Duhuo Jisheng Decoction inhibits SDF-1-induced inflammation and matrix degradation in human degenerative nucleus pulposus cells in vitro through the CXCR4/NF-κB pathway.

Lower back pain (LBP) is the most common disease in orthopedic clinics world-wide. A classic Fangji of traditional Chinese medicine, Duhuo Jisheng Decoction (DHJSD), has been proven clinically effective for LBP but its therapeutic mechanisms remain unclear. We hypothesized that DHJSD might relieve LBP through inhibiting the exaggerated proinflammatory cytokines and extracellular matrix (ECM) degradation. Thus, we studied the effects of DHJSD on stromal cell-derived factor-1 (SDF-1)-induced inflammation and ECM degradation in human nucleus pulposus cells (hNPCs). The primary hNPCs were isolated from either degenerated human intervertebral disc (HID) of LBP patients or normal HID of lumbar vertebral fracture patients, and cultured in vitro. The cells were treated with SDF-1 (10 ng/mL) and subsequently with different concentrations (100-500 µg/mL) of DHJSD for 24 h, respectively. We found that application of DHJSD significantly antagonized the SDF-1-induced production of proinflammatory cytokines and reduction of aggrecan and type II collagen in the hNPCs. DHJSD also markedly reduced the SDF-1-induced increase of CXCR4 and p-p65 and inhibited the nuclear translocation of p65 in the hNPCs. DHJSD, CXCR4-siRNA, and NF-κB inhibitor (BAY11-7082) caused the same inhibition of exaggerated proinflammatory cytokines in the SDF-1-treated hNPCs. These results provided compelling evidence that DHJSD may inhibit the generation of proinflammatory mediators and ECM degradation of HID through an orchestrated targeting at multiple molecules in the SDF-1/CXCR4/NF-κB pathway, thus offered novel mechanistic insights into the clinical effectiveness of DHJSD on LBP.

[Mechanism of Duhuo Jisheng decotion in delaying degeneration of nucleus pulposus cells in human intervertebral disc].

This paper aimed to investigate the role of Duhuo Jisheng decotion (DHJSD) in delaying human disc degeneration and its possible molecular mechanism. The intervertebral disc specimens were divided into normal and degenerated groups according to Pfirrmann classfication. The expressions of TNF-α, IL-1ß, MMP-3 and MMP-13 in intervertebral disc tissue were detected by Western blot and PCR. Then degenerated human primary NPCs were cultured in vitro, the viability of NPCs treated with stromal cell-derived factor-1 (SDF-1，10 µg·L⁻¹)and various concentrations of DHJSD was assessed by the CCK-8 assay, and the appropriate concentration was screened. The experiment was divided into three groups, control group, SDF-1 group and DHJSD plus SDF-1 group. The levels of TNF-α, IL-1ß, Agg, coIⅡ, MMP-3 and MMP-13 were detected. The levels of CXCR4, NF-κB major groups P65 phosphorylation (p-P65) and nuclear translocation, after treated with CXCR4 siRNA and NF-κB inhibitor (BAY11-7082) were measured by Western blot and immunofluorescence. At the same time, the expression of cell inflammatory factors and extracellular matrix were also measured. The expressions of TNF-α, IL-1ß, MMP-3 and MMP-13 in the degenerated intervertebral disc tissue were significantly increased. In vitro study, the results of CCK-8 indicated that the viability of NPCs was significantly increased when DHJSD concentration was 300 mg·L⁻¹. After the experiment was divided into three groups, compared with SDF-1 group, the expressions of TNF-α, IL-1ß, MMP-3 and MMP-13 in DHJSD group were significantly decreased, but the expressions of Agg, coIⅡ were significantly increased. When CXCR4-siRNA was transfected into NPCs, SDF-1 increased expressions of CXCR4 and p-P65 and inhibited nuclear translocation of P65, whose effect was suppressed by CXCR4-siRNA and DHJSD. In addition, when BAY11-7082 was used to treat NPCs, the expression of TNF-α, IL-1ß, MMP-3 and MMP-13 were significantly decreased. DHJSD could inhibit the production of inflammatory factors and promote the synthesis of extracellular matrix. The potential mechanism may be related to the SDF-1/CXCR4/NF-κB signaling pathway.

The Prognostic Value of the Combination of the Prognostic Nutritional Index and the Lymphocyte:Monocyte Ratio for the Prediction of Patients with Muscle-Invasive Bladder Cancer.

