The primary therapeutic approach for Brucella species infections has mainly been based on antibiotic treatment. However, the development of vaccines for brucellosis control remains controversial. Furthermore, there is currently no licensed vaccine available for human brucellosis. This study aims to evaluate the effect of a combination of recombinant protein vaccines against Brucella (B.) abortus infection using a mouse model. Two B. abortus genes, namely dapB and gpm, were cloned and expressed in competent Escherichia (E.) coli DH5α using the pCold-TF vector. Successfully cloned vectors were subjected to PCR amplification using specific primer pairs. The apparent sizes of dapB and gpm were detected at 807 bp and 621 bp, respectively. Besides, the purified recombinant proteins dapB and gpm were detected using SDS-PAGE electrophoresis with correct sizes of 82.86 kDa and 87.61 kDa, respectively. These recombinant proteins were used to immunize mice as a combined subunit vaccine (CSV) to elicit host immunity against B. abortus infection. Mice immunized with CSV exhibited increased proliferation of CD4+ and/or CD8+ T cells at week 7th and 9th before sacrifice, in comparison to the control group. Notably, CSV immunization showed a significant decrease in bacterial burden in the spleen compared to the control group. Altogether, CSV using dapB and gpm induced host adaptive immune response against Brucella infection, suggesting its potential as an effective new subunit vaccine candidate.

Canine mammary tumors account for ~30% of all tumors in the female dogs and approximately 50% of the tumors are malignant. Exosomes have been the focus of great interest, as they appear to be involved in numerous important cellular processes. In this study, we examined the anti-tumor effects of canine mesenchymal stem cells-derived exosomes (MSC-exosomes) in an experimental murine mammary tumor model using canine mammary carcinoma cells, REM134. The MSC-exosomes were injected tumor site and tail vein of REM134 xenografted mice. We found that tumor size of the MSC-exosomes-treated group decreased compared to those of the only tumor group in REM134-driven tumorigenic mouse model. In addition, the MSC-exosomes-treated tumor group showed meaningfully reduced expression levels of the MMP-3, IL-1β, IL-6, and TNF-α compared to those in the tumor group. Specifically, we confirmed that the expression level of the CD133, potent cancer stem cell (CSC) markers, decreased in the MSC-exosomes-treated tumor group compared to the tumor group. This study suggests that the MSC-exosomes exhibited anti-tumor effects through downregulating CSC-related markers in the canine mammary tumor murine model. Further study is needed in the future, and we are conducting research on the detailed anti-tumor mechanism of the MSC-exosomes.

Amyotrophic lateral sclerosis (ALS) is progressive neurological disease that results in the death of motor neurons in the brain and spinal cord, leading to a decrease in skeletal muscle size and muscle weakness, wasting, or paralysis. Most research on ALS has focused on motor neuron death, and the underlying mechanisms are not well understood. This study examined the molecular mechanisms underlying muscle degeneration. We compared the protein and cytokine profiles of gastrocnemius muscle in ALS model hSOD1G93A mice at pre-symptomatic and symptomatic stages by western blotting. Pro-inflammatory factors including tumor necrosis factor-α, interleukin (IL)-1β and IL-6, and cluster of differentiation 11b were upregulated in the muscle of symptomatic as compared to pre-symptomatic mice. Additionally, the levels of oxidative stress-related proteins, heme oxygenase-1 and ferritin, were increased in muscle from symptomatic as compared to pre-symptomatic mice. We also observed increased autophagy dysfunction and metabolic dysregulation in the muscles of symptomatic hSOD1G93A as compared to non-Tg and pre-symptomatic hSOD1G93A mice, which was accompanied by upregulation of thrombospondin- 1, Prospero-related homeobox 1, glial fibrillary acidic protein, and DNA-damage-inducible 45α. Increased inflammation, oxidative stress, and autophagy contribute to motor neuron death and muscle atrophy in ALS, and the factors involved in these processes are potential therapeutic targets for treatment of this disease.

