• Parasites, Hosts and Diseases
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• v.60 no.2
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• pp.117-126
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• 2022

Cystatin, a cysteine protease inhibitor found in many parasites, plays important roles in immune evasion. This study analyzed the molecular characteristics of a cystatin from Fasciola hepatica (FhCystatin) and expressed recombinant FhCystatin (rFhcystatin) to investigate the immune modulatory effects on lipopolysaccharide-induced proliferation, migration, cytokine secretion, nitric oxide (NO) production, and apoptosis in mouse macrophages. The FhCystatin gene encoded 116 amino acids and contained a conserved cystatin-like domain. rFhCystatin significantly inhibited the activity of cathepsin B. rFhCystatin bound to the surface of mouse RAW264.7 cells, significantly inhibited cell proliferation and promoted apoptosis. Moreover, rFhCystatin inhibited the expression of cellular nitric oxide, interleukin-6, and tumor necrosis factor-α, and promoted the expression of transforming growth factor-β and interleukin-10. These results showed that FhCystatin played an important role in regulating the activity of mouse macrophages. Our findings provide new insights into mechanisms underlying the immune evasion and contribute to the exploration of potential targets for the development of new drug to control F. hepatica infection.

• Nutrition Research and Practice
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• v.15 no.3
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• pp.319-328
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• 2021

BACKGROUND/OBJECTIVES: Curcuma zedoaria R. (Zingiberaceae) has been used to treat headache, fever, and hypertension-related symptoms in Asian countries, including Korea, China, and Japan. We investigated whether dietary intake of a C. zedoaria extract (CzE) affected atherosclerosis in vivo. MATERIALS/METHODS: Apolipoprotein E-deficient (ApoE^-/-) mice (n = 32) were fed a normal diet (ND), a high-cholesterol diet (HCD), an HCD containing CzE (100 mg/kg/day), or an HCD containing simvastatin (10 mg/kg/day) for 12 weeks. The anti-atherosclerotic effects were evaluated by observing changes in fatty streak lesions, immunohistochemical analysis, ex vivo fluorescence imaging, lipid profiles, and western blot analysis. RESULTS: The CzE-fed group showed a 41.6% reduction of atherosclerosis. Furthermore, CzE significantly reduced the levels of serum triglyceride, high-density lipoprotein, the chemokine (C-X3-C-motif ) ligand 1, the adhesion molecules vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin; down-regulation of tumor necrosis factor-α, interleukin-6, high mobility group box-1, and cathepsin levels in the aortic sinuses and aortas of ApoE^-/- mice were also observed. CONCLUSIONS: The results suggest that the inclusion of a water extract of C. zedoaria in a HCD is closely correlated with reducing the risk of vascular inflammatory diseases in an ApoE mouse model.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.7 no.1
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• pp.33-40
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• 2021

Recently, antibody-drug conjugates (ADCs) are used to deliver efficient cytotoxic payloads selectively in cancer cells. In the designing of an ADC, the antibody is connected to a toxic payload via a covalent linker, which helps to solubilizes the typical hydrophobic payload as well as stabilizes the linkage over circulation. The development of the linkers for the antibody drug conjugate is still in demand. Initially, the acid, disulfide, and cathepsin-sensitive ADCs attracted considerable attention for the delivery of a potent cytotoxic payload but suffer from instability in human and mouse plasma with a short half-life. In addition, It also suffer from a solubility issue that induces aggregation, which is the major problem in their development. ADCs associated with sulfatase and phosphatase cleavable linker are highly soluble due to the anionic nature of sulfate and phosphate groups. The ADCs also showed high stability in human and mouse plasma. Therefore, to overcome these limitations, sulfatase and phosphatase cleavable linkers were developed. This review focuses on the recently reported advantages of sulfatase and phosphatase cleavable linkers for ADCs.

• Food Science of Animal Resources
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• v.42 no.2
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• pp.266-279
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• 2022

This study was conducted to evaluate the proteolysis trends and change in meat quality during 10 days of cold storage in duck M. pectoralis major (PM) and M. iliotibialis (IL). Duck IL had a higher pH and greater degree of lightness but lower cooking loss than PM (p<0.05). During the 10-day cold storage, the pH value of PM declined significantly (p<0.05), while the meat quality traits of IL were not affected by cold storage (p>0.05). In PM, the redness increased from day 1 to day 5, while cooking loss was lower on day 10 compared to day 5 (p<0.05). There were no significant differences in the activities of cathepsin B and proteasome 20S during cold storage (p>0.05). The activity of calpains declined gradually during 10 days of storage (p<0.05), and the activity of calpains in PM was higher than that in IL (p<0.05). A total of 5,155 peptides were detected and derived from 34 proteins of duck PM muscle, whereas 4,222 peptides derived from 32 proteins were detected from duck IL muscle. Duck PM muscle was composed only of fast type of muscle fiber, whereas IL muscle was composed of both slow and fast types. The proteins responsible for glycolysis or myofibrillar proteins were closely related to changes in meat color or water-holding capacity during cold storage. These results suggest that changes in meat quality characteristics during cold storage are closely related to protein degradation, which is also related to the distribution of muscle fiber types.

• Biomolecules & Therapeutics
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• v.32 no.2
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• pp.205-213
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• 2024

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.

• BMB Reports
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• v.57 no.6
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• pp.293-298
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• 2024

Microtubule acetylation has been shown to regulate actin filament dynamics by modulating signaling pathways that control actin organization, although the precise mechanisms remain unknown. In this study, we found that the downregulation of microtubule acetylation via the disruption ATAT1 (which encodes α-tubulin N-acetyltransferase 1) inhibited the expression of RhoA, a small GTPase involved in regulating the organization of actin filaments and the formation of stress fibers. Analysis of RHOA promoter and chromatin immunoprecipitation assays revealed that C/EBPβ is a major regulator of RHOA expression. Interestingly, the majority of C/EBPβ in ATAT1 knockout (KO) cells was found in the nucleus as a 27-kDa fragment (referred to as C/EBPβ^p27) lacking the N-terminus of C/EBPβ. Overexpression of a gene encoding a C/EBPβ^p27-mimicking protein via an N-terminal deletion in C/EBPβ led to competitive binding with wild-type C/EBPβ at the C/EBPβ binding site in the RHOA promoter, resulting in a significant decrease of RHOA expression. We also found that cathepsin L (CTSL), which is overexpressed in ATAT1 KO cells, is responsible for C/EBPβ^p27 formation in the nucleus. Treatment with a CTSL inhibitor led to the restoration of RHOA expression by downregulation of C/EBPβ^p27 and the invasive ability of ATAT1 KO MDA-MB-231 breast cancer cells. Collectively, our findings suggest that the downregulation of microtubule acetylation associated with ATAT1 deficiency suppresses RHOA expression by forming C/EBPβ^p27 in the nucleus through CTSL. We propose that CTSL and C/EBPβ^p27 may represent a novel therapeutic target for breast cancer treatment.

• Food Science of Animal Resources
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• v.44 no.5
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• pp.1055-1068
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• 2024

The differences in the proteolytic patterns and shear force of pork and beef during aging were evaluated. Pork and beef semitendinosus muscles were obtained at 24 and 48 h postmortem, respectively, and aged at 4℃ for 0 (Day 0), 7 (Day 7), and 14 days (Day 14). Changes in the electrical conductivity were observed in pork on Day 7 and beef on Day 14. The calpain activity increased in pork (p<0.05) after 14 days of aging, whereas that of beef decreased on Day 7 (p<0.05). The cathepsin B activity in pork and beef increased between Day 7 and 14 (p<0.05). The content of α-amino group in the 10% trichloroacetic acid-soluble fraction increased between Day 7 and 14 in pork (p<0.05), but increased steadily in beef throughout aging (p<0.05). The electrophoretogram of the myofibrillar proteins revealed a 30 kDa protein band only in the beef lane on Day 14. The cooked pork had no significant changes in the shear force during aging periods (p>0.05), while the gradual decrease in the shear force with the increasing aging periods was shown in the cooked beef (p<0.05). Circular dichroism analysis of myosin extracts from pork and beef revealed thermal denaturation temperatures of 55℃ and 58℃, respectively. This study highlights the different post-mortem proteolytic patterns and thermal denaturation temperatures of myosin in pork and beef semitendinosus muscles, which contribute to distinct changes in the shear force during aging between pork and beef.

• International Journal of Molecular Medicine
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• v.44 no.3
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• pp.913-926
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• 2019

Leonurus sibiricus L. (LS) is a medicinal plant used in East Asia, Europe and the USA. LS is primarily used in the treatment of gynecological diseases, and recent studies have demonstrated that it exerts anti-inflammatory and antioxidant effects. To the best of our knowledge, the present study demonstrated for the first time that LS may promote osteoblast differentiation and suppress osteoclast differentiation in vitro, and that it inhibited lipopolysaccharide (LPS)-induced bone loss in a mouse model. LS was observed to promote the osteoblast differentiation of MC3T3-E1 cells and upregulate the expression of runt-related transcription factor 2 (RUNX2), a key gene involved in osteoblast differentiation. This resulted in the induction of the expression of various osteogenic genes, including alkaline phosphatase (ALP), osteonectin (OSN), osteopontin (OPN), type I collagen (COL1) and bone sialoprotein (BSP). LS was also observed to inhibit osteoclast differentiation and bone resorption. The expression levels of nuclear factor of activated T-cells 1 (NFATc1) and c-Fos were inhibited following LS treatment. NFATc1 and c-Fos are key markers of osteoclast differentiation that inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced mitogen-activated protein kinase (MAPKs) and nuclear factor (NF)-κB. As a result, LS suppressed the expression of osteoclast-associated genes, such as matrix metallopeptidase-9 (MMP-9), cathepsin K (Ctsk), tartrate-resistant acid phosphatase (TRAP), osteoclast-associated immunoglobulin-like receptor (OSCAR), c-src, c-myc, osteoclast stimulatory transmembrane protein (OC-STAMP) and ATPase H+ transporting V0 subunit d2 (ATP6v0d2). Consistent with the in vitro results, LS inhibited the reduction in bone mineral density and the bone volume/total volume ratio in a mouse model of LPS-induced osteoporosis. These results suggest that LS may be a valuable agent for the treatment of osteoporosis and additional bone metabolic diseases.

• The Korean Journal of Pharmacology
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• v.18 no.2
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• pp.15-25
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• 1982

It was undertake to investigate the factors involved in the micro thrombus formation in the plasma from the patients with cerebrovascular disease(CVD) and the in vitro actions of sodium nitroprusside on the platelet aggregate formation. 1) The microthrombus formation in the plasma from CVD was significantly enhanced, in comparison with that from the healthy volunteers. 2) Both lipid peroxide and cathepsin D in the plasma from CVD were higher than those levels from the healthy volunteers. 3) Whereas the platelets from healthy individuals showed less aggregation activity in response to ADP in the second phase those from CVD revealed the enhanced aggregating response to ADP. 4) When the bovine basilar artery, rabbit aorta and human umbilical artery were pretreated with $K^+-free$ PSS, ouabain, 13-hydroperoxylinoleic acid(13-HPLA) and cadmium they markedly enhanced the platelet aggregability respectively. 5) Platelet aggregation induced by $K^+-free$ PSS-treated bovine basilar artery was decreased by sodium nitroprusside in a dose-dependent manner, but not by either hydralazine. 6) Both dibutyryl cyclic AMP and 8-bromo cyclic GMP had the inhibitory action on the platelet aggregation. However, the latter had more prominent action than former. The antiaggregating effect by sodium nitroprusside was antagonized by pretreatment with methylene blue, but not by hemoglobin. These results provide the evidences for the therapeutic use of sodium nitroprusside in the emergency of cerebrovascular disease and in remains the further study of the clinical therapy with it.

• Journal of Acupuncture Research
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• v.19 no.2
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• pp.51-64
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• 2002

We have compared(using the same series of experimental tissue samples) the levels of proteolytic enzyme activities and free radical-induced protein damage in synovial fluid from RA and CPH cases. Many protease types showed significantly increased (typically by a factor of approximately 2-3-fold) activity in RA, compared to normal rats. However, CPH significantly reduced the cytoplasmic enzyme activities of arginyl aminopeptidase, leucyl aminopeptidase, pyroglutamyl aminopeptidase, tripeptidyl aminopeptidase, and proline endopeptidase to almost about 1/10 each. For the Iysosomal proteases, synovial fluid samples from RA rats, CPH significantly reduced the enzyme activities of cathepsin B, dipeptidyl aminopeptidase I and dipeptidyl aminopeptidase II. In extracellular matrix degrading(collagenase, tissue elastase) and leukocyte as sociated proteases (leukocyte elastase, cathepsin G), CPH decreased these enzyme activities of collagenase, tissue elastase and leukocyte associated elastase in RA. In cytoplasmic and lysosomal protease activities in plasma from RA. CPH and normal plasma samples were not significantly different, suggesting that altered activity of plasma proteases (particularly those enzymes putatively involved in the immune response) is not a contributory factor in the pathogenesis of RA. In addition, the level of free radical induced damage to synovial fluid proteins was approximately twice that in RA, compared with CPH. CPH significantly decreased the level of ROS induced oxidative damage to synovial fluid proteins (quantified as protein carbonyl derivative). Therefore we conclude that both proteolytic enzymes and free radicals are likely to be of equal potential importance as damaging agents in the pathogenesis of inflammatory joint disease, and that the design of novel therapeutic strategies for patients with the latter disorder should include both protease inhibitory and free radical scavenging elements. In addition, the protease inhibitory element should be designed to inhibit the action of a broad range of protease mechanistic types (i.e. cysteine-, metallo- and serine- proteinases and peptidases). However, increased protein damage induced by ROS could not be rationalised in terms of compromised antioxidant total capacity, since the latter was not significantly altered in RA synovial fluid or plasma compared with CPH.