CDC5L promotes early chondrocyte differentiation and proliferation by modulating pre-mRNA splicing of SOX9, COL2A1, and WEE1.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a common pathological condition that causes intractable myelopathy and radiculopathy, mainly the result of an endochondral ossification-like process. Our previous genome-wide association study identified six susceptibility loci for OPLL, including the cell division cycle 5-like (CDC5L) gene region. Here, we found CDC5L to be expressed in type II collagen-producing chondrocyte-like fibroblasts in human OPLL specimens, as well as in differentiating ATDC5 chondrocytes. Cdc5l siRNA transfection in murine chondrocytes decreased the expression of the early chondrogenic genes Sox9 and Col2a1, diminished the cartilage matrix production, and enhanced the expression of parathyroid-hormone-related protein (a resting chondrocyte marker). We also showed that Cdc5l shRNA suppressed the growth of cultured murine embryonal metatarsal cartilage rudiments and that Cdc5l knockdown suppressed the growth of ATDC5 cells. Fluorescence-activated cell sorting analysis revealed that the G2/M cell cycle transition was blocked; our data showed that Cdc5l siRNA transfection enhanced expression of Wee1, an inhibitor of the G2/M transition. Cdc5l siRNA also decreased the pre-mRNA splicing efficiency of Sox9 and Col2a1 genes in both ATDC5 cells and primary chondrocytes; conversely, loss of Cdc5l resulted in enhanced splicing of Wee1 pre-mRNA. Finally, an RNA-binding protein immunoprecipitation assay revealed that Cdc5l bound directly to these target gene transcripts. Overall, we conclude that Cdc5l promotes both early chondrogenesis and cartilage growth and may play a role in the etiology of OPLL, at least in part by fine-tuning the pre-mRNA splicing of chondrogenic genes and Wee1, thus initiating the endochondral ossification process.

Host lactosylceramide enhances Edwardsiella tarda infection.

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-ß-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.

Assessment of Early Biological Fixation of Cementless Tapered-Wedge Stems Using Digital Tomosynthesis.

BACKGROUND: We aimed to compare radiographic and digital tomosynthesis assessments of early biological fixation of a cementless stem in primary total hip arthroplasty and to investigate the factors associated with early biological fixation. METHODS: Seventy-three patients underwent total hip arthroplasty using cementless short tapered-wedge stems. Both radiography and digital tomosynthesis were performed at 6 weeks and 3, 6, 12, and 24 months after surgery. The presence of spot welds (SW) was evaluated at each postoperative period to assess biological fixation between the stem and the femur. The area of contact between the femur and the stem was divided into seven zones based on Gruen's zone classification. RESULTS: All 73 patients had no SW 6 weeks after surgery on radiography and digital tomosynthesis. Three months postoperatively, there was no SW on radiography; however, digital tomosynthesis revealed SW in 31 (42%) patients. Six months postoperatively, radiography showed 22 SW in 18 (24.7%) patients and digital tomosynthesis showed 94 SW in 48 patients (65.8%). CONCLUSION: Digital tomosynthesis detected biological fixation between the stem and femur earlier than radiography; biological fixation may appear within 3 months after surgery.

Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness.

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2-3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Regulation of triglyceride metabolism in medaka (Oryzias latipes) hepatocytes by Neu3a sialidase.