OBJECTIVE: To explore the efficacy of combining the prognostic nutritional index (PNI) and the lymphocyte:monocyte ratio (LMR) for patients with muscle-invasive bladder cancer (MIBC). METHODS: Of 172 patients who were diagnosed with MIBC in our hospital, 94 were eligible for the study. The clinical data of the 94 patients with MIBC were collected. The patients were divided according to the optimal cut-off values for the preoperative PNI and LMR into a low-PNI subgroup (PNI <44.15, 52 patients), a high-PNI subgroup (PNI ≥44.15, 42 patients), a low-LMR subgroup (LMR <2.98, 50 patients) and a high-LMR subgroup (LMR ≥2.98, 44 patients). The area under the receiver operating characteristic (ROC) curve (AUC) was used to analyse the efficacy of the PNI and the LMR in predicting the prognosis of patients with MIBC. Univariate and multivariate logistic regression analyses were performed to evaluate prognostic factors for patients with MIBC. Kaplan-Meier (K‒M) survival analysis was used for overall survival (OS) analysis to explore the ability of the PNI combined with the LMR to predict the prognosis of patients with MIBC. RESULTS: The optimal cut-off values for the preoperative PNI and the preoperative LMR were 44.15 and 2.98, respectively, on the basis of ROC curves. ROC curve analysis revealed that the PNI (AUC = 0.720, sensitivity 65.9%, specificity 74.50%, Youden index 0.399) and the LMR (AUC = 0.724, sensitivity 65.9%, specificity 70.0%, Youden index 0.395) both had good prognostic efficacy for patients with MIBC. The results of univariate and multivariate logistic regression analyses showed that preoperative PNI <44.15 was an independent risk factor for OS in patients with MIBC (p = 0.027). Based on K‒M survival curve analysis, patients with PNI <44.15 and LMR <2.98 had the shortest OS (p = 0.00002). CONCLUSIONS: Low preoperative PNI and LMR values are indicative of poor prognosis in patients with MIBC. The efficacy of their combination was better than that of the factors independently.

Ferroptosis: A New Direction in the Treatment of Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is one of the most common musculoskeletal disorders in middle-aged and elderly people, and lower back pain (LBP) is the main clinical symptom [1, 2], which often causes significant pain and great economic burden to patients [3]. The current molecular mechanisms of IVDD include extracellular matrix degradation, cellular pyroptosis, apoptosis, necrotic apoptosis, senescence, and the newly discovered ferroptosis [4, 5], among which ferroptosis, as a new hot spot of research, has a non-negligible role in IVDD. Ferroptosis is an iron-dependent cell death caused by lipid peroxide accumulation [6]. Its main mechanism is cell death caused by lipid peroxidation by oxygen radicals due to iron overload and inhibition of pathways such as SLC7A11-GSH-GPX4. Currently, more and more studies have found a close relationship between IVDD and ferroptosis [7]. In the process of ferroptosis, the most important factors are abnormal iron metabolism, increased ROS, lipid peroxidation, and abnormal proteins such as GSH, GPX4, and system XC-. Our group has previously elucidated the pathogenesis of IVDD in terms of extracellular matrix degradation, myeloid cell senescence and pyroptosis, apoptosis, and inflammatory immunity. Therefore, this time, we will use ferroptosis as an entry point to discover the new mechanism of IVDD and provide guidance for clinical treatment.

Cloning and primary characterizations of rLcn9, a new member of epididymal lipocalins in rat.

Lipocalins are a structurally conserved and diversely functional family of proteins that are of potential importance in epididymis functions. The rat Lcn9 gene was cloned by in silico methods and genome walking based on homology to the rhesus monkey epididymal ESC513 and its polyclonal antisera were prepared. The rat Lcn9 gene is located on chromosome 3p13 spanning 7 exons, contains 2.3 kb and encodes 179 amino acids with a 17-amino acid signal peptide. Northern blot, western blot, and immunohistochemical staining analysis revealed that rat Lcn9 was a novel epididymis-specific gene, expressed selectively in the proximal caput region, influenced by luminal fluid testicular factors. Moreover, Lcn9 protein was modified by N-glycosylation and bound on the postacrosomal domain of caput sperm. In conclusion, the rat Lcn9 exhibited tissue-, region-, and temporal-specific expression patterns and its expression was regulated by luminal testicular factors. Its potential roles in sperm maturation are discussed.

[Effectiveness of proximal femoral nail anti-rotation and cerclage fixation for complicated femoral subtrochanteric fractures].

