Physically separated soil organic matter pools as indicators of carbon and nitrogen change under long-term fertilization in a Chinese Mollisol.

Isolation and quantification of soil organic matter (SOM) pools under the influence of management practices is needed for assessing the changes in soil fertility. However, the knowledge on how the active, slow and passive pools of SOM respond to long-term fertilization is scarce. Therefore, the present study was designed to isolate the active, slow, and passive pools of soil organic matter through physical fractionation under long-term fertilization. The treatments included; inorganic fertilization (NPK) either alone or combined with a normal dose of manure (MNPK) or a high dose of manure (1.5MNPK) with an unfertilized control (CK) for comparison. The isolated pools were analyzed and compared for their sizes, SOC and TN storage and their contribution to total SOC and TN sequestration. The results revealed that the fertilization enhanced the active, slow and passive pools of SOC and TN and their storage under applied treatments was patterned as 1.5MNK > MNPK > NPK > CK. The highest SOC and TN storage was observed in the active pool, while, greater response to fertilization (in terms of response ratio) was associated with the slow pool. Results show that fertilization enhanced the proportion of SOC and TN stocks to bulk SOC and TN stocks in active and slow pools, while a diminishing trend was found for passive pools. Moreover, the highest response ratio was found for TN sequestration in each pool as compared to SOC, suggesting preferential accumulation of TN over SOC in the studied soil. Nevertheless, the highest SOC and TN storage took place in the active pool. The slow pool showed greater response to applied fertilizer, with the highest values being observed under 1.5MNPK. This study concluded that long-term manure + inorganic fertilization is crucial for enhancing C and N sequestration by altering the size and response of SOM pools.

Stability of soil organic carbon under long-term fertilization: Results from 13C NMR analysis and laboratory incubation.

Long-term fertilization has shown a high relevance as regards soil organic carbon (SOC) sequestration, but the degree of stability of the sequestered SOC has not been widely studied up to now. Using physical fractionation combined with laboratory incubation and NMR spectroscopy, we evaluated the differences in SOC stability caused by long-term fertilization. Four SOC fractions were isolated and examined for contents and chemical composition and cumulative amount of CO2-C respired from the fractions under six fertilization treatments: control (CK); balanced inorganic fertilization (NPK); NPK combined with pig manure (MNPK); NPK combined 1.5 times of pig manure (1.5MNPK); and NPK combined with high amount of manure (M2NPK). The highest contents of SOC were recorded for the coarse particulate organic carbon (cPOC) fraction, ranging from 17.25 to 30.47 g kg-1 under CK and M2NPK. The highest cumulative amount of CO2-C was released from the cPOC fraction under manure treatments (M2NPK and 1.5NPKM), which was 56 and 43% higher than that from CK, whereas the lowest amount of CO2-C was released from the mineral associated-C (MOC) fraction under the same treatments, being 65 and 49% higher than that released from CK, suggesting low SOC stability in cPOC and high SOC stability in MOC fractions. However, manure treatments (M2NPK and 1.5NPKM) greatly lowered the specific amount of C-mineralized (C-mineralized per unit total SOC) in fractions and whole soil, suggesting the ability of manure to accumulate more SOC by reducing SOC losses. Moreover, carbonyl-C was found to be the form of SOC experiencing major degree of sequestration under current fertilization practices. The SOC stability indices; aromaticity index (AI), hydrophobicity index (HI) and alkyl-C/O-alkyl-C were found to be higher in manure treated plots further suggesting higher stability of SOC under manure addition. Thus, long-term manure combined with mineral fertilizers would enhance SOC stability through minimizing SOC losses and promoting accumulation of stable C forms in a Chinese Mollisol.

Identification and Characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage (Brassica rapa L.).

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9-16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2-4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.

Soil aggregation and soil aggregate stability regulate organic carbon and nitrogen storage in a red soil of southern China.