Fish store triglycerides (TGs) in the liver, muscle, and adipose tissue and TGs constitute an energy source upon metabolic demand. The liver generally plays important roles in lipid metabolism. Recent studies have suggested the possibility of hepatic lipid metabolic regulation by ganglioside in mammals; however, ganglioside-mediated regulation of lipid metabolism is unclear in fish. This study aimed to clarify the role of ganglioside in fish TG metabolism, with particular reference to Neu3a, a ganglioside-specific sialidase expressed in the fish liver. Under fasting conditions, there was a decrease in hepatic TG contents, and neu3a mRNA level was significantly up-regulated in the medaka liver. To determine the role of Neu3a in hepatic lipid metabolism, Neu3a stable transfectants were generated using fish liver Hepa-T1 cells. After treating Neu3a cells with oleic acid, reduction of TG was detected in comparison with the mock cells. Furthermore, lipase activity was greater in Neu3a cells than in mock cells. To examine which ganglioside regulates these events, alterations of ganglioside composition in Neu3a cells were analyzed. Neu3a cells exhibited increased level of lactosylceramide (LacCer), a Neu3 enzymatic product originating from GM3. In addition, exposure of LacCer toward Hepa-T1 cells resulted in an increase of neutral lipase activity. The present results suggest that Neu3a up-regulation in medaka under fasting condition accelerates hepatic TG degradation for energy production via GM3 desialylation.

Anatomical relationship between insertion sites, tunnel placement, and lateral meniscus anterior horn injury during single and double bundle anterior cruciate ligament reconstructions: A comparative macroscopic and histopathological evaluation in cadavers.

PURPOSE: The influence of tunnel extension outside the anatomical anterior cruciate ligament (ACL) insertion in single-bundle (SB) or double-bundle (DB) ACL reconstruction is unclear. This study aimed to investigate the anatomical relationship between ACL insertion and tunnel extension in SB and DB ACL reconstruction, and the impact of tibial tunnel extension to the insertion of anterior horn of lateral meniscus in terms of injury. METHODS: Forty-six paired cadaver knees (mean age, 82.7 ± 10.7 years) were used. Right and left knees were used for SB (10 mm) and DB tunnel reaming (6 mm for the anteromedial and posterolateral bundles). Tibial and femoral tunnels were created to aim at the center of the ACL insertion by arthroscopic visualization. The relationship between tunnel extension and ACL insertion was evaluated macroscopically, and there ratio in two groups were compared by chi-square test. Further, the relative risk for meniscus injury based on tunnel placement was estimated. Coronal section of tibia and parallel section to Blumensaat line in femur were prepared to evaluate the relationship among tunnel position, ACL insertion, and anterior horn of the meniscus histologically. RESULTS: Tibial tunnel extension out of the ACL insertion was observed macroscopically in 9 (39.1%) knees of the SB group, and 3 (13.0%) of the DB group (p = 0.045). In femoral tunnels, extension out of the ACL insertion was seen in 8 (34.8%) knees of the SB group and 1 (4.3%) of the DB group (p = 0.011). Partial injuries of the lateral meniscus anterior horn (LMAH) were observed in 5 (21.7%) knees of the SB group and 1 (4.3%) knee of the DB group (p = 0.091). The relative risk for LMAH injury was calculated as 5.0 (odds ratio, 6.1). Microscopically, SB tunnels appeared to expand out of ACL insertion, both in the femur and tibia. CONCLUSIONS: The incidence of tunnel extension out of the ACL insertion in femur and tibia were higher with SB than with DB reconstruction. Furthermore, injury rate of the LMAH in the DB group was lower.

Recombinant sialidase NanA (rNanA) cleaves α2-3 linked sialic acid of host cell surface N-linked glycoprotein to promote Edwardsiella tarda infection.

