real-time PCR. Histological analysis showed that K284 treatment suppressed PA-induced epidermal thickening and infiltration of mast cells. K284 treatment also reduced PA-induced release of inflammatory cytokines. In addition, K284 treatment inhibited the expression of NF-κB activity in PA-treated skin tissues and TNF-α and IFN-γ-treated HaCaT cells. Protein-association network analysis indicated that CHI3L1 is associated with lactoferrin (LTF). LTF was elevated in PA-treated skin tissues and TNF-α and IFN-γ-induced HaCaT cells. However, this expression was reduced by K284 treatment. Knockdown of LTF decreased the expression of inflammatory cytokines in TNF-α and IFN-γ-induced HaCaT cells. Moreover, anti-LTF antibody treatment alleviated AD development in PA-induced AD model. Our data demonstrate that CHI3L1 targeting K284 reduces AD-like skin inflammation and K284 could be a promising therapeutic agent for AD by inhibition of LTF expression.

• IMMUNE NETWORK
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• 제20권5호
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• pp.38.1-38.12
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• 2020

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) that initiate both T-cell responses and tolerance. Tolerogenic DCs (tDCs) are regulatory DCs that suppress immune responses through the induction of T-cell anergy and Tregs. Because lactoferrin (LF) was demonstrated to induce functional Tregs and has a protective effect against inflammatory bowel disease, we explored the tolerogenic effects of LF on mouse bone marrow-derived DCs (BMDCs). The expression of CD80/86 and MHC class II was diminished in LF-treated BMDCs (LF-BMDCs). LF facilitated BMDCs to suppress proliferation and elevate Foxp3⁺ induced Treg (iTreg) differentiation in ovalbumin-specific CD4⁺ T-cell culture. Foxp3 expression was further increased by blockade of the B7 molecule using CTLA4-Ig but was diminished by additional CD28 stimulation using anti-CD28 Ab. On the other hand, the levels of arginase-1 and indoleamine 2,3-dioxygenase-1 (known as key T-cell suppressive molecules) were increased in LF-BMDCs. Consistently, the suppressive activity of LF-BMDCs was partially restored by inhibitors of these molecules. Collectively, these results suggest that LF effectively causes DCs to be tolerogenic by both the suppression of T-cell proliferation and enhancement of iTreg differentiation. This tolerogenic effect of LF is due to the reduction of costimulatory molecules and enhancement of suppressive molecules.

• 미생물학회지
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• 제51권3호
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• pp.271-279
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• 2015

락토페린(LF)는 철이온과 결합하는 당 단백질로서 항균, 항바이러스, 항진균 등의 기능을 가지고 있으며, 생체의 각종 체액으로부터 분비되는 다기능성 단백질이다. 본 연구에서는 사람의 락토페린(hLF)으로부터 유래된 N-lobe의 유전자를 분리하고 산업용 균주로서 많이 사용되는 메탄올자화 효모인 Pichia pastoris에서 발현시켰다. 재조합 사람 락토페린 N-lobe (rhLF-N)는 배양액으로 분비 발현되었으며, 3L 발효조에서 약 $458{\mu}g/ml$이 수준으로 생성되었다. rhLF-N을 정제한 다음 SDS-PAGE와 western blot으로 분석하여 분자량 35 kDa 단백질을 확인하였으며, hLF에 대한 항체를 이용하여 면역확산법으로 면역성을 확인하였다. rhLF-N의 mRNA 발현양상을 qRT-PCR로 분석한 결과 메탄올 첨가에 의한 발현 유도 후 2-3일째에 발현율이 가장 높았으며, 4일째에는 점차적으로 감소하였다. 정제한 rhLF-N을 이용하여 항균활성을 조사한 결과 Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhimurium과 같은 병원성 균에 대해 광범위한 항균활성을 보였으나, LF유래 항균 peptide들과 항균활성을 비교하였을 때, 항균력이 상대적으로 매우 떨어지는 것으로 나타났다. 비록 본 연구에서 발현한 rhLF-N은 항균력은 떨어지나, hLF에 비해 그 크기가 작고 배양조건 연구로 P. pastoris에서 대량 생산이 가능하며, 배양액으로 분비시킬 수 있기 때문에 정제 비용 등을 고려 할 때 산업적 응용에는 보다 유리할 것으로 사료된다.