Soil aggregation plays a critical role in the maintenance of soil structure, as well as in its productivity. Fertilization influences soil aggregation, especially by regulating soil organic carbon (SOC) and total nitrogen (TN) contents in aggregate fractions. The present study evaluated the influence of three contrasting fertilizer regimes (unfertilized control -CK-, mineral fertilization -NPK- and manure combined with NPK -NPKM) on soil aggregate stability, aggregate-associated organic carbon and total nitrogen sequestration and mineralization of SOC. Soil samples from (20 cm) depth were collected from a long-term fertilization experiment and analysed for size distribution ranging (>250 µm, 250-53 µm and <53 µm sizes), SOC and TN contents, as well as for mineralization of bulk and aggregate associated-SOC. Both NPK and NPKM fertilizations significantly enhanced SOC and TN contents in bulk soil and its constituent aggregates of >250 µm, 250-53 µm and <53 µm sizes, as compared to CK. Long-term NPK and NPKM increased SOC and TN stock in bulk soil by 45 and 98%, and by 70 and 144%, respectively, as compared to CK. Similarly, higher values of SOC and TN stock in all aggregate fractions was observed with the application of NPKM. Application of NPK and NPKM for 26 years significantly increased aggregate stability, which was positively correlated with total SOC contents in terms of mean weight diameter (MWD) (Adj. R2 = 0.689, p < 0.03) and geometric mean diameter (GMD) (Adj. R2 = 0.471, p < 0.24). Moreover, higher scores regarding cumulative mineralization for bulk soil and aggregate associated OC were observed with the application of NPK and NPKM. Irrespective of treatments, higher cumulative C-mineralization was observed for macro-aggregates (>250 µm size) followed by 250-53 µm and <53 µm size aggregates. Interestingly, a highly positive correlation was observed between aggregate stability and the cumulative amount of mineralization for bulk soil and aggregate fractions, with R2 ranging from 0.84 to 0.99. This study evidenced that long-term fertilization of NPK and NPKM can improve soil aggregation, stability and associated OC and TN stock in aggregates, as well as aggregate-associated OC mineralization, which was further governed by aggregate size.

LPS enhances CTB-INSULIN induction of IDO1 and IL-10 synthesis in human dendritic cells.

Autoantigen-specific immunotherapy promises effective treatment for devastating tissue specific autoimmune diseases like multiple sclerosis (MS) and type 1 diabetes (T1D). Because activated dendritic cells (DCs) stimulate the differentiation of autoreactive T cells involved in the initiation of autoimmunity, blocking the activation of DCs may be an effective strategy for inhibiting tissue specific autoimmunity. Following this approach, immature DCs were shown to remain inactive after treatment with chimeric fusion proteins composed of the cholera toxin B subunit adjuvant linked to autoantigens like proinsulin (CTB-INS). Mass spectrometer analysis of human DCs treated with CTB-INS suggest that upregulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) is responsible for inhibiting DC activation thereby resulting in a state of immunological tolerance within the DC. Here we show that the fusion protein CTB-INS inhibits human monocyte derived DC (moDC) activation through stimulation of IDO1 biosynthesis and that the resultant state of DC tolerance can be further enhanced by the presence of residual E. coli lipopolysaccharide (LPS) present in partially purified CTB-INS preparations. Additional experiments showed that LPS enhancement of DC tolerance was dependent upon stimulation of IDO1 biosynthesis. LPS stimulation of increased levels of IDO1 in the DC resulted in increased secretion of kynurenines, tryptophan degradation products known to suppress DC mediated pro-inflammatory T cell differentiation and to stimulate the proliferation of regulatory T cells (Tregs). Further, the presence of LPS in CTB-INS treated DCs stimulated the biosynthesis of costimulatory factors CD80 and CD86 but failed to upregulate maturation factor CD83, suggesting CTB-INS treated DCs may be maintained in a state of semi-activation. While treatment of moDCs with increasing amounts of LPS free CTB-INS was shown to increase DC secretion of the anti-inflammatory cytokine IL-10, the presence of residual LPS in partially purified CTB-INS preparations dramatically increased IL-10 secretion, suggesting that CTB-INS may enhance DC mediated immunological tolerance by stimulating the proliferation of anti-inflammatory T cells. While the extraction of LPS from bacterial generated CTB-INS may remove additional unknown factors that may contribute to the regulation of IDO1 levels, together, our experimental data suggest that LPS stimulates the ability of CTB-INS to induce IDO1 and IL-10 important factors required for establishment of a state of functional immunological tolerance in human DCs. Regulation of the ratio of LPS to CTB-INS may prove to be an effective method for optimization of readily available "off the shelf" CTB-INS mediated immune-therapy for tissue specific autoimmune diseases including type 1 diabetes.

Production of functional recombinant cyclic citrullinated peptide monoclonal antibody in transgenic rice cell suspension culture.