OBJECTIVE: To investigate the effectiveness of proximal femoral nail anti-rotation (PFNA) and cerclage fixation for complicated femoral subtrochanteric fractures. METHODS: A clinical data of 74 patients with complicated femoral subtrochanteric fractures, who were admitted between March 2016 and March 2019 and met the criteria, was retrospectively analyzed. Among them, 39 patients were treated with limited open reduction and PFNA combined with cerclage fixation (observation group) and 35 patients were treated with closed reduction and PFNA fixation (control group). There was no significant difference in gender, age, cause of injury, side and type of fracture, and the time from injury to operation ( P>0.05). The ratio of postoperative hemoglobin (1, 3, and 5 days) to the preoperative hemoglobin, the operation time, the first weight-bearing time after operation, and the hospital stay were recorded. X-ray films were taken to observe fracture healing in the two groups and bone resorption around the cerclage in the observation group, and the fracture healing time was recorded. Hip function was evaluated by Harris scoring. RESULTS: The operation time of the observation group was significantly longer than that of the control group ( P<0.05), but the first weight-bearing time and hospital stay were significantly shorter ( P<0.05). All patients were followed up 12 months. There was no significant difference in the ratios of post- to pre-operative hemoglobin (1, 3, and 5 days) between the two groups ( P>0.05). X-ray film reexamination showed that the fractures of the two groups healed smoothly, and the fracture healing time of the observation group was significantly shorter than that of the control group ( t=-12.989, P=0.000). No bone resorption around the cerclage occurred in the observation group. The Harris scores of the observation group were better than those of the control group at 7 days and 1, 2, and 3 months after operation ( P<0.05), and there was no significant difference between the two groups at 6 months after operation ( t=1.329, P=0.180). CONCLUSION: Compared with PFNA fixation, PFNA combined with cerclage fixation for the complicated femoral subtrochanteric fractures has a shorter operation time, and can obtain immediate stability after fixation, which can meet the needs of patients for early functional exercise.

New member of the guanosine triphosphatase activating protein family in the human epididymis.

The effect of the guanosine triphosphatase activating proteins (GAPs) on spermatogenesis has been studied for years, though no GAPs have been explored in epididymis, an essential organ for sperm maturation. In this study, a new GAP member, designated as MacGAP, was cloned in human epididymis. The MacGAP gene encodes a protein of 618 amino acids with a putative size of 70 kDa and harbors the conserved RhoGAP domain. The N-terminal and C-terminal peptides of MacGAP were expressed and their corresponding antisera were prepared. The antisera against N-terminal peptide could detect antigen as low as 0.3 ng, and its specificity was also confirmed. However, the antisera against C-terminal peptide failed to detect its antigen because of its low sensitivity. Immunohistochemistry showed that the MacGAP protein was dependent on epididymis and had a region-specific expression pattern, with high expression in the epithelial cells'basal section in the caput region. The results have created a foundation for further interpretation of the biological effects of GAPs in sperm maturation.

The spatiotemporal expression changes of 16 epididymis-specific genes induced by testosterone, heat, and combination treatment in cynomolgus monkey.

The experimental infertility model of treatments involving testicular warming, testosterone implant, and a combination of the two was developed to confirm a synergistic action induced by the combination treatment on germ cell apoptosis in cynomolgus monkey testis. Using this model, the spatiotemporal expression changes of 16 reported or novel genes in epididymis were investigated to examine the treatment's effect on epididymal genes. It was demonstrated that these region-specific genes, some of which were not regionally fixed, changed greatly with these treatments. The expression levels of these epididymal genes fluctuated, and the expression of most of the genes returned to nearly normal level at the end of treatments. Moreover, the expression changes resulting from the combination treatment were not more significant than those resulting from the single treatment. This suggests that the combination treatment has an antagonistic action on the expression of epididymal genes and that its effect is not as adverse on epididymis as that of the two single treatments.

Detection of antimicrobial resistance genes of pathogenic Salmonella from swine with DNA microarray.

A glass-based microarray was developed to detect 11 antimicrobial resistance genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, and chloramphenicols. The target genes for microarray were generated from Salmonella isolates by PCR and confirmed by sequencing. The specificity of the microarray was tested using 11 positive DNA probes. The sensitivity was tested with tetA gene and Salmonella isolates. Using detection threshold of signal-to-noise ratio (S/N) > or = 1.5 or median pixel intensity > or =1000, antimicrobial resistance genes carried by 30 Salmonella isolates were detected. Common genes included sul I(76.7%, 23/30), aph(3')-IIa (60%, 18/30), tetC (60%, 18/30), cat] (43.3%, 13/30), tetA (40%, 12/30) and aadA1 (36.7%, 11/30), and the results were confirmed to be correct by PCR.

Bioassay-guided fractionation of antifertility components of castorbean (Ricinus communis L.) seed extracts.