본 연구는 알츠하이머(Alzheimer’s disease: AD) 형질전환 생쥐를 대상으로 저항성 운동 (resistance exercise: RE)이 해마의 베타 아밀로이드(β-amyloid: Aβ) 단백질 대사, 신경세포사멸 및 인지기능에 미치는 영향을 확인하는데 목적이 있다. AD 비 형질전환 생쥐(non-transgenic: non-tg, n=14) 와 형질전환 생쥐(transgenic: Tg, n=14)를 무선 배정하여 비 형질전환 생쥐 대조군 (non-tg-control: NTC, n=7), 비 형질전환 생쥐 저항성 운동군(non-tg-RE: NTRE, n=7), 형질전환 대조군(tg-control: TC, n=7) 및 형질전환 저항성 운동군(tg-RE: TRE, n=7)으로 구분하였다. RE는 특수 제작한 사다리 저항성 운동 기구를 사용하여 점진적으로 set 수를 증가시켜 총 8주간 실시하였다. 운동 후 인지기능 능력을 평 가하기 위한 수중미로검사와 Aβ 단백질 대사, 신경세포사멸 지표 및 SIRT1/PGC-1α 단백질 발현 수준 을 확인하였다. 수중미로검사 결과 거리와 시간 모두 TC 집단에서 유의하게 증가 되었지만 RE를 실시한 TRE 집단에서 거리와 시간이 감소 되어 인지능력이 개선된 것으로 확인되었다. 또한, TC 집단에서 증가 된 Aβ 단백질 발현은 RE를 통해 감소하는 것으로 나타났다. 신경세포사멸 관련 단백질인 Bcl-2/Bax ratio는 TC 집단에서 유의하게 감소되어 신경세포사멸이 증가 된 것으로 나타났지만 RE는 Bcl-2/Bax ratio을 증가시켜 신경세포사멸을 감소시킨 것으로 확인되었다. TC 집단에서 증가된 BACE1 및 ROCK1 과 감소된 ADAM10과 RARβ 단백질 발현은 RE를 통해 감소되거나 증가 된 것으로 나타났고, SIRT1/ PGC-1α 단백질 발현은 TC 집단에서 감소 되었지만 RE를 통해 증가 된 것으로 나타났다. 따라서 8주간 의 RE는 AD의 병리학적 특징인 Aβ 단백질 발현을 감소시키고 관련 생성 기전들을 조절하여 (SIRT1/PGC-1α 기전 활성, 아밀로이드 생성기전 억제, 비-아밀로이드 생성기전 활성) 신경세포사멸 억제시키고 결과적으로 인지기능을 개선 시킬 수 있는 효과적인 운동 방법이라고 생각된다.

In previous studies, we found the production of antibodies against cross-reactive bovine serum albumin (BSA) in D-galactose (D-gal) induced aging mouse models. We performed immunoblot analysis with mouse tissue lysates to investigate the changes in the overall autoantibody production in this animal model. And we were able to see the possibility of altering the activity of mouse natural antibodies in this process. In this study, we examined changes in existing natural antibodies in a D-gal-induced aging mouse model. Serum samples were collected from 3-week-old mice (3w), 13-week-old mice (13w), and 13-week-old mice that were treated with D-gal for 6 weeks (13wDG), beginning at the age of 8 weeks. Levels of immunoglobulins (IgM, IgG, and IgA) were quantitatively analyzed in serum samples. Tissue samples were obtained from skin, spleen, and ovary for Western blotting analyses. Natural antibody activity was examined by enzyme-linked immunosorbent assay analyses of anti- double-stranded DNA (dsDNA) antibody. Western blotting analyses using mouse tissue lysates showed that several protein bands detected by serum antibodies from 3w mice became increasingly thicker when detection was performed with serum samples from 13w and 13wDG mice, indicating quantitative increases in levels of natural antibodies. Relative amounts of total IgG, IgM, and IgA immunoglobulins sequentially increased in serum samples from 3w, 13w, and 13wDG mice. A similar tendency was observed regarding the levels of IgM and IgG antibodies against dsDNA. These results indicate increased levels of natural antibodies in the D-gal-induced aging mouse model. Therefore, this animal model could be useful for future natural antibody research.

Recent studies showed that tight junctions (TJs) integrity and assembly are required for blastocyst development in mouse and pig models. However, the biological functions of TJs associated with embryo implantation and maintenance of pregnancy were not investigated yet. To examine whether disrupted TJs affect further embryo development, we employed RNAi approach and inhibitor treatment. The embryos were injected with Cxadr (Coxsackievirus and adenovirus receptor) siRNA for knock down (KD) and treated with Adam10 (A Disintegrin and Metalloproteinase specific inhibitor 10; GI254023X; SI). We compared blastocyst development and paracellular sealing assay using FITC dextran uptake between control and KD or SI embryos. Finally, we transferred control and Cxadr KD or Adam 10 SI treated blastocyst to uteri of recipients. Cxadr KD and Adam 10 SI showed lower blastocyst development and more permeable to FITC-dextran. Moreover, we observed that half of KD and inhibited embryos failed to maintain pregnancies after the second trimester. Our findings suggested that TJs integrity is required for the maintenance of pregnancy and can be used as a selective marker for the successful application of assisted reproduction technologies.