Cyclic citrullinated peptide (CCP) antibody has been shown recently to be a promising marker for early detection and diagnosis of rheumatoid arthritis (RA). In order to exploit newly developed therapies for RA, early intervention is crucial in preventing irreversible joint damage. Here, we describe use of a plant expression system to produce a CCP antibody that could be used in the early diagnosis of RA. Heavy and light chain gene sequences of a CCP monoclonal antibody (CCP mAb) were cloned from the hybridoma cell (12G1) and introduced into two separate plant expression vectors under the control of the rice α-amylase 3D (RAmy3D) promoter system. The vectors were introduced into rice calli (Oryza sativa L. cv. Dongjin) using Agrobacterium tumefaciens mediated transformation. Integration of the CCP mAb genes into rice chromosomes was confirmed by a genomic DNA polymerase chain reaction and expression was verified by northern blot analysis of mRNA. The in vivo assembly and secretion of CCP mAb occurred in transgenic rice cell suspension culture under the RAmy3D expression system; accumulated CCP mAbs in the medium were purified by protein G affinity chromatography. Immunoblot assays and ELISA showed these plant-produced CCP mAbs successfully bound to a synthetic CCP antigen. Taken together, our results suggest that CCP mAb produced in a transgenic rice suspension culture were easily purified and biologically active against their antigen in the RA, and thus may be used a specific serological marker, which is present very early in the RA.

Production of recombinant human acid ß-glucosidase with high mannose-type N-glycans in rice gnt1 mutant for potential treatment of Gaucher disease.

Gaucher disease is an inherited metabolic disease caused by genetic acid ß -glucosidase (GBA) deficiency and is currently treated by enzyme replacement therapy. For uptake into macrophages, GBA needs to carry terminal mannose residues on their N-glycans. Knockout mutant rice of N-acetylglucosaminyltransferase-I (gnt1) have a disrupted N-glycan processing pathway and produce only glycoproteins with high mannose residues. In this study, we introduced a gene encoding recombinant human GBA into both wild-type rice (WT) and rice gnt1 calli. Target gene integration and mRNA expression were confirmed by genomic DNA PCR and Northern blotting, respectively. Secreted rhGBAs in culture media from cell lines originating from both WT (WT-GBA) and rice gnt1 (gnt1-GBA) were detected by Western blotting. Each rhGBA was purified by affinity and ion exchange chromatography. In vitro catalytic activity of purified rhGBA was comparable to commercial Chinese hamster ovary cell-derived rhGBA. N-glycans were isolated from WT-GBA and gnt1-GBA and analyzed by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The amounts of high mannose-type N-glycans were highly elevated in gnt1-GBA (100%) compared to WT-GBA (1%).

Identification of Anti-Long Chain Saturated Fatty Acid IgG Antibodies in Serum of Patients with Type 2 Diabetes.

High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1ß. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1ß, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used for in vitro characterization of avidity and specificity. A cohort participating in En Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1ß secretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients.

Agomelatine: An Astounding Sui-generis Antidepressant?

Major depressive disorder (MDD) is one of the foremost causes of disability and premature death worldwide. Although the available antidepressants are effective and well tolerated, they also have many limitations. Therapeutic advances in developing a new drug's ultimate relation between MDD and chronobiology, which targets the circadian rhythm, led to a renewed focus on psychiatric disorders. In order to provide a critical analysis about antidepressant properties of agomelatine, a detailed PubMed (Medline), Scopus (Embase), Web of Science (Web of Knowledge), Cochrane Library, Google Scholar, and PsycInfo search was performed using the following keywords: melatonin analog, agomelatine, safety, efficacy, adverse effects, pharmacokinetics, pharmacodynamics, circadian rhythm, sleep disorders, neuroplasticity, MDD, bipolar disorder, anhedonia, anxiety, generalized anxiety disorder (GAD), and mood disorders. Agomelatine is a unique melatonin analog with antidepressant properties and a large therapeutic index that improves clinical safety. Published articles revealed that agomelatine is a melatonin receptors (MT1 and MT2) agonist and 5HT2C receptor antagonist. The effects receptors' on melatonin receptors enable the resynchronization of irregular circadian rhythms with beneficial effects on sleep architectures. In this way, agomelatine is accredited for its unique mode of action, which helps to exert antidepressant effects and resynchronize the sleep-wake cycle. To sum up, an agomelatine has not only antidepressant properties but also has anxiolytic effects.