In the present study, the aether extracts of castorbean seed (AEC), which possessed antifertility activity and contraceptive efficacy in female and male rodents, were evaluated for their in vitro inhibitory activity in the primary cultured rat decidual stromal cells (DSC). A bioassay-guided fractionation technique was used to separate active components from crude extracts. A colorless crystal showed a significant inhibitory activity (IC(50) = 63.84 +/- 3.04 microg mL(-1), r = +0.9478). The chemical analysis of the colorless crystal by capillary gas chromatography-mass spectrometry (GC-MS) revealed that the colorless crystal was a mixture of five components: four phytosterols which were ergost-5-en-3-ol (6.10%), stigmasterol (35.80%), gamma-sitosterol (44.77%), and fucosterol (8.40%); and one probucol analog (4.93%). It was presumed that gamma-sitosterol may be the main component contributing to inhibit the viability of DSC.

[Role and mechanism of stromal cell derived factor 1 on proliferation of vascular endothelial cells].

Objective: To investigate the role and relative mechanism of stromal cell derived factorl (SDF-1) secreted by nucleus pulposus cells (NPCs) on the proliferation of vascular endothelial cells (VECs). Methods: The NPCs were isolated from the degenerated disc specimens after discectomy. NPCs at passage 1 were transfected with lentivirus-mediated SDF-1 over-expression; transfected and untransfected NPCs at passage 2 were cultured in the three-dimensional alvetex® scaffold, then they were co-cultured with HMEC-1 cells. The morphology of NPCs was observed by scanning electron microscope (SEM), and the apoptosis of HMEC-1 cells was detected by Annexin V/propidiumiodide staining after 72 hours co-culutre. The proliferation of HMEC-1 cells was detected by cell counting kit 8 at 12, 24, 48, and 72 hours in transfected group and untransfected group, respectively. ELISA was used to measure the vascular endothelial growth factor (VEGF) expression level. The virus transfection efficiency and relative Akt pathway were determined by Western blot. Results: The NPCs maintained cell phenotype and secreted much extracellular matrix in three-dimensional-culture by SEM observation. In the co-culutre system, after NPCs were transfected with SDF-1 over-expression lentivirus, the proliferation of HMEC-1 cells was significantly increased, while the apoptosis was decreased obviously. The ELISA results demonstrated that the amount of VEGF was remarkably increased in the culture medium. Furthermore, SDF-1 promoted the up-regulation of phosphorylate Akt expression; after inhibition of Akt expression by GSK690693, the proliferation rate of VECs decreased significantly. Conclusion: Over-expression of SDF-1 by NPCs is beneficial for VECs proliferation, which is involved in SDF-1-Akt signalling pathway.

Genome-wide transcriptional analysis of temperature shift in L. interrogans serovar lai strain 56601.

BACKGROUND: Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptation to a range of new environmental conditions in the organs and tissues of the infected host. Several studies have shown that a shift in culture temperature from 28 degrees C to 37 degrees C, similar to that encountered during infection of a host from an environmental source, is associated with differential synthesis of several proteins of the outer membrane, periplasm and cytoplasm. The whole genome of the Leptospira interrogans serogroup Icterohaemorrhagiae serovar lai type strain #56601 was sequenced in 2003 and microarrays were constructed to compare differential transcription of the whole genome at 37 degrees C and 28 degrees C. RESULTS: DNA microarray analyses were used to investigate the influence of temperature on global gene expression in L. interrogans grown to mid-exponential phase at 28 degrees C and 37 degrees C. Expression of 106 genes differed significantly at the two temperatures. The differentially expressed genes belonged to nine functional categories: Cell wall/membrane biogenesis genes, hemolysin genes, heat shock proteins genes, intracellular trafficking and secretion genes, two-component system and transcriptional regulator genes, information storage and processing genes, chemotaxis and flagellar genes, metabolism genes and genes with no known homologue. Real-time reverse transcription-PCR assays confirmed the microarray data. CONCLUSION: Microarray analyses demonstrated that L. interrogans responds globally to temperature alteration. The data delineate the spectrum of temperature-regulated gene expression in an important human pathogen and provide many new insights into its pathogenesis.

Acaricidal activity against Panonychus citri of a ginkgolic acid from the external seed coat of Ginkgo biloba.