Edwardsiella tarda is one of the major pathogenic bacteria affecting both marine and freshwater fish species. Sialidase NanA expressed endogenously in E. tarda is glycosidase removing sialic acids from glycoconjugates. Recently, the relationship of NanA sialidase activity to E. tarda infection has been reported, however, the mechanism with which sialidase NanA aids the pathogenicity of E. tarda remained unclear. Here, we comprehensively determined the biochemical properties of NanA towards various substrates in vitro to provide novel insights on the potential NanA target molecule at the host cell. GAKS cell pretreated with recombinant NanA showed increased susceptibility to E. tarda infection. Moreover, sialidase inhibitor treated E. tarda showed a significantly reduced ability to infect GAKS cells. These results indicate that NanA-induced desialylation of cell surface glycoconjugates is essential for the initial step of E. tarda infection. Among the natural substrates, NanA exhibited the highest activity towards 3-sialyllactose, α2-3 linked sialic acid carrying sialoglycoconjugates. Supporting this finding, intact GAKS cell membrane exposed to recombinant NanA showed changes of glycoconjugates only in α2-3 sialo-linked glycoproteins, but not in glycolipids and α2-6 sialo-linked glycoproteins. Lectin staining of cell surface glycoprotein provided further evidence that α2-3 sialo-linkage of the N-linked glycoproteins was the most plausible target of NanA sialidase. To confirm the significance of α2-3 sialo-linkage desialylation for E. tarda infection, HeLa cells which possessed lower amount of α2-3 sialo-linkage glycoprotein were used for infection experiment along with GAKS cells. As a result, infection of HeLa cells by E. tarda was significantly reduced when compared to GAKS cells. Furthermore, E. tarda infection was significantly inhibited by mannose pretreatment suggesting that the bacterium potentially recognizes and binds to mannose or mannose containing chains following desialylation. Together, these results suggest that E. tarda may employ endogenous NanA to desialylate α2-3 glycoproteins on host cells, thus revealing one of the potential binding molecules during infection.

Mobile bearing shows larger rollback motion than fixed bearing in total knee arthroplasty using a medial stabilising technique with a navigation system.

Purpose: This study aimed to investigate the intraoperative knee kinematics of cruciate-retaining total knee arthroplasty with a medial stabilising technique (MST-TKA) and compare the kinematics between mobile- and fixed-bearing MST-TKAs. We hypothesised that mobile-bearing MST-TKA would result in greater physiological kinematic motion than fixed-bearing MST-TKA. Methods: Twenty-one and 20 knees underwent mobile- and fixed-bearing MST-TKAs using a navigation system (Orthopilot® ver. 6.0; B. Braun Aesculap), respectively. In the preoperative and postoperative kinematic analysis, the knee was moved manually from 0° to 120°, and femoral anteroposterior translations of the medial femoral condyle (MFC) and lateral femoral condyle (LFC) were recorded every 0.1 s from 0° to 120°. Data were subsequently extracted from the software every 10° of flexion and compared between the two groups, and the correlation coefficients between preoperative and postoperative kinematics were calculated. Results: In the postoperative analysis, the MFC in the mobile-bearing group showed significant posterior translation at 100°, 110° and 120° compared to the fixed-bearing group (p < 0.01). Similarly, the LFC in the mobile-bearing group showed significant posterior translation at 100°, 110° and 120° compared to the fixed-bearing group (p < 0.05, p < 0.01 and p < 0.05, respectively). In the mobile-bearing group, the preoperative and postoperative anteroposterior translations of the MFC and LFC were correlated (p < 0.01), while in the fixed-bearing group, there was no correlation. Conclusion: The femoral rollback motion in the mobile-bearing MST-TKA correlated with the preoperative kinematics and was larger than that in the fixed-bearing group. Level of Evidence: Level II, therapeutic prospective cohort study.

Bilateral Sleeve Fracture of the Patella in a Healthy 11-Year-Old Male: A Case Report.

Bilateral sleeve fracture of the patella (SFP) in skeletally immature children is a rare injury. We report the case of a healthy 11-year-old male who suffered bilateral SFP while playing tag. The avulsed fragments of his left patella were highly comminuted. Open reduction and internal fixation (ORIF) were performed using suture anchors, and the knees were immobilized using a cylinder cast for three weeks. At the one-year follow-up assessment, both knees were found to have regained full strength with no extension lag. However, we observed malunion due to lateral shift of the avulsed fragment, cystic lesions, and clicking in the patella, and the patient experienced residual pain in the left knee. Based on this, we conclude that the sleeve fracture of the patella with comminuted cartilaginous fragments was difficult to treat and might have led to poor clinical results if anatomical reduction and fixation had not been performed.

The Relationship between LRP6 and Wnt/ß-Catenin Pathway in Colorectal and Esophageal Cancer.

High expression of low-density lipoprotein receptor-related protein 6 (LRP6), a key component of the Wnt/ß-catenin signaling pathway, is reported to be associated with malignant potential in some solid tumors including breast cancer and hepatocellular carcinoma. Few reports, however, have examined its function and clinical significance in colorectal cancers (CRC) demonstrating constitutive activation of Wnt signaling. Here, we compared the expression level and function of LRP6 in CRC with that of esophageal squamous cell carcinoma (ESCC) bearing few Wnt/ß-catenin pathway mutations. On immunohistochemical staining, high LRP6 expression was noted in three of 68 cases (4.4%), and high ß-catenin in 38 of 67 cases (56.7%) of CRC. High LRP6 expression was found in 21 of 82 cases (25.6%), and high ß-catenin expression in 29 of 73 cases (39.7%) of ESCC. In our in vitro studies, LRP6 knockdown hardly changed Wnt signaling activity in CRC cell lines with mutations in Wnt signaling downstream genes. In contrast, in ESCC cell lines without Wnt signaling-related mutations, LRP6 knockdown significantly decreased Wnt signaling activity. LRP6 function may depend on constitutive activation of Wnt signaling.

Effectiveness of photofunctionalized titanium alloy on osseointegration in rats with type 2 diabetes.

BACKGROUND: Ultraviolet (UV) light-mediated photofunctionalization improves the osseointegration of pure titanium and titanium alloy (Ti6Al4V). However, little is known about the effect of UV irradiation on Ti6Al4V, used frequently in orthopedic surgery, in diabetic patients. We examined the effect of UV irradiation on Ti6Al4V in rats with type 2 diabetes. METHODS: Cylinder Ti6Al4V implants were used. Half the animals were Sprague Dawley rats (the control group), and the other half were Spontaneously Diabetic Torii fatty rats (the diabetes mellitus model). For radiological analysis, bone density was observed and calculated using 3D microcomputed tomography. Histological analysis was performed to calculate the bone-implant contact (BIC) ratio. We used Pearson correlation to analyze the correlation between average blood glucose level and BIC ratio, and between average blood glucose level and bone volume (BV) ratio. RESULTS: In the UV light-treated group, the BIC ratios of the normal and diabetic rats increased significantly compared with those in the untreated group at 2 weeks; at 4 weeks, the BIC ratio of the diabetic rats increased significantly, but there was no significant increase in the control animals. In both the control and diabetic groups, there was no significant difference in the BV ratios between the UV-treated and untreated implants at 2 or 4 weeks. The average blood glucose level in the 4-week group negatively correlated with the BIC and BV ratios. The average blood glucose level in the UV-treated group negatively correlated with the BIC ratio. CONCLUSION: Photofunctionalization of Ti6Al4V implants may promote osseointegration in the early stages in rats with type 2 diabetes.

Radiographic assessment of radiolucent lines around a highly porous titanium cup (Tritanium) using digital tomosynthesis, after total hip arthroplasty.

BACKGROUND: The objectives of this study were to assess radiolucent lines around a highly porous titanium cup (Tritanium) using digital tomosynthesis and to investigate the clinical and radiographic factors associated with radiolucent lines on tomosynthesis. METHODS: Fifty-five patients underwent total hip arthroplasty using a Tritanium cup, and digital tomosynthesis and plain radiography were performed at 1 week, 6 months, 1 year, and 2 years after surgery. The radiolucent lines around the cup were measured on both DTS and plain radiography at each postoperative period. Clinical evaluations were performed by the Japanese Orthopaedic Association hip disease evaluation questionnaire (JHEQ), and revision surgeries were examined. Based on the presence of radiolucent lines on digital tomosynthesis at 2 years postoperatively, patients were divided into RL (+) and RL (-) groups and investigated for related factors. RESULTS: There were 20 cases in the RL (+) group and 35 cases in the RL (-) group, and no revision surgeries were required. Statistically, there were more cases with radiolucent lines on digital tomosynthesis (45.4% at 1 week and 36.3% at 2 years) than on plain radiography (9.1% at 1 week and 9.1% at 2 years) at each postoperative point. Logistic analysis showed no significant associations between the presence of radiolucent lines at 2 years on digital tomosynthesis, and the JHEQ parameters of pain (p = 0.937), movement (p = 0.266), or mental status (p = 0.404). CONCLUSION: In a short-term evaluation up to 2 years, digital tomosynthesis detected more radiolucent lines around the titanium cups than plain radiography. The occurrence of radiolucent lines was not related to the postoperative clinical evaluation.

Possibility of inhibiting arthritis and joint destruction by SSEA-3 positive cells derived from synovial tissue in rheumatoid arthritis.

AIM: Joint destruction progresses irreversibly once they occur in rheumatoid arthritis (RA), even with the recent development of anti-rheumatic drugs. Cells positive for stage-specific embryonic antigen-3 (SSEA-3), a marker of human embryonic stem cell, act as stem cells in the blood. The aim of this study is to investigate the effectiveness of SSEA-3 positive cells for the treatment for RA. METHODS: Synovial tissues were harvested at the time of joint surgery in RA patients. Cultured synovial cells were sorted by anti-SSEA-3 antibody using flow cytometry and were analyzed in in vitro. To investigate inhibitory effects on arthritis by SSEA-3 positive cells, collagen antibody-induced arthritis (CAIA) mice were used and transplanted with labeled cells intravenously. RESULTS: Presence of SSEA-3 positive cells was confirmed with approximately 1% in RA synovial cells. SSEA-3 positive cells were negative for CD34 and positive for CD44, CD90 and CD105. Multipotency of SSEA-3 positive cells was higher than that of SSEA-3 negative cells. Arthritis of the group transplanted with SSEA-3 positive cells in CAIA mice decreased over time. CONCLUSIONS: SSEA-3 positive cells derived from RA synovial tissue might have the inhibitory effect on arthritis and would be one of cell source for new RA treatment.

High Osteogenic Potential of Adipose- and Muscle-derived Mesenchymal Stem Cells in Spinal-Ossification Model Mice.

STUDY DESIGN: Basic experiments in a mouse model of ossification of the posterior longitudinal ligament (OPLL). OBJECTIVE: To assess the osteogenic potential of mesenchymal stem cells (MSCs) obtained from muscle and adipose tissue in Tiptoe-walking (ttw) mice, in which cervical OPLL compresses the spinal cord and causes motor and sensory dysfunction. SUMMARY OF BACKGROUND DATA: In humans, MSCs have been implicated in the pathogenesis of cervical OPLL. Cervical OPLL in ttw mice causes chronic compression of the spinal cord. Few studies have compared the MSC osteogenic potential with behavioral changes in an OPLL animal model. METHODS: We compared the osteogenic potential and behavioral characteristics of MSCs from ttw mice (4 to 20 weeks old) with those from control wild-type mice (without hyperostosis). Ligament ossification was monitored by micro-computed tomography and pathology; tissues were double stained with fluorescent antibodies against markers for MSCs (CD45 and CD105), at 8 weeks. The Basso Mouse Scale was used to assess motor function, and heat and mechanical tests to assess sensory function. The osteogenic potential of adipose and muscle MSCs was assessed by Alizarin Red S absorbance, staining for osteogenic mineralization, and real-time quantitative polymerase chain reaction for osteogenesis-related genes. RESULTS: Spinal-ligament ossification began in ttw mice at 8 weeks of age, and the ossified area increased with age. Immunofluorescence staining identified MSCs in the ossification area. The ttw mice became hyposensitive at 8 weeks of age, and Basso Mouse Scale scores showed motor-function deficits starting at 12 weeks of age. Alizarin Red S staining for mineralization showed a higher osteogenic potential in the adipose- and muscle-derived MSCs from ttw mice than from wild-type mice at 4, 8, and 20 weeks of age. Real-time quantitative polymerase chain reaction showed that ttw MSCs strongly expressed osteogenesis-related genes. CONCLUSION: MSCs derived from muscle and adipose tissue in ttw mice had a high osteogenic potential. LEVEL OF EVIDENCE: N/A.