Transcriptome and QTL mapping analyses of major QTL genes controlling glucosinolate contents in vegetable- and oilseed-type Brassica rapa plants.

Glucosinolates (GSLs) are secondary metabolites providing defense against pathogens and herbivores in plants, and anti-carcinogenic activity against human cancer cells. Profiles of GSLs vary greatly among members of genus Brassica. In this study, we found that a reference line of Chinese cabbage (B. rapa ssp. pekinensis), 'Chiifu' contains significantly lower amounts of total GSLs than the oilseed-type B. rapa (B. rapa ssp. trilocularis) line 'LP08'. This study aimed to identify the key regulators of the high accumulation of GSLs in Brassica rapa plants using transcriptomic and linkage mapping approaches. Comparative transcriptome analysis showed that, in total, 8,276 and 9,878 genes were differentially expressed between 'Chiifu' and 'LP08' under light and dark conditions, respectively. Among 162 B. rapa GSL pathway genes, 79 were related to GSL metabolism under light conditions. We also performed QTL analysis using a single nucleotide polymorphism-based linkage map constructed using 151 F5 individuals derived from a cross between the 'Chiifu' and 'LP08' inbred lines. Two major QTL peaks were successfully identified on chromosome 3 using high-performance liquid chromatography to obtain GSL profiles from 97 F5 recombinant inbred lines. The MYB-domain transcription factor gene BrMYB28.1 (Bra012961) was found in the highest QTL peak region. The second highest peak was located near the 2-oxoacid-dependent dioxygenase gene BrGSL-OH.1 (Bra022920). This study identified major genes responsible for differing profiles of GSLs between 'Chiifu' and 'LP08'. Thus, our study provides molecular insights into differences in GSL profiles between vegetative- and oilseed-type B. rapa plants.

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures.

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.

Long-term fertilization affects functional soil organic carbon protection mechanisms in a profile of Chinese loess plateau soil.

Crop productivity and soil health are limited by organic carbon (OC), however, the variations in the mechanisms of SOC preservation in a complete soil profile subjected to long-term fertilization remains unclear. The objective of the study was to examined the content and profile distribution of the distinctive SOC protection mechanisms on a complete profile (0-100 cm) of Eumorthic Anthrosols in Northwest China after 23 years of chemical and manure fertilization. The soil was fractionated by combined physical-chemical and density floatation techniques. Throughout the profile, significant variations were observed among fractions. In the topsoil (0-20 and 20-40 cm), mineral coupling with the fertilization of manure (MNPK) enhanced total SOC content and recorded for 29% of SOC in the 0-20 and 20-40 cm layers. Moreover, MNPK increased the SOC content of the unprotected cPOC fraction by 60.9% and 61.5% in the 0-20 and 20-40 cm layer, while SOC content was low in the subsoil layers (40-60, 60-80 and 80-100 cm, respectively) compared with the control (C). The highest OC under MNPK in physically protected micro-aggregates (µagg) (6.36 and 6.06 g C kg-1), and occluded particulate organic carbon (iPOC) (1.41 and 1.29 g C kg-1) was found in the topsoil layers. The unprotected cPOC fraction was the greatest C accumulating fraction in the topsoil layers, followed by µagg and H-µSilt fractions in the soil profile, implying that these fractions were the most sensitive to the fertilization treatments. Overall, the unprotected, physically protected, and physico-chemically protected fractions were the dominant fractions for the sequestration of carbon across fertilization treatments and soil layers.

The BrGI Circadian Clock Gene Is Involved in the Regulation of Glucosinolates in Chinese Cabbage.