An acaricidal substance extracted from the external seed coat of Ginkgo biloba L. was identified by UV (ultraviolet), IR (infrared), EI-MS (electron impact ion source mass spectrometry), (1)H NMR (nuclear magnetic resonance) and (13)C NMR as 6-[(Z)-10-heptadecenyl]-2-hydroxybenzoic acid (compound 1). Laboratory bioassay on citrus red mite, Panonychus citri (Mcg), showed that compound 1 possessed the following properties. (i) Powerful contact toxicity with an LC(50) of 5.2 mg litre(-1) after 24 h that was similar to that of pyridaben (LC(50) = 3.4 mg litre(-1)) and significantly superior to that of omethoate (LC(50) = 122 mg litre(-1)). Furthermore, its LC(90) was 13.4 mg litre(-1) after 24 h, which is significantly superior to both pyridaben (LC(90) = 69.6 mg litre(-1)) and omethoate (LC(90) = 453 mg litre(-1)). (ii) Quick-acting acaricidal activity. At identical concentrations, compound 1 was much faster-acting than pyridaben or omethoate. (iii) Compound 1 had strong corrosive action on the cuticle of P. citri but no phytotoxicity to plants.

Stomatal movement in response to long distance-communicated signals initiated by heat shock in partial roots of Commelina communis L.

The systematic or long-distance signal transmission plays crucial roles in animal lives. Compared with animals, however, much less is known about the roles of long-distance signal communication in plant lives. Using the model plant Commelina communis L., we have probed the root to shoot communication mediated by heat-shock signals. The results showed that a heat shock of 5 min at 40 degrees C in partial roots, i.e. half or even 1/4 root system, could lead to a significant decrease in stomatal conductance. The regulation capability depends on both heat shock temperature and the amount of root system, i.e. with higher temperature and more roots stressed, the leaf conductance would decrease more significantly. Interestingly, the stomatal regulation by heat shock signal is in a manner of oscillation: when stomata conductance decreased to the lowest level within about 30 min, it would increase rapidly and sometimes even exceed the initial level, and after several cycles the stomata conductance would be finally stabilized at a lower level. Feeding xylem sap collected from heat-shocked plants could lead to a decrease in stomata conductance, suggesting that the heat shock-initiated signal is basically a positive signal. Further studies showed that heat shock was not able to affect ABA content in xylem sap, and also, not able to lead to a decrease in leaf water status, which suggested that the stomatal regulation was neither mediated by ABA nor by a hydraulic signal. Heat shock could lead to an increase in xylem sap H2O2 content, and moreover, the removal of H2O2 by catalase could partially recover the stomatal inhibition by xylem sap collected from heat-shocked plants, suggesting that H2O2 might be able to act as one of the root signals to control the stomatal movement. Due to the fact that heat-shock and drought are usually two concomitant stresses, the stomatal regulation by heat-shock signal should be of significance for plant response to stresses. The observation for the stomatal regulation in an oscillation manner by presently identified new signals should contribute to further understanding of the mystery for the pant systematic signaling in response to stresses.

Cloning and expression of a chitinase gene from Sanguibacter sp. C4.

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank database. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.

[Isolation and identification of a Streptomyces strain against ginger bacterial wilt caused by Pseudomonas solanacearum Smith].

Streptomyces SR-11 was isolated from the soil of ginger in Sichuan Province against ginger bacterial wilt caused by Pseudomonas solanacearum Smith. It had distinctively inhibitive effect on gram-positive bacterium, gram-negative bacterium and some kinds of pathogenic fungi. It's morphological, cultural physiological, biochemical characteristics, chemotaxonomy and 16S rDNA sequences analysis were studied. The substrate mycelium have no partition, the aerial mycelium are ramose; The spore-bearing filaments are spiral, the spores are oval and the surface are smooth. Cell wall type I, Sugar type C. When SR-11 is matured, the aerial mycelium are gray and the strain can give out an earthy smell. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related bacteria species. In the phylogenetic tree the overall similarity value between strain SR-11 and 12 type Streptomyces sp are 96.5-98.3%.

[Identification of a pathogenic strain of locusts and its toxicity and pathology].

A pathogen was isolated from naturally dead grasshoppers. Its pathogenicity was testified by law of KOCH. It was identified as Serratia marcescens by physiological, biochemical test and molecular systematic analysis. Toxicity of HR-3 to grasshoppers was assayed by means of oral infection. The results show that the linear regression relationship between the logarithm(y) of HR-3 concentration and the probability (x) of corrected grasshopper morality is y = 1.067 + 0.809x, and the median lethal concentration is LC50 = 1.164 x 10(8) cfu/mLo The infection mechanism of HR-3 to grasshoppers were also studied. The histopathological studies show that midgut epithelia are damaged at the beginning of infection, and then partial denaturalization and putrescence occurr in this area. After 48h infection, vacuoles appear in most regions of midgut epithelia.

[The effect of entrapment of CpG sequence with cationic PLG nanoparticles on the immune