A rapid approach to capture the potential bioactive compounds from Rhizoma Drynariae, utilizing disease-associated mutation in calcium sensing receptor to alter the binding affinity for agonists.

Rhizoma Drynariae (RD) was used clinically to treat osteoporosis in China due to stimulating bone formation and inhibiting bone resorption, however, the bioactive constituents with the dual effect on bone are still unknown exactly. Disease-causing mutations in calcium sensing receptor (CaSR) can alter parathyroid hormone secretion and affect Ca2+ release from bone and Ca2+ reabsorption from kidney, which gives an indication that CaSR is a potential target for developing therapeutics to manage osteoporosis. Herein, a chromatographic approach was established, by immobilizing the mutant CaSR onto the surface of silica gels as stationary phase in a one-step procedure and then adding the different amino acids into mobile phase as competitors, for exploring the binding features of the known agonists and further screening ligands from RD. The mutant CaSR-coated column was prepared rapidly without the complicated purification and separation of the receptor, which had the large capacity of 13.1 mg CaSR /g silica gels and kept a good stability and specificity for at least 35 days. The CaSR mutation can weaken the binding affinities for three agonists, and the largest decreases occurred on the mutational site Thr151Met for neomycin, on the two sites of Asn118Lys and Glu191Lys for gentamicin-C, and on the site Phe612Ser for kanamycin, which gained new insights into their structure-function relationship. The potential bioactive compounds from RD were screened using the mutant CaSR-coated column and were recognized as coumaric acid 4-O-ß-D-glucopyranoside, caffeic acid, and naringin using UPLC-MS. Among them, naringin targeting CaSR gives a possible explanation that RD could manage osteoporosis. These results indicated that, such a rapid and simple method, utilizing disease-associated mutation in CaSR to alter the binding affinity for agonists, can be applied in capturing the potential bioactive compounds efficiently from complex matrices like herb medicines.

[Community of Benthic Diatoms and Their Relationship with Aquatic Environmental Factors in the Tangwang River, China].

To reasonably evaluate the eco-environmental health of the Tangwang River, which is a tributary of the Songhua River in China, community structures of periphyton and cleanliness of the benthic diatom at 24 sampling sites were investigated using McNaughton's dominance index, clustering, and ecotype analysis, while the relationship between the environmental factors and the diatom communities were studied by principal component analysis, Spearman correlation test, and redundancy analysis, in August 2018 (flood season). A total of 99 species or variants of benthic diatoms have been identified, indicating that there were abundant diatoms in the Tangwang River. Achnanthidium minutissimum and other diatoms that can be used as clean water indicators were dominant species in the Tangwang River, which indicates that the eco-environmental quality of the Tangwang River was relatively healthy in the flood season. Of these, the dominant degree of A. minutissimum was 0.32, making it the absolute dominant species in Tangwang River. Sampling sites can be divided into three groups based on clustering analysis. The dominant species of group 1 and group 2 were mainly clean species, indicating that the two groups were in a relatively healthy state. Nitzschia palea, Ulnaria ulna, and other diatoms that can be used as eutrophication indicators were the dominant species of group 3, indicating that group 3 was less healthy than the other two groups. From groups 1 and 2, the results from ecotype analysis showed a decrease in the proportion of polyoxybiontic diatoms and an increase in the proportion of α-mesosaphrobe diatoms, polysaprobe diatoms, oligo-mesotrophic diatoms, mesotrophic diatoms, meso-eutrophic diatoms, and eutrophic diatoms. Compared to the other two groups, the results from ecotype analysis showed a significant increase in the proportion of α-mesosaphrobe diatoms, polysaprobe diatoms, eutrophic diatoms and hypereutrophic diatoms in group 3. The predominant aquatic influencing factors of diatom community structures for the Tangwang River were permanganate index, total nitrogen (TN), and ammonia nitrogen (NH4+-N), of which permanganate index was the main factor for group 2, while TN and NH4+-N were the main factors for group 3. As a result, the eco-environmental quality of the Tangwang River was good, and the benthic diatom was found to be an effective indicator of the nutritional conditions and saprophytic status.

[Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and its possible mechanisms].

OBJECTIVE: To investigate the effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocyte, and study the possible mechanisms. METHODS: 3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of 3T3-L1 preadipocytes that were treated with different concentrations of ghrelin were evaluated by MTT methods. The levels of c-myc and thymidine kinase mRNA were detected using RT-PCR. 3T3-L1 preadipocytes were differentiated into the matured adipocytes with insulin (INS) or ghrelin. The morphological changes of 3T3-L1 adipocytes were observed and the differentiation rate was assayed by oil-red O staining. Total RNA was extracted from adipocytes at various times, and the levels of peroxisome proliferation activated receptor gamma (PPARgamma) and CAAT/enhancer binding protein(C/EBPalpha) mRNA expressions were detected using RT-PCR. RESULTS: Ghrelin at concentrations of 10(-7) to 10(-15) mol/L significantly stimulated preadipocyte proliferation (p<0.05). The levels of c-myc and thymidine kinase mRNA significantly increased in 3T3-L1 preadipocytes with 10(-9) mol/L and 10(-11) mol/L ghrelin treatment (p<0.01). The 3T3-L1 preadipocytes treated with 10(-11) mol/L ghrelin had lots of lipid droplets in the cytoplasma, but the differentiation rate was lower than those treated with INS. Ghrelin of 10(-11) mol/L significantly increased the mRNA expression of PPARgamma and C/EBPalpha in the course of 3T3-L1 preadipocyte differentiation, compared with the normal control group (p<0.05). The PPARgamma and C/EBPalpha mRNA expression increased with the prolonged differentiation of preadipocytes induced by ghrelin or INS. There were significant differences in the levels of PPARgamma and C/EBPalpha mRNA expression between the 2nd and 8th days of differentiation(p<0.01). CONCLUSIONS: Ghrelin promotes the proliferation and differentiation of 3T3-L1 preadipocytes. The proliferation of 3T3-L1 preadipocytes induced by ghrelin may be associated with increased c-myc levels. Ghrelin may promote differentiation of 3T3-L1 preadipocytes by increasing mRNA expression of PPARgamma and C/EBPalpha, thus enhances the sensitivity of adipocytes to INS.

[Expression of fibrillin-1 in congenital bicuspid aortic valves].

OBJECTIVE: To detect the expression of fibrillin-1 in congenital bicuspid aortic valves, and to investigate the molecular mechanism of congenital bicuspid aortic valves. METHODS: Specimens of aortic valve were obtained from 12 pediatric patients with congenital bicuspid aortic valve, 11 boys and 1 girl, aged 16.7 (10 - 18), including 5 cases of aortic stenosis (AS), 8 of aortic insufficiency (AI), and 1 of AS and AI, undergoing valve replacement, 8 children who died accidentally without cardiovascular system and collagen system diseases, 6 boys and 2 girls, aged 9.1 (1 - 17), collected in autopsy [normal (tricuspid) aortic valve controls], and 18 pediatric patients of rheumatic valvular heart disease with diseased tricuspid aortic valves who underwent aortic valve replacement, 13 boys and 5 girls, aged 16.5 (12 - 18) (rheumatic valvular heart disease controls). HE staining and light microscopy were conducted. Immunohistochemistry was used to detect the expression of fibrillin-1 in the aortic valves. RESULTS: Microscopy showed that the tissue structure of the congenital bicuspid aortic valves was unclear with hyperplasia of fibrous tissue. The grey degree value of fibrillin-1 of the congenital bicuspid aortic valve group was 170 +/- 10, significantly lower than those of normal aortic valve group and diseased tricuspid aortic valve group (126 +/- 8 and 73 +/- 16 respectively, both P < 0.05). There were not significant difference in the grey degree value of fibrillin-1 among the patients of congenital bicuspid aortic valves with AS, AI, and AS + AI (167 +/- 6, 171 +/- 8, and 168 +/- 6 respectively). CONCLUSION: The expression of fibrillin-1 is significantly reduced in congenital bicuspid aortic valves which may contribute to the morphological changes of the aortic valve leaflets and their resultant functional failure in congenital bicuspid aortic valves.

Lack of association between mannose-binding lectin gene polymorphisms and juvenile idiopathic arthritis in a Han population from the Hubei province of China.

Many studies have reported that polymorphisms of the mannose-binding lectin (MBL) gene are associated with autoimmune disease. Here, we investigate the relationship between MBL gene polymorphisms and susceptibility to juvenile idiopathic arthritis (JIA) in a Han-nationality population from the Hubei province of China. PCR-restriction fragment length polymorphism was used to investigate polymorphisms of codons 54 and 57 in exon 1 of the MBL gene in 93 patients with JIA and 48 control children. Neither group showed codon 57 polymorphisms. There was no significant difference in the genotypic frequencies of codon 54 between patients with JIA and healthy controls (wild type, 71.0% versus 75.0%, respectively; heterozygous type, 25.8% versus 25.0%, respectively; and homozygous type, 3.2% versus 0.0%, respectively). In addition, no association was found between the subgroups of patients with JIA and control individuals. Our results provide no evidence for a relationship between MBL gene mutation and susceptibility to JIA.

[Macrophage activation syndrome in children with systemic onset juvenile idiopathic arthritis: analysis of 13 patients].

OBJECTIVE: Macrophage activation syndrome (MAS) is a rare but life-threatening complication in children with rheumatic diseases, particularly systemic-onset juvenile idiopathic arthritis (SOJIA). Because of the potential fatality of this condition, prompt recognition and immediate therapeutic intervention are important. This study reviewed the data of MAS in 13 cases with SOJIA. METHODS: Retrospective review was performed on the precipitating events, clinical manifestations, laboratory data, treatment, and outcome of macrophage activation syndrome in 13 children with SOJIA seen from 1996 to 2005. RESULTS: Over the past 10 years the unit has had 90 new patients with SOJIA. Thirteen of those patients (14.4%) developed MAS during the course of their primary SOJIA, of whom ten were male. All patients were noted to have active SOJIA prior to developing MAS; 3 patients had medications, which were considered as trigger factors; 8 had infections prior to MAS, in two of them the infections were possible triggers. All the patients had high grade fever; 12 cases (92.3%) had hepatomegaly; 10 patients (76.9%) had coagulopathy, and eight patients (61.5%) had central nervous system dysfunction. The counts of platelet, white blood cells and the mean erythrocyte sedimentation rate fell dramatically in all patients; hyperferritinemia was identified in 8 patients, in 5 of whom serum ferritin (SF) was >or= 10,000 microg/L; in 8 (72.7%) of 11 cases fibrinogen was or= 2.5 mmol/L in 9 (69.2%) of 13 cases. CONCLUSION: MAS is a rare and potentially fatal complication of children with SOJIA. Primary disease activity, medications and infections preceding MAS were all important triggers. The strongest clinical discriminators were hepatomegaly, hemorrhages and central nervous system dysfunction. The strongest laboratory tests were decreased counts of platelet and white blood cells, decreased ESR and fibrinogen, dramatically increased SF and TG. It calls for the immediate treatments, particularly with cyclosporin A, which are often effective.

[Therapeutic effectiveness of intravenous immunoglobulin at 1 g/kg and 2 g/kg on Kawasaki disease: a comparative and follow-up study].

OBJECTIVE: To evaluate the effectiveness of intravenous immunoglobulin IVIG, 1 g/kg single intravenous injection in treating and preventing cardiac consequences of Kawasaki disease (KD) in children. METHODS: A total of 242 children with KD disease were enrolled in the study. In the randomized controlled trial, they were randomly divided into two groups: IVIG 1 g/kg group and IVIG 2 g/kg group, with aspirin administered within the first 7 to 10 days of illness. The occurrence and restoration of coronary artery lesion (CAL) in these two groups as well as the clinical and laboratory indexes including total fever duration, restoration of cervical lymphadenopathy, white blood cells count, platelet count, serum immunoglobulin, C reactive protein, erythrocyte sedimentation rate and EKG were observed. The clinical effectiveness of the groups before and after the treatment was analyzed. RESULTS: The age of the 242 children with KD disease ranged from 3 months to 14 years (mean 4.0 +/- 2.8 years old). Male to female ratio was 1.66:1, 83.1% of KD patients were blow 5 years old, 93.4% patients were followed up with echocardiography at the end of the first year and the follow-up period was (38 +/- 18) months, ranging from 4 months to 5.4 years; 86.9% of the cases in 1 g/kg group and 91.7% of the cases in 2 g/kg group had their fever controlled within 48 hours. The difference was not significant (P > 0.05). Serum immunoglobulin level was markedly enhanced after IVIG. Serum immunoglobulin levels in the patients of 2 g/kg group and 1 g/kg group were (26.9 +/- 7.4) g/L and (18.3 +/- 6.9) g/L, respectively (P < 0.01). The average duration of fever in IVIG 1 g/kg group was 10.6 days. After the treatment with 1 g/kg of IVIG, the abnormal white blood cells count, platelet count, C reactive protein, erythrocyte sedimentation rate and abnormal EKG findings were greatly improved (P < 0.001). However, there was no significant difference in the above-mentioned improvement between IVIG 1 g/kg group and IVIG 2 g/kg group (P > 0.05). In IVIG 1 g/kg group the occurrence of CAL was 29.5%. After the one-year follow-up, 87.5% CAL restored, but 12.5% did not, among which 9.4% were those of IVIG non-responders. In IVIG 2 g/kg group the incidence of CAL was 24.2%. After the one-year follow-up, 89.3% CAL restored, but 10.7% did not, all of which were those of IVIG non-responders. There was no significant difference in the incidence of CAL between the two groups (P > 0.05). CONCLUSION: Single intravenous injection of IVIG at 1 g/kg could effectively alleviate the clinical symptoms, decrease the incidence of CAL and reduce the complication of cardiovascular system. In the treatment of KD, the therapeutic effectiveness of IVIG at 1 g/kg was not significantly different from that of single intravenous injection of IVIG at 2 g/kg.