Circadian clocks integrate environmental cues with endogenous signals to coordinate physiological outputs. Clock genes in plants are involved in many physiological and developmental processes, such as photosynthesis, stomata opening, stem elongation, light signaling, and floral induction. Many Brassicaceae family plants, including Chinese cabbage (Brassica rapa ssp. pekinensis), produce a unique glucosinolate (GSL) secondary metabolite, which enhances plant protection, facilitates the design of functional foods, and has potential medical applications (e.g., as antidiabetic and anticancer agents). The levels of GSLs change diurnally, suggesting a connection to the circadian clock system. We investigated whether circadian clock genes affect the biosynthesis of GSLs in Brassica rapa using RNAi-mediated suppressed transgenic Brassica rapa GIGENTEA homolog (BrGI knockdown; hereafter GK1) Chinese cabbage. GIGANTEA plays an important role in the plant circadian clock system and is related to various developmental and metabolic processes. Using a validated GK1 transgenic line, we performed RNA sequencing and high-performance liquid chromatography analyses. The transcript levels of many GSL pathway genes were significantly altered in GK1 transgenic plants. In addition, GSL contents were substantially reduced in GK1 transgenic plants. We report that the BrGI circadian clock gene is required for the biosynthesis of GSLs in Chinese cabbage plants.

[Comparative study of different therapeutic methods on autoimmune premature ovarian failure in mice].

OBJECTIVE: To investigate the effects of different methods of treatment, including tonifying the kidney, activating blood and soothing the liver, on mice with autoimmune premature ovarian failure (POF). METHODS: After preparing ovarian antigen with the ovarian tissue of female BALB/C mice, POF in female BALB/C mice was induced by immunization injection of the ovarian antigen on multiple subcutaneous sites and two posterior soles. The mice were treated with Liuwei Dihuang Pill, Shaofu Zhuyu Granule, Chaihu Shugan Pill respectively after the third immunization. Levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E(2)), prolactin (PRL), testosterone (T), anti-ovarian antibodies (AOAb) and anti-zona pellucida antibody (aZP) in sera were analyzed by enzyme-linked immunosorbent assay. RESULTS: Compared with the normal group, the levels of serum FSH and LH were increased and the level of E(2) was decreased in the untreated group. AOAb and aZP positive rates in the untreated group were obviously higher than those in the normal group. Liuwei Dihuang Pill could significantly reduce the levels of FSH and LH and increase the level of E(2) as compared with the untreated group, but there were no significant differences in the AOAb and aZP positive rates. Shaofu Zhuyu Granule and Chaihu Shugan Pill could significantly reduce the positive rates of AOAb and aZP as compared with the untreated group, but no significant effect on the levels of FSH, LH and E(2) were observed. The drugs had no effects on serum contents of PRL and T. CONCLUSION: Tonifying the kidney method can recover the ovarian function in POF mice mainly by regulating the hormone levels. Activating blood and soothing the liver methods can improve the humoral immune function in POF mice.

Expression and purification of active recombinant human bikunin in Pichia pastoris.

Bikunin is a proteoglycan exhibiting broad-spectrum inhibitory activity against serine proteases and could potentially suppress tumor cell invasion and metastasis. Here, we have successfully expressed recombinant human bikunin (rh-bikunin) in the methylotrophic yeast Pichia pastoris and established the purification procedure. The cDNA encoding human bikunin was cloned by PCR and inserted into the expression vector pPICZalphaC. After expressed in shake flask, rh-bikunin was produced in an 80-L fermenter and purified by cation exchange chromatography and reverse phase chromatography. The rh-bikunin was active by trypsin inhibition test. The final expression levels were 55 mg/L and we got totally 1.44 g (5600 inhibitor units/mg) of purified rh-bikunin (purity is 95%) from 40 L of fermentation broth. The rh-bikunin consists of two forms with molecular masses of 24 and 21 kDa, respectively. Both forms were immunoreactive by Western blotting and N-terminals were correctly processed by amino-terminal sequencing. This study provided a new method for expression and purification of active rh-bikunin.

[Preventive and therapeutic effects of Bushen Huoxue Recipe on autoimmune premature ovarian failure in mice].

OBJECTIVE: To investigate the preventive effect of Bushen Huoxue Recipe (BSHXR), a compound traditional Chinese herbal medicine, on autoimmune premature ovarian failure (POF) in mice. METHODS: Ovarian antigen was prepared with the ovarian tissue of female BALB/c mice. A mouse model of POF was established by immunization injection of the ovarian antigen of isotype female mice on multiple subcutaneous sites and two posterior soles. The POF mice were treated with BSHXR after the first and third immunization. The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) in peripheral blood were detected by enzyme linked immunosorbent assay. RESULTS: Lymphocyte infiltration was appeared in ovarian stroma of POF mice. The levels of FSH and LH were evaluated and the E(2) level was decreased significantly (P<0.05). BSHXR could reduce the increased levels of FSH and LH, increase the level of E(2) and the number of growing and mature follicles. The efficacy of early treatment was better than that of late treatment. CONCLUSION: BSHXR can recover ovarian function in POF mice mainly by regulating the indiscriminate hormone level, and BSHXR has preventive effect on autoimmune POF in mice.

Spontaneous pepsin C-catalyzed activation of human pepsinogen C in transgenic rice cell suspension culture: Production and characterization of human pepsin C.

A human pepsinogen C (hPGC) gene was synthesized with rice-optimized codon usage and cloned into a rice expression vector containing the promoter, signal peptide, and terminator derived from the rice α-amylase 3D (Ramy3D) gene. In addition, a 6-His tag was added to the 3' end of the synthetic hPGC gene for easy purification. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. The integration of the hPGC gene into the chromosome of the transgenic rice callus and hPGC expression in transgenic rice cell suspensions was verified via genomic DNA polymerase chain reaction amplification and Northern blot analysis. Western blot analysis indicated both hPGC and its mature form, human pepsin C, with masses of 42- and 36-kDa in the culture medium under sugar starvation conditions. Human pepsin C was purified from the culture medium using a Ni-NTA agarose column and the NH2-terminal 5-residue sequences were verified by amino acid sequencing. The hydrolyzing activity of human pepsin C was confirmed using bovine hemoglobin as a substrate. The optimum pH and temperature for pepsin activity were 2.0 and 40°C, respectively.

Production of recombinant human acid α-glucosidase with high-mannose glycans in gnt1 rice for the treatment of Pompe disease.

Lysosomal storage diseases are a group of inherited metabolic disorders. Patients are treated with enzyme replacement therapy (ERT), in which the replacement enzymes are required to carry terminal mannose or mannose 6-phosphate residues to allow efficient uptake into target cells and tissues. N-acetylglucosaminyltransferase-I (GnTI) mediates N-glycosylation in the cis cisternae of the Golgi apparatus by adding N-acetylglucosamine to the exposed terminal mannose residue of core N-glycan structures for further processing. Mutant rice lacking GnTI produces only high mannosylated glycoproteins. In this study, we introduced a gene encoding recombinant human acid α-glucosidase (rhGAA), which is used in ERT for Pompe disease, into gnt1 rice callus by particle bombardment. Integration of the target gene into the genome of the gnt1 rice line and its mRNA expression were confirmed by PCR and Northern blot, respectively. Western blot analysis was performed to confirm secretion of the target proteins into the culture media. Using an indirect enzyme linked immunosorbent assay, we determined the maximum expression of rhGAA to be approximately 45mg/L, 13days after induction. To assay the enzymatic activity and determine the N-glycan profile of rhGAA, we purified the protein using a 6×histidine tag. The in vitro α-glucosidase activity of rhGAA from gnt1 rice callus (gnt1-GAA) was 3.092U/mg, similar to the activity of the Chinese hamster ovary cell-derived GAA (3.154U/mg). N-glycan analysis revealed the presence of high-mannose N-glycans on gnt1-GAA. In addition, the production of high-mannose GAA using gnt1 rice calli as an expression host was characterized, which may aid the future development of therapeutic enzymes for the treatment of Pompe disease.

Production and characterization of recombinant human acid α-glucosidase in transgenic rice cell suspension culture.

Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid α-glucosidase (GAA), a glycogen-degrading lysosomal enzyme. In this study, the human GAA cDNA gene was synthesized from human placenta cells and cloned into a plant expression vector under the control of the rice α-amylase 3D (RAmy3D) promoter. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin) mediated by Agrobacterium tumefaciens. Genomic DNA PCR and Northern blot analysis were used to determine the integration and mRNA expression of the hGAA gene in the putative transgenic rice cells. SDS-PAGE and Western blot analysis showed that the glycosylated precursor recombinant hGAA had a molecular mass of 110kDa due to the presence of seven N-glycosylation sites. The accumulation of hGAA protein in the culture medium was approximately 37mg/L after 11 days of culturing in a sugar depletion medium. The His tagged-hGAA protein was purified using an Ni-NTA column and confirmed as the precursor form of hGAA without the signal peptide encoded by the cDNA on the N-terminal amino acid sequence. The acid alpha-glucosidase activity of hGAA produced in transgenic rice cells gave results similar to those of the enzyme produced by CHO cells.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.