The Knee injury and Osteoarthritis Outcome Score reflects the severity of knee osteoarthritis better than the revised Knee Society Score in a general Japanese population.

PURPOSE: The purposes of this study were to examine population-based reference data for sex- and age-related differences between the 2011 revised Knee Society Score (KSS2011) and the Knee injury and Osteoarthritis Outcome Score (KOOS), to assess the correlation between those scores and radiographic knee osteoarthritis (OA), and to validate the use of the scores in a general Japanese population. METHODS: This cross-sectional study included 963 volunteers (368 males, 595 females; mean age: 54.7years). Participants were classified into five subgroups by age: under 40, 40s, 50s, 60s, and over 70years old. The KSS2011 and KOOS were determined using self-administered questionnaires. Weight-bearing radiographs of the bilateral knee were taken and graded according to the Kellgren-Lawrence (KL) scale. The mean KSS2011 and KOOS were compared among age groups. Correlations between the severity of knee OA and each score were assessed using multiple regression analysis. RESULTS: The overall KSS2011 tended to gradually decrease with age. Most subscales of the KSS2011 did not show sex-related differences. Similarly, the overall KOOS and all its subscales steadily decreased by approximately 20 points per decade with age. Most subscales of the KOOS were significantly decreased in females over 50. The KL grade was significantly related to both the overall KOOS (ß=-0.42, p<0.001) and KSS2011 (ß=-0.13, p=0.001), though the correlation to the KOOS was stronger. CONCLUSION: The overall KSS2011 and KOOS appear to decrease with age. In this population, the KOOS reflects the severity of knee OA better than the KSS2011.

Lysosomal localization of Japanese medaka (Oryzias latipes) Neu1 sialidase and its highly conserved enzymatic profiles with human.

Desialylation in the lysosome is a crucial step for glycoprotein degradation. The abnormality of lysosomal desialylation by NEU1 sialidase is involved in diseases of mammals such as sialidosis and galactosialidosis. Mammalian Neu1 sialidase is also localized at plasma membrane where it regulates several signaling pathways through glycoprotein desialylation. In fish, on the other hand, the mechanism of desialylation in the lysosome and functions of Neu1 sialidase are still unclear. Here, to understand the significance of fish Neu1 sialidase, neu1 gene was cloned from medaka brain and the profiles of its polypeptides were analyzed. Open reading frame of medaka neu1 consisted 1,182 bp and the similarity of its deduced amino acids with human NEU1 was 57%. As this recombinant polypeptide did not show significant sialidase activity, medaka cathepsin A, known in mammals as protective protein activating Neu1, was cloned and then co-expressed with medaka Neu1 to examine whether medaka cathepsin A activates Neu1 activity. As a result, Neu1/cathepsin A showed a drastic increase of sialidase activity toward MU-NANA. Major substrate of medaka Neu1 was 3-sialyllactose and its optimal pH was 4.0. With immunofluorescence analysis, signal of overexpressed medaka Neu1 was found to coincide with Lysotracker signals (organelle marker of lysosome) and co-localized with medaka cathepsin A in fish hepatic Hepa-T1 cells. Furthermore, part of medaka Neu1 was also detected at plasma membrane. Medaka Neu1 possessed signal peptide sequence at N-terminal and incomplete lysosomal targeting sequence at C-terminus. Medaka neu1 gene was ubiquitously expressed in various medaka tissues, and its expression level was significantly higher than other sialidase genes such as neu3a, neu3b and neu4. The present study revealed the profiles of fish Neu1 sialidase and indicated its high conservation with human NEU1 for the first time, suggesting the presence of similar desialylation system in the medaka lysosome to human. Moreover, the present study showed the possibility of medaka as a model animal of human NEU1 sialidase.

NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth.