Reports of Biochemistry and Molecular Biology最新文献

Background: Romiplostim is a thrombopoietin receptor agonist approved for the treatment of immune thrombocytopenia. It is produced by recombinant DNA technology in Escherichia coli. Many researchers have studied the periplasmic or extracellular production of recombinant proteins in E. coli by using signal peptide sequences due to its advantages compared to intracellular production. In this study, the effect of the pelB signal peptide on Romiplostim production was analyzed.

Methods: The nucleotide sequence of Romiplostim was codon optimized for expression in E. coli BL21. For analysis of the effect of the pelB signal peptide, pET-22b (+) and pET-15b plasmids were used. The probability of signal peptide cleavage and pathway was predicted by using the SignalP 5.0 program, and expression, purification, and biological activity of the recombinant protein were analyzed.

Results: In-silico analysis predicted the correct cleavage of the pelB signal peptide. However, the experimental results showed intracellular accumulation of the protein in fusion with this signal peptide without any detectable protein band in periplasmic or extracellular spaces. The in-vivo experiment of purified protein without signal peptide exhibited a significant increment in platelets compared to the control group.

Conclusions: Romiplostim was expressed in E. coli with and without signal peptide. The latest one showed suitable in-vivo bioactivity. Despite the results of in-silico prediction, the pelB signal peptide could not transport the protein into the periplasm or extracellular environment in the experimental condition. Trying different signal peptides and more in-silico analysis might be helpful for the efficient secretion of the Romiplostim protein.

Background: Autophagy has been proven to contribute to maintaining eukaryotic cells' normal intracellular homeostasis, whereas autophagy malfunction may predispose to Behcet Disease (BD). The accumulation of the products of autophagic degradation as well as impairment in autophagic flux in cases with BD, may be attributed to dysregulated miRNA-155 expression. This study attempts to determine the contribution of circRNA-0067835 in miRNA-155-mediated modulation of the autophagy axis as well as to investigate its impact on the production of pro-inflammatory cytokines in BD.

Methods: This study was carried out on 40 cases with BD and 40 healthy control subjects. The collection of serum samples was done before performing a real-time PCR to estimate the relative gene expression of ATG1, AKT, miRNA-155, mTOR, TAB2, and circRNA-0067835. Additionally, IL-1β, IL-17, and TNF-α serum levels were determined by ELISA.

Results: Behcet Disease (BD) patients had significantly upregulated circRNA-0067835, with subsequent downregulation of its target gene, miRNA-155 than controls (P<0.05). In addition, decreased miRNA-155 gene expression was correlated with significantly increased TAB2 gene expression levels in BD patients compared to the controls (P<0.05). Furthermore, enhanced production of pro-inflammatory cytokines was detected in cases with BD than in controls.

Conclusion: The correlation between circRNA-0067835 and miRNA-155 fairly contributes to the regulation of cytokine production in BD via the modulation of autophagy. The investigation of the circRNA-0067835 and the microRNA-155 and their downstream adaptor molecules could be a potential therapeutic agent for BD.

Background: Diabetes mellitus (DM) is a metabolic disease, characterized by hyperglycemia resulting from defects in insulin secretion and/or insulin action. The current study was designed to assess the therapeutic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) alone and in combination with pioglitazone (Pz) or exendin-4 (Ex) in high-fat diet/streptozotocin (HFD/STZ)-induced diabetes in rats.

Methods: The rats were subjected to the HFD for three weeks before being injected with a single low dosage of STZ (35 mg/kg bw). The animals were assigned to different treatment groups after type II diabetes mellitus (T2DM) induction was confirmed.

Results: Severe insulin resistance was verified in untreated HFD/STZ T2DM rats, along with the exaggeration of oxidative stress, inflammation, apoptosis, and autophagy suppression in the adipose tissues. Monotherapy of HFD/T2DM rats with BM-MSCs and Pz or Ex alleviated diabetic complications by increasing insulin sensitivity, decreasing apoptosis and inflammation as evidenced by a decrease in serum tumor necrosis factor-alpha, caspase-3, and nuclear factor-kappa B (NF-κB) genes expression and Janus kinase (JNK) protein expression, and enhancing autophagy as revealed by upregulation in beclin and LC3, as well as peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1α) genes expression in the adipose tissues. An augmented ameliorative efficacy was recorded in combined treatments. The biochemical and molecular results were confirmed by histological investigation of pancreatic tissues.

Conclusions: Combining Pz or Ex with BM-MSCs is a synergistic therapeutic option that reduces insulin resistance and subsequent complications in T2DM via multiple molecular mechanisms.

Background: The incidence of prostate cancer (PC) exhibits geographical heterogeneity. However, the metabolic mechanisms underlying this geographic heterogeneity remain unclear. This study aimed to reveal the metabolic mechanism of the geographic heterogeneity in the incidence of PC.This study aimed to investigate the anti-cancer effects of different gum extracts on metabolic changes and their impact on gene expression in HT-29 cell.

Methods: Transcriptomic data from public databases were obtained and analyzed to screen geographic-differentially expressed genes and metabolic pathways. Associations between these differentially expressed genes and the incidence of PC were determined to identify genes that were highly associated with PC incidence. A co-expression network analysis was performed to identify geographic-specific regulatory pathways.

Results: A total of 175 differentially expressed genes were identified in four countries and were associated with the regulation of DNA replication and the metabolism of pyrimidine, nucleotides, purines, and galactose.Additionally, the expression of the genes CLVS2, SCGB1A1, KCNK3, HHIPL2, MMP26, KCNJ15, and PNMT was highly correlated with the incidence of PC. Geographic-specific differentially expressed genes in low-incidence areas were highly correlated with KCNJ15, MMP26, KCNK3, and SCCB1A1, which play a major role in ion channel-related functions.

Conclusions: This study suggests that geographic heterogeneity in PC incidence is associated with the expression levels of genes associated with amino acid metabolism, lipid metabolism, and ion channels.

Background: Smad4 regulates the expression of the genes required for heart homeostasis. Regarding the central role of microRNAs in cardiac biology, we investigated the expression of the three Smad4-targeting miRNAs, namely miR-18a-5p, miR-19a-3p, and miR-20a-5p, as well as Smad4 during differentiation of human endometrium-derived mesenchymal stem cells (hEMSCs) into cardiomyocytes (CMs).

Methods: To evaluate mesenchymal phenotype and multi-lineage differentiation ability of hEMSCs, immunophenotyping by flow cytometry and differentiation into osteoblasts and adipocytes were performed, respectively. For transdifferentiation into CMs, hEMSCs were exposed to a cardiomyogenic medium composed of 5-aza and bFGF for 30 days. The comparison between transcriptional expression levels of Nkx2-5, GATA4, Smad4, TNNT2, TBX5, miR-18a-5p, miR-19a-3p, and miR-20a-5p by qRT-PCR, as well as protein levels of Nkx2-5, Smad4, and cTnT by immunofluorescence staining, was conducted in every 6 days.

Results: In vitro, the mesenchymal stem cell phenotype of hEMSCs and their potency for differentiation into other MSCs were confirmed. Differentiated hEMSCs had morphological characteristics of CMs. The percentage of positive cells for Nkx2-5, Smad4, and cTnT proteins was increased following induction and culminated on the 24th day. Also, mRNA levels of Nkx2-5, GATA4, Smad4, TNNT2, and TBX5 exhibited the same trend. The expression of investigated miRNAs was significantly decreased sequentially. A significant negative correlation between expressions of Smad4 and investigated miRNAs was observed.

Conclusion: Our results indicate that miR-18a-5p, miR-19a-3p, and miR-20a-5p are involved in the cardiac differentiation propensity of hEMSCs potentially by regulation of Smad levels. Although, more mechanistic experiments are required to confirm this idea.

Background: To examine the impact of aging on the response of rats to pentylenetetrazole (PTZ)-induction of epilepsy and the possible role of oxidative stress and nuclear factor erythroid 2-related factor 2 (Nrf2)/ heme oxygenase (HO)-1 pathway in this response.

Methods: Forty male albino rats were equally allocated into 4 groups; 1) Young control (YC) group, aged 8-12 weeks, 2) Old control (OC) group, aged 24 months, 3) PTZ-Young group: young rats received PTZ (50 mg/Kg, i.p. every other day) for 2 weeks and 4) PTZ-Old group: as group 3 but rats were old. The seizure score stage and latency to the first jerk were recorded in rats. Redox state markers in brain tissues including malondialdehyde (MDA), catalase and total antioxidant capacity (TAC) were evaluated. Also, the expression of Nrf2 and HO-1 genes were measured in the brain tissues.

Results: Old rats showed an early and a significant rise in the seizure score with PTZ administration and a significant drop in the seizure latency compared to young rats (P <0.01). Also, old rats showed a significantly higher MDA concentration and a significantly lower TAC and catalase activity than young rats (P <0.01). Moreover, the expression of Nrf2 and HO-1 was significantly lowered in old rats compared to young rats with PTZ administration (P < 0.01).

Conclusion: Aging increases the vulnerability of rats to PTZ-induced epilepsy. An effect might come down to the up-regulation of oxidative stress and the down regulation of antioxidant pathways including Nrf2 and HO-1.

Background: Dental caries is an oral disease that is widely suffered by the population of Aceh caused by Streptococcus mutans. S. mutans serotypes c and d are widely isolated in the human oral cavity. This research was focused on detecting the presence and variability of S. mutans in supragingival dental plaque of caries teenager and young adults' patients.

Methods: Subjects involved in this study were patients who treated at the Rumah Sakit Gigi dan Mulut of Dentistry Faculty of Universitas Syiah Kuala. The approach used in this research was molecular microbiology technique. To determine the presence of S. mutans, supragingival plaque from caries patients was cultivated in TYS20B. The culture findings were utilized to detect the presence of bacteria using PCR. The primers utilized in the PCR were S. mutans specific primers, GTFB (517 bp) for S. mutans serotype c and GTFI (712 bp) for S. mutans serotype d.

Results: Culture results on TYS20B media showed the growth of S. mutans colonies isolated from the supragingival plaque of research subjects. PCR results also revealed the presence of S. mutans in the supragingival plaques of caries patients, with the variability of S. mutans discovered to be a serotype c and a serotype d.

Conclusion: Based on the findings of this study, it can be concluded that S. mutans can be found in the supragingival plaques of caries patients with the serotypes c and d variability.

Background: Oxidative stress is involved in the pathogenesis of preeclampsia (PE). Dysregulation of SOD1 may be involved in the pathogenesis of PE. We examined and compared the methylation level of the promoter region (PMR) of the SOD1, gene expression, and enzyme activity of superoxide dismutase (SOD) in both placenta and maternal blood in PE women.

Methods: A total of 140 blood samples and 40 placental tissue samples from PE and healthy pregnant controls were studied. The PMR of the SOD1 (Methylight PCR method), the expression (Real-time PCR), and its enzyme activity were investigated and compared in two groups.

Results: The PMR of the SOD1 gene in the placental tissue of the patients was significantly increased compared to the control group (P= 0.008); this result was accompanied by a decrease in the expression of the gene and a decrease in the activity of the SOD enzyme. Meanwhile, the PMR of the SOD1 gene did not significantly change in the blood samples of the patients (P= 0.95), while a significant decrease in the expression of SOD1 (without a significant change in the SOD activity) was observed.

Conclusion: The results showed significant changes in the PMR of the SOD1 gene and gene expression in placenta tissue. The results highlight the role of the placenta in complications during pregnancy and also revealed epigenetics as an important regulatory pathway in the pathogenesis of preeclampsia.

Background: Mutations in the receptor tyrosine kinase KIT are the major cause of gastrointestinal stromal tumors. KIT-mediated activation of the RAS/RAF/MEK/ERK and PI3 kinase/AKT pathways plays an important role in KIT mutant-mediated cell transformation.

Methods: The frequently seen primary KIT mutations W557K558del and V560D, and the secondary KIT mutations V654A and N822K, in gastrointestinal stromal tumors were stably transfected into Ba/F3 cells. Cell proliferation was examined with a CCK kit, and cell survival and cell cycle were examined by flow cytometry. Cell signaling was examined by western blot.

Results: We found that farnesyltransferase inhibitors tipifarnib and lonafarnib, which inhibit RAS activity, inhibited ERK activation mediated by both wild-type and KIT mutants, which often occur in gastrointestinal stromal tumors. Correspondingly, both wild-type and KIT mutant-mediated cell survival and proliferation were inhibited by both inhibitors. Imatinib is used as the first-line targeted therapy for gastrointestinal stromal tumors in the clinic. In our study, both inhibitors increased imatinib-mediated inhibition of cell survival and proliferation induced by both wild-type and KIT mutants. Similar to the primary KIT mutations, secondary mutations of KIT-induced ERK activation and cell response were inhibited by both inhibitors.

Conclusions: Our results suggested the potential benefit of farnesyltransferase inhibitors either alone or combined with imatinib in the treatment of gastrointestinal stromal tumors carrying KIT mutations.