Journal of Biomedical Science - Latest Articles

MicroRNA-488 regulates zinc transporter SLC39A8/ZIP8 during pathogenesis of osteoarthritis

Jinsoo Song — 2013-05-20T00:00:00Z

Background: Even though osteoarthritis (OA) is the most common musculoskeletal dysfunction, there are no effective pharmacological treatments to treat OA due to lack of understanding in OA pathology. To better understand the mechanism in OA pathogenesis and investigate its effective target, we analyzed miRNA profiles during OA pathogenesis and verify the role and its functional targets of miR-488. Results: Human articular chondrocytes were obtained from cartilage of OA patients undergoing knee replacement surgery and biopsy samples of normal cartilage and the expression profile of miRNA was analyzed. From expression profile, most potent miR was selected and its target and functional role in OA pathogenesis were investigated using target validation system and OA animal model system. Among miRNAs tested, miR-488 was significantly decreased in OA chondrocytes Furthermore, we found that exposure of IL-1beta was also suppressed whereas exposure of TGF-beta3 induced the induction of miR-488 in human articular chondrocytes isolated from biopsy samples of normal cartilages. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Target validation study showed that miR-488 targets ZIP8 and suppression of ZIP8 in OA animal model showed the reduced cartilage degradation. Conclusions: miR-488 acts as a positive role for chondrocyte differentiation/cartilage development by inhibiting MMP-13 activity through targeting ZIP-8.

H-rev107 regulates prostaglandin D2 synthase-mediated suppression of cellular invasion in testicular cancer cells

Rong-Yaun Shyu — 2013-05-20T00:00:00Z

Background: H-rev107 is a member of the HREV107 type II tumor suppressor gene family which includes H-REV107, RIG1, and HRASLS. H-REV107 has been shown to express at high levels in differentiated tissues of post-meiotic testicular germ cells. Prostaglandin D2 (PGD2) is conjectured to induce SRY-related high-mobility group box 9 (SOX9) expression and subsequent Sertoli cell differentiation. To date, the function of H-rev107 in differentiated testicular cells has not been well defined. Results: In the study, we found that H-rev107 was co-localized with prostaglandin D2 synthase (PTGDS) and enhanced the activity of PTGDS, resulting in increase of PGD2 production in testis cells. Furthermore, when H-rev107 was expressed in human NT2/D1 testicular cancer cells, cell migration and invasion were inhibited. Also, silencing of PTGDS would reduce H-rev107-mediated increase in PGD2, cAMP, and SOX9. Silencing of PTGDS or SOX9 also alleviated H-rev107-mediated suppression of cell migration and invasion. Conclusions: These results revealed that H-rev107, through PTGDS, suppressed cell migration and invasion. Our data suggest that the PGD2-cAMP-SOX9 signal pathway might play an important role in H-rev107-mediated cancer cell invasion in testes.

Control of post-translational modifications in antithrombin during murine post-natal development by miR-200a

Raúl Teruel — 2013-05-16T00:00:00Z

Background: Developmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant. Results: In this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control. Conclusions: These data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.

Role of tissue engineered collagen based tridimensional implant on the healing response of the experimentally induced large Achilles tendon defect model in rabbits: a long term study with high clinical relevance

Abdolhamid Meimandi-Parizi — 2013-05-14T00:00:00Z

Background: Tendon injury is one of the orthopedic conditions poses with a significant clinical challenge to both the surgeons and patients. The major limitations to manage these injuries are poor healing response and development of peritendinous adhesions in the injured area. This study investigated the effectiveness of a novel collagen implant on tendon healing in rabbits. Results: Seventy five mature White New-Zealand rabbits were divided into treated (n = 55) and control (n = 20) groups. The left Achilles tendon was completely transected and 2 cm excised. The defects of the treated animals were filled with collagen implants and repaired with sutures, but in control rabbits the defects were sutured similarly but the gap was left untreated. Changes in the injured and normal contralateral tendons were assessed weekly by measuring the diameter, temperature and bioelectrical characteristics of the injured area. Clinical examination was done and scored. Among the treated animals, small pilot groups were euthanized at 5, 10, 15, 20, 30, 40 and 60 (n = 5 at each time interval) and the remainder (n = 20) and the control animals at 120 days post injury (DPI). The lesions of all animals were examined at macroscopic and microscopic level and the dry matter content, water delivery and water uptake characteristics of the lesions and normal contralateral tendons of both groups were analyzed at 120 DPI.No sign of rejection was seen in the treated lesions. The collagen implant was invaded by the inflammatory cells at the inflammatory phase, followed by fibroplasia phase in which remnant of the collagen implant were still present while no inflammatory reaction could be seen in the lesions. However, the collagen implant was completely absorbed in the remodeling phase and the newly regenerated tendinous tissue filled the gap. Compared to the controls, the treated lesions showed improved tissue alignment and less peritendinous adhesion, muscle atrophy and fibrosis. They also showed significantly better clinical scoring, indices for water uptake and water absorption, and bioelectrical characteristics than the controls. Conclusion: This novel collagen implant was biodegradable, biocompatible and possibly could be considered as a substitute for auto and allografts in clinical practice in near future.

Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients

Hong-How Chang — 2013-05-04T00:00:00Z

Background: Antibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). Anti-Sm autosera have been reported to specifically recognize Sm D1 and D3 with symmetric di-methylarginines (sDMA). We investigated if anti-Sm sera from local SLE patients can differentially recognize Sm proteins or any other proteins due to their methylation states. Results: We prepared HeLa cell proteins at normal or hypomethylation states (treated with an indirect methyltransferase inhibitor adenosine dialdehyde, AdOx). A few signals detected by the anti-Sm positive sera from typical SLE patients decreased consistently in the immunoblots of hypomethylated cell extracts. The differentially detected signals by one serum (Sm1) were pinpointed by two-dimensional electrophoresis and identified by mass spectrometry. Three identified proteins: splicing factor, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein D-like (hnRNP DL) and cellular nucleic acid binding protein (CNBP) are known to contain methylarginines in their glycine and arginine rich (GAR) sequences. We showed that recombinant hnRNP DL and CNBP expressed in Escherichia coli can be detected by all anti-Sm positive sera we tested. As CNBP appeared to be differentially detected by the SLE sera in the pilot study, differential recognition of arginine methylated CNBP protein by the anti-Sm positive sera were further examined. Hypomethylated FLAG-CNBP protein immunopurified from AdOx-treated HeLa cells was less recognized by Sm1 compared to the CNBP protein expressed in untreated cells. Two of 20 other anti-Sm positive sera specifically differentiated the FLAG-CNBP protein expressed in HeLa cells due to the methylation. We also observed deferential recognition of methylated recombinant CNBP proteins expressed from E. coli by some of the autosera. Conclusion: Our study showed that hnRNP DL and CNBP are novel antigens for SLE patients and the recognition of CNBP might be differentiated dependent on the level of arginine methylation.

New insights of P2X7 receptor signaling pathway in alveolar functions

Amarjit Mishra — 2013-05-01T00:00:00Z

Purinergic P2X7 receptor (P2X7R), an ATP-gated cation channel, is unique among all other family members because of its ability to respond to various stimuli and to modulate pro-inflammatory signaling. The activation of P2X7R in immune cells is absolutely required for mature interleukin -1beta (IL-1beta) and IL-18 production and release. Lung alveoli are lined by the structural alveolar epithelial type I (AEC I) and alveolar epithelial type II cells (AEC II). AEC I plays important roles in alveolar barrier protection and fluid homeostasis whereas AEC II synthesizes and secrete surfactant and prevents alveoli from collapse. Earlier studies indicated that purinergic P2X7 receptors were specifically expressed in AEC I. However, their implication in alveolar functions has not been explored. This paper reviews two important signaling pathways of P2X7 receptors in surfactant homeostatsis and Acute Lung Injury (ALI). Thus, P2X7R resides at the critical nexus of alveolar pathophysiology.

CoDP: predicting the impact of unclassified genetic variants in MSH6 by the combination of different properties of the protein

Hiroko Terui — 2013-04-28T00:00:00Z

Background: Lynch syndrome is a hereditary cancer predisposition syndrome caused by a mutation in one of the DNA mismatch repair (MMR) genes. About 24% of the mutations identified in Lynch syndrome are missense substitutions and the frequency of missense variants in MSH6 is the highest amongst these MMR genes. Because of this high frequency, the genetic testing was not effectively used in MSH6 so far. We, therefore, developed CoDP (Combination of the Different Properties), a bioinformatics tool to predict the impact of missense variants in MSH6. Methods: We integrated the prediction results of three methods, namely MAPP, PolyPhen-2 and SIFT. Two other structural properties, namely solvent accessibility and the change in the number of heavy atoms of amino acids in the MSH6 protein, were further combined explicitly. MSH6 germline missense variants classified by their associated clinical and molecular data were used to fit the parameters for the logistic regression model and to assess the prediction. The performance of CoDP was compared with those of other conventional tools, namely MAPP, SIFT, PolyPhen-2 and PON-MMR. Results: A total of 294 germline missense variants were collected from the variant databases and literature. Of them, 34 variants were available for the parameter training and the prediction performance test. We integrated the prediction results of MAPP, PolyPhen-2 and SIFT, and two other structural properties, namely solvent accessibility and the change in the number of heavy atoms of amino acids in the MSH6 protein, were further combined explicitly. Variants data classified by their associated clinical and molecular data were used to fit the parameters for the logistic regression model and to assess the prediction. The values of the positive predictive value (PPV), the negative predictive value (NPV), sensitivity, specificity and accuracy of the tools were compared on the whole data set. PPV of CoDP was 93.3% (14/15), NPV was 94.7% (18/19), specificity was 94.7% (18/19), sensitivity was 93.3% (14/15) and accuracy was 94.1% (32/34). Area under the curve of CoDP was 0.954, that of MAPP for MSH6 was 0.919, of SIFT was 0.864 and of PolyPhen-2 HumVar was 0.819. The power to distinguish between pathogenic and non-pathogenic variants of these methods was tested by Wilcoxon rank sum test (p < 8.9 × 10-6 for CoDP, p < 3.3 × 10-5 for MAPP, p < 3.1 × 10-4 for SIFT and p < 1.2 × 10-3 for PolyPhen-2 HumVar), and CoDP was shown to outperform other conventional methods. Conclusion: In this paper, we provide a human curated data set for MSH6 missense variants, and CoDP, the prediction tool, which achieved better accuracy for predicting the impact of missense variants in MSH6 than any other known tools. CoDP is available at http://cib.cf.ocha.ac.jp/CoDP/.

Association of CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes

Akio Nakashima — 2013-04-18T00:00:00Z

Background: mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. Results: CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. Conclusion: The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8.

Remodeling of calcium signaling in tumor progression

Yih-Fung Chen — 2013-04-17T00:00:00Z

Intracellular Ca2+ is one of the crucial signalings that modulate various cellular functions. The dysregulation of Ca2+ homeostasis has been suggested as an important event in driving the expression of the malignant phenotypes, such as proliferation, migration, invasion, and metastasis. Cell migration is an early prerequisite for tumor metastasis that has a significant impact on patient prognosis. During cell migration, the exquisite spatial and temporal organization of intracellular Ca2+ provides a rapid and robust way for the selective activation of signaling components that play a central role in cytoskeletal reorganization, traction force generation, and focal adhesion dynamics. A number of known molecular components involved in Ca2+ influx pathways, including stromal interaction molecule (STIM)/Orai-mediated store-operated Ca2+ entry (SOCE) and the Ca2+-permeable transient receptor potential (TRP) channels, have been implicated in cancer cell migration and tumor metastasis. The clinical significance of these molecules, such as STIM proteins and the TRPM7 channel, in tumor progression and their diagnostic and prognostic potentials have also been demonstrated in specific cancer types. In this review, we summarize the recent advances in understanding the important roles and regulatory mechanisms of these Ca2+ influx pathways on malignant behaviors of tumor cells. The clinical implications in facilitating current diagnostic and therapeutic procedures are also discussed.

Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients

Tiffany Shi Yeen — 2013-04-16T00:00:00Z

Background: Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods. Results: All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations. Conclusions: Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS.