Production of bioactive peptides in an in vitro system

An in vitro system for the preparation of bioactive peptides is described. This system couples three different posttranslational modification enzymes, prohormone convertases (PCs), carboxypeptidase E, and peptidyl alpha-amidating enzyme, to transform recombinant precursors into bioactive peptides. Three different precursors, mouse proopiomelanocortin (mPOMC), rat proenkephalin (rPE), and human proghrelin, were used as model systems. The conversion of mPOMC and rPE to smaller peptide products was measured by radioimmunoassay. After optimization of the system, excellent efficiency was obtained: about 85% of starting mPOMC was converted to des-acetyl alpha-melanocyte-stimulating hormone (alpha-MSH). For proenkephalin, 75 and 96% yields were obtained for the opioid peptides Met-RGL and Met-enk, respectively. Cell-based assays demonstrated that in-vitro-generated des-acetyl alpha-MSH successfully activated the melanocortin 4 receptor. Proghrelin digestion was used to screen the specificity of PC cleavage and to confirm the cleavage site by mass spectroscopy. Mature ghrelin was produced by human furin, mouse prohormone convertase 1, and human prohormone convertase 7 but not by mouse prohormone convertase 2. These results demonstrate that our in vitro system (1) can produce peptides in quantities sufficient to carry out functional analyses, (2) can be used to determine the specificity of proprotein convertases on recombinant precursors, and (3) has the potential to identify novel peptide functions on both known and orphan G-protein-coupled receptors.     

Application of matrix solid-phase dispersion methodology to the extraction of endogenous peptides from porcine hypothalamus samples for MS and LC-MS analysis

In this study, we investigated a novel application of matrix solid-phase dispersion (MSPD) methodology for the extraction of endogenous peptides from porcine hypothalamus tissue samples. Several experimental factors of the MSPD procedure were examined. Finally, silica-based octadecyl was chosen as dispersing material and blended with 0.25 g porcine hypothalamus at a ratio of 5, and 10 mL of 60% acetonitrile with 0.2% formic acid in water was chosen as the extraction and elution solvent. This MSPD extraction method was compared to the classic acid extraction method. More peaks were observed in the MSPD extracts (74±5) by MALDI-TOF MS than in acid extracts (34±5). Moreover, 14 potential endogenous peptides were identified in the MSPD extracts after nanoLC-MS/MS analysis, while only 2 endogenous peptides in the acid extracts. These results indicated that MSPD could be employed as a simple and efficient method for the extraction of endogenous peptides from tissues.     

Pesticide Residues in Agricultural Produce in Hubei Province,PR China

Pesticide residues in agricultural produce were examined in two villages of Qianjiang municipality, Hubei province, PR China. Six food groups were sampled from the fields prior to harvesting from the farmers' market and from farmers' homes. Organochlorine residues were detected in almost all examined food groups, with residue levels significantly lower than the national tolerances (MRL). Total BHC residues were on average 31.7?µg/kg and DDT 102.5 (µg/kg. Organophosphorus residues were detected in vegetable samples. Mean residue levels of phoxim and methamidophos in vegetables were 89.9?µg/kg and 36.5?µg/kg, respectively, both exceeding the MRL. Estimated daily intakes (EDIs) of pesticide residues per person, based on the six food groups sampled, were 4.88?mg for total DDT, 2.04 (µg for total BHC, and 19.33 (µg for methamidophos. The overall results show that the increased use and misuse of pesticides for crop protection in Qianjiang municipality, notably in vegetable production, have led to worrisome levels of pesticide contamination of agricultural produce that may carry certain risks for human health.     

Maohuoside A promotes osteogenesis of rat mesenchymal stem cells via BMP and MAPK signaling pathways

Osteoporosis is becoming a more prevalent health problem with the aging of the population around the world. Epimedium koreanum Nakai is one of the most used herbs in East Asia for curing osteoporosis, with its major ingredient, icariin, mostly explored by researchers. In this article, maohuoside A (MHA), a single isolated compound from the herb, was identified to be more potent than icariin in promoting osteogenesis of rat bone marrow-derived mesenchymal stem cells (rMSCs) (increasing by 16.6, 33.3, and 15.8% on D3, D7, and D11, respectively). Alkaline phosphatase (ALP) assay and calcium content measurement were assigned to quantify the promoted osteogenesis and alizarin red S (ARS) staining was conducted to visualize it. Quantitative real-time PCR (Q-PCR) was assayed to evaluate the mRNA expression of marker genes in osteogenesis and master regulators in BMP pathway. Moreover, PD98059 (PD) and SB203580 (SB), inhibitor of ERK1/2 and p38 MAPK pathway, were administered to assess the involvement of MAPK pathway in the promotion process. In conclusion, MHA pronouncedly enhanced the osteogenesis of rMSC, plausibly via the BMP and MAPK signaling pathways.     

Overexpression of a small GTP-binding protein Ran1 in Arabidopsis leads to promoted elongation growth and enhanced disease resistance against P. syringae DC3000

Plants resist infection through an innate immune response, which is usually associated with slowing of growth. The molecular mechanisms underlying the trade-off between plant growth and defense remain unclear. The present study reveals that growth/defense trade-offs mediated by gibberellin (GA) and salicylic acid (SA) signaling pathways are uncoupled during constitutive overexpression of transgenic AtRAN1 and AtRAN1_Q72L (active, GTP-locked form) Arabidopsis plants. It is well known that the small GTP-binding protein Ran (a Ras-related nuclear protein) functions in the nucleus–cytoplasmic transport of proteins. Although there is considerable evidence indicating that nuclear–cytoplasmic partitioning of specific proteins can participate in hormone signaling, the role of Ran-dependent nuclear transport in hormone signaling is not yet fully understood. In this report, we used a combination of genetic and molecular methods to reveal whether AtRAN1 is involved in both GA and SA signaling pathways. Constitutively overexpressed AtRAN1 promoted both elongation growth and the disease resistance response, whereas overexpression of AtRAN1_Q72L in the atran2atran3 double mutant background clearly inhibited elongation growth and the defense response. Furthermore, we found that AtRAN1 coordinated plant growth and defense by promoting the stability of the DELLA protein RGA in the nucleus and by modulating NPR1 nuclear localization. Interestingly, genetically modified rice (Oryza sativa) overexpressing AtRAN1 exhibited increased plant height and yield per plant. Altogether, the ability to achieve growth/defense trade-offs through AtRAN1 overexpression provides an approach to maximizing crop yield to meet rising global food demands.     

Increased Expression of IL-22 Is Associated with Disease Activity in Behcet’s Disease

Objective

Interleukin (IL)-22 has been reported to be involved in the development of autoimmune diseases. This study aimed to analyze the expression and potential role of IL-22 in the pathogenesis of Behcet’s disease (BD).

Methods

The levels of IL-22 in patient sera or supernatants of cultured peripheral blood mononuclear cells (PBMCs) and CD4⁺T cells were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to evaluate the frequency of IL-22–producing CD4⁺ T cells. IL-22 mRNA from erythema nodosum skin lesions was examined using real time quantitative RT-PCR.

Results

BD patients with active uveitis showed a significantly higher expression of IL-22 in the supernatants of stimulated PBMCs and CD4⁺T cells compared with BD patients without active uveitis and normal controls. An increased frequency of IL-22-producing CD4⁺T cells was also found in BD patients with active uveitis. IL-22 mRNA expression was elevated in erythema nodosum skin lesions. In BD patients, a high IL-22 level in the supernatant of stimulated PBMCs correlated with the presence of retinal vasculitis and erythema nodosum.

Conclusions

IL-22 was associated with disease activity in BD and correlated with the presence of small vessel inflammation, suggesting that it may be involved in its pathogenesis.     

星球藻属一新种

本文发表了星球藻属一新种,即高山星球藻Asterocapsa alpina Cheng et Cai,sp.nov.。     

Increasing Lysine Content of Waxy Maize through Introgression of Opaque-2 and Opaque-16 Genes Using Molecular Assisted and Biochemical Development

The low lysine content of waxy maize cannot meet the nutritional requirements of humans, livestock, or poultry. In the present study, the high-lysine genes o2 and o16 were backcrossed into wx lines using the maize high-lysine inbreds TAIXI19 (o2o2) and QCL3021 (o16o16) as donors and the waxy maize inbred line QCL5019 (wxwx) as a receptor. In the triple-cross F₁, backcross, and inbred generations, the SSR markers phi027 and phi112 within the wx and o2 genes and the SSR marker umc1121 linked to the o16 gene were used for foreground selection. Background selection of the whole-genome SSR markers was performed for the selected individuals. The grain lysine content was determined using the dye-binding lysine method. The waxiness of the grain was determined with the I₂-KI staining and dual-wavelength spectrophotometric analysis. The BC₂F₂ generation included 7 plants of genotype wxwxo2o2O16_, 19 plants of genotype wxwxo16o16O2_, and 3 plants of genotype wxwxo2o2o16o16. In these seeds, the average amylopectin content was 96.67%, 96.87%, and 96.62%, respectively, which is similar to that of QCL5019. The average lysine content was 0.555%, 0.380%, and 0.616%, respectively, representing increases of 75.1%, 19.9%, 94.3%, respectively, over QCL5019. The average genetic background recovery rate of the BC₂F₃ families was 95.3%, 94.3%, 94.2%, respectively. Among these 3 wxwxo2o2O16O16 families, 4 wxwxo2o2O16o16 families, and 3 wxwxo2o2o16o16 families, the longest imported parent donor fragment was 113.35 cM and the shortest fragment was 11.75 cM. No significant differences in lysine content were found between the BC₂F₄ seeds and the BC₂F₃ seeds in these 10 families. This allowed us to increase the lysine content of waxy corn and produce seeds with excellent nutritional characteristics suitable for human consumption, animal feed, and food processing. This may be of significance in the breeding of high-quality corn and in improvement of the nutrition of humans, livestock, and poultry.     

MicroRNA-30c serves as an independent biochemical recurrence predictor and potential tumor suppressor for prostate cancer

MicroRNA-30c (miR-30c) acts as a tumor suppressor or a tumor promoter in various human malignancies. However, the involvement of miR-30c in prostate cancer (PCa) is still unclear. The aim of this study was to investigate the molecular function and the clinical significance of miR-30c in PCa. Expression levels of miR-30c in PCa tissues and cells were detected by quantitative real-time-PCR (qRT-PCR). Additionally, the associations of miR-30c expression with clinicopathological features and prognosis in PCa patients were analyzed. The potential role of miR-30c in tumorigenesis of PCa cells was further evaluated by in vitro cell assays. MiR-30c was significantly down-regulated in PCa tissues and cells compared with the corresponding controls (P < 0.05). In addition, the downregulation of miR-30c in PCa tissues was significantly associated with higher Gleason score (P = 0.009), advanced pathological stage (P = 0.016) and biochemical recurrence (P = 0.034). Moreover, Kaplan–Meier survival analysis showed that the reduced expression of miR-30c was correlated with shorter biochemical recurrence-free survival (P = 0.023). The multivariate analysis also identified miR-30c as an independent prognostic predictor for biochemical recurrence-free survival in patients with PCa. Furthermore, the enforced expression of miR-30c suppressed proliferation, migration and invasion of PCa cells in vitro. Our data indicated the involvement of miR-30c in PCa progression and suggested its potential role as an independent predictor of biochemical recurrence in PCa. On cellular level, miR-30c may function as a tumor suppressor for PCa cells by inhibiting tumor cell proliferation, migration and invasion.