폐경은 여성비만의 중요한 원인이다. 본 연구는 폐경여성의 동물모델인 난소절제 암컷 쥐에서 몸무게와 혈청 속 지질 성분의 조절에 대한 제니스테인의 농도 의존적 영향을 수영운동과 비교함으로써, 비만 조절에 대한 제니스테인의 효과적인 농도를 조사하였다. 난소절제 암컷 쥐는 대조군, 수영 운동군, 제니스테인 농도별(0.005%, 0.05%, 0.1% wt/wt) 처리군으로 나누고, 모든 쥐는 고지방식 사료를 8주 동안 섭취하였다. 고지방식 사료를 섭취한 대조군에 비해 수영운동을 실시한 군과 제니스테인이 농도별로 처리된 군 모두 몸무게, 백색지방조직의 무게, 혈청 속 지질 성분 농도 및 간조직의 지질 성분 축적이 감소되었다. 이러한 몸무게, 백색지방조직의 무게, 혈청 속 지질 성분 농도 및 간조직의 지질 성분 축적에 대한 제니스테인의 감소효과는 제니스테인 처리농도에 의존적이었고 제니스테인 농도 0.1%에서 가장 효과적이었으며 1시간 수영운동을 실시한 경우와 유사한 효과를 나타내었다. 본 연구결과들은 난소가 절제된 암컷 쥐에서 적정농도의 제니스테인 처리는 비만개선에 대해 수영운동과 유사한 효과를 나타낸다는 것을 제시한다. 제니스테인 보충제 식이의 섭취는 페경기 여성의 비만예방에 도움을 줄 것이다.

Soy isoflavones have been reported to possess many physiological activities such as antioxidant activity and inhibition of cancer cell proliferation. This study investigated the photoprotective effects of soybean extract in human fibroblast cell line and hairless mice model. Human fibroblast was treated with soybean extract before and after ultraviolet B (UVB; 290-302 nm) irradiation. In the soybean extract treated group, the cells showed better resistance to ultraviolet (UV) than control group. The amount of type I collagen recovered from the soybean treated group was higher than the vehicle group exposed to UV-induced damage. Moreover, increased expression of metalloproteinases-1 as a result of UV irradiation was suppressed by the soybean extract. Female mice were orally administered soybean extract and irradiated with UVB light for 8 weeks. The effects of the soybean extract on the skin appearance, collagen deposition and epidermal thickness in the UV-damaged mouse skin were analyzed using histopathological methods. In soybean extract treated group, the skin had a better morphology than that of the control group. Furthermore, the amount of type I collagen was increased and overexpression of MMP-1 was reduced in the soybean extract group compared to vehicle group. Additionally, up-regulation of pro-inflammatory cytokines induced by UV irradiation was suppressed by dietary soybean extract treatment. It appears that soybean extract had a photoprotective effect, including anti-aging and anti-inflammatory effect, from UV-induced damage in not only human fibroblast, but also hairless mice. We confirmed that these effects were possibly due to promotion of collagen synthesis and inhibition of MMP-1 expression.

Mammalian fetal ovaries contains numerous primordial germ cells, however fewer ones can yield mature oocytes due to apoptosis and follicle atresia. Successful in vitro reconstitution of primordial germ cells has recently had a significant effect in the field of assisted reproductive technologies. However, the regulatory mechanisms underlying oogenesis remain unknown and recapitulation of oogenesis in vitro remains unachieved. Therefore, development of methods for obtaining mature oocytes by culturing the fetal ovaries in vitro could contribute to clarify these mechanisms. We adapt an in vitro system for culturing mouse fetal ovaries that support successful follicle assembly and improve oocyte growth and maturation. Ovarian tissues from 12.5 days postcoitum (dpc) fetal mice were cultured in vitro and the matured oocytes were differentiated from primordial germ cells after a 31 days culture period. Our results demonstrate that mouse fetal germ cells are able to form primordial follicles with artificial ovarian cells, and that oocytes within the growing follicles are able to mature normally in vitro. Taken together, this in vitro culture system is expected to aid in the development of new strategies to identify the reasons behind failure of follicle assembly and offer a platform for innovative research into preservation of female germ cells and conservation of endangered species.

Muscle atrophy is characterized by a decrease in the mass of the muscle. With an increase in life expectancy and chronic illnesses, the incidence of muscle atrophy is increasing and the quality of life of patients is decreasing. Thus, reducing muscle atrophy is of high clinical and socio-economic importance. Mistletoe is a semi-parasitic plant that has been used as a traditional medicine in many countries to treat various human illnesses. It has been reported that Korean mistletoe extract (KME) has diverse biological functions including anti-tumor, anti-oxidant, anti-diabetic, anti-obesity properties, and extension of lifespan. Especially, we have recently reported that KME improves exercise endurance in mice, indicating its beneficial roles in enhancing the capacity of skeletal muscle. In this study, we investigated whether KME could activate the signaling pathway related to protein synthesis in a mouse model of muscle atrophy. Interestingly, KME efficiently activated the Akt/mTOR pathway, and Akt and mTOR are important signaling hub molecules for the acceleration of protein synthesis in muscle cells. In addition, KME also increased the activity of S6 kinase which is involved in the regulation of muscle cell size. Moreover, the ERK activity, required for transcription of ribosomal RNA for protein synthesis, was also enhanced in KME-treated mouse muscle. These data support the idea that KME increases muscle mass via increased protein synthesis. Our findings also suggest that Korean mistletoe might be a promising candidate for the development of functional foods that are beneficial for preventing muscle atrophy.

Bitter melon (Momordica charantia) is used in traditional herbal medicine in many Asian countries for the treatment of several diseases such as diabetes, eczema, night blindness, psoriasis, and rheumatism. Especially, most reports concerning the biological activities of bitter melon have focused on its effects on diabetes and hyperglycemia. Also, bitter melon is regarded as a longevity food, suggesting that it has several beneficial effects on anti-aging and the maintenance of a healthy state. Thus, we investigated whether bitter melon could increase the capacity of exercise in this study. Interestingly, bitter melon fruit extract activated AMP-activated protein kinase (AMPK), which is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. In addition, bitter melon extract increased the expression of enzymes involved in fatty acid oxidation such as mitochondrial uncoupling protein 3 (UCP3), carnitine palmitoyl transferase 1b (CPT1b), and pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4). Moreover, exercise tolerance was much more enhanced in bitter melon treated animals compared to the non-treated control group. These results suggest that bitter melon is a promising candidate for the development of functional foods beneficial for physical strength and the enhancement of exercise capacity.

The objective of the present study was to examine the effects of β-glucan originating from Aureobasidium on full-thickness skin wound healing in diabetic C57BL/KsJ-db/db mouse models. In the diabetic C57BL/KsJ-db/db model, test articles were topically applied twice a day for 20 days starting from 1 day after wounding. The results were compared to that of MadecassolTM ointment (madecassol; 1% Centella asiatica extracts) topically applied at a concentration of 100 mg/kg. Treatment with β-glucan resulted in significant (p<0.01 or p<0.05) and dose-dependent decreases in wound size compared with that of vehicle control showing increased wound size (WS, %). In addition, 50% contraction time (CT50) was dramatically and dose-dependently reduced, and inflammatory cells in granulation tissues of the wound area were significantly (p<0.01 or p<0.05) and dose-dependently reduced compared with that of vehicle control showing increased numbers of micro-vessels and fibroblasts as well as re-epithelialization. In the madecassol group, similar changes in inflammatory cells and fibroblasts with re-epithelialization were also observed, but madecassol did not influence angiogenesis. No meaningful changes in body weight were detected in all tested groups compared with the vehicle control. Therefore, these data suggest that β-glucan has a beneficial effect on diabetic delayed skin wound healing and may be useful to manage incurable skin wounds in diabetic animals.

Calcium exerts antiproliferative effects on cellular targets through the promotion of differentiation and apoptosis. We investigated the influence of calcium on the formation of colonic aberrant crypt foci (ACFs), which were induced by exposure to azoxymethane (AOM) followed by dextran sodium sulfate (DSS), in ICR mice. Six-week-old ICR mice received 3 (weeks 0–2) intraperitoneal injections of AOM (10 mg/kg BW), followed by treatment with 2% DSS via drinking water for a week to induce preneoplastic lesions. The mice were then divided into 3 groups: the control (AOM/DSS), AOM/DSS + 1.0% Ca, and AOM/DSS + 2.0% Ca groups. Calcium (1.0 or 2.0%) was administered via drinking water for 12 weeks. After sacrificing the mice, the total numbers of aberrant crypts (ACs) and ACFs were measured in the colonic mucosa after methylene blue staining. The control group displayed 11.58 ± 2.43 ACFs/colon, which were composed of a total of 30.42 ± 5.18 ACs/colon. The number of ACFs with more than 3 ACs, which are likely to progress to colon cancer, was 2.37 ± 0.68. Compared to the control, 1.0% or 2.0% calcium treatment significantly decreased the number of total ACFs and ACs in a concentration-dependent manner. The decrease in ACFs or ACs after calcium treatment was associated with decreases in cell proliferation and β-catenin expression and an increase in apoptosis in colonic mucosal cells. These results suggest that calcium may exert a protective effect against colon cancer by inhibiting the development of ACFs/ACs in ICR mice.

Recombinant thymosin β4 (rTβ4) has been reported to migrate and promote vascularization, wound-healing, and hair growth in a mouse hindlimb ischemia model of peripheral vascular disease. C57BL/6 mice (11-weeks-old) were anesthetized and an ischemic model was made by cutting the right aorta femoralis. The ischemic group was intraperitoneally administered with saline (300 μL/mouse) and the muscular administration group received rTβ4 (150 μg in 300 μL of saline) or rTβ4 (150 μg in 300 μL saline) to the abdominal cavity at 3-day intervals for 21 days. Myoatrophy of the ischemic group was observed compared to the normal control group. Generation of adjacent vessels was carried out in the rTβ4 administration group compared to the ischemic group. The biopsy results showed significant fibrosis around the muscular undersurface and perimysium in the musculus quadriceps femoris of the ischemic group, whereas partial fibrosis was observed in the perimysium and endomysium in the rTβ4 administration group. Immunostaining indicated that expression levels of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor-1 (VEGF-1), and endothelial nitric oxide synthase (eNOS) in the rTβ4 group were higher than those of the ischemic group. Western blotting showed that expression levels of HIF-1α, VEGF-1, and eNOS in the rTβ4 group were higher than those of the ischemic group. In conclusion, rTβ4 increases expression levels of HIF-1α, VEGF-1, and eNOS, resulting in angiogenesis.

Human natural killer (NK) cells are major players in innate immune response. The functions of these cells as a scavenger of cancer cells are enhanced by cytokines such as interleukin-2 (IL-2), which play an important role in immune response in both tumors and virally infected cells. Liver cancer has a high incidence rate and is a major cause of death in Korea. We provide evidence that human NK cells inhibit tumor growth of the hepatocellular carcinoma cell line SNU-354. NK cells were cultured with human IL-2 for 14 days, yielding an enriched NK cell population containing 35% CD8+ cells, 6% CD4+ cells, and 51% CD16+ /CD56+ cells. Intravenous injection of NK cells at doses from 2.5 to 10 million cells/mouse was administered once per week in a nude mouse model that retains human liver tumor induced by implantation of SNU-354 cells. The results showed that human NK cells were recruited within tumor tissue and inhibited SNU-354 tumor growth by 32%, 58%, and 65%. The current data suggest the potential for use of NK cell-based immunotherapy for treatment of human liver cancer.

Phosphoprotein Enriched in Astrocytes (PEA15) is a 15kD-sized intracellular signaling protein, highly expressed in astrocytes and constitutively expressed in peripheral tissues. Recently it was found that PEA15 expression was elevated in patients suffering type 2 diabetes and suggested to be involved in the syndrome of insulin resistance. To investigate the functional role of PEA15 for the control of blood glucose level, we produced a transgenic pig over-expressing mouse PEA15 (mPEA15). As a model animal, pig has many advantages. They have a higher fecundity and a short generation time and are physiologically similar to human. Using the transgenic pig, we carried out a series of experiments to establish a link between PEA15 expression and the insulin resistance. Our results suggested that, compared with control pig, mPEA15 pig has, (1) a higher blood resistin level, (2) a lower cell membrane-embeded GLUT4 level, and (3) a lower glucose clearing ability based on oral glucose tolerance test (OGTT). When our results combined, it can be concluded that mPEA15 over-expressing pig has many symptoms of insulin resistance and these pigs will become a useful disease model to investigate diabetes mellitus in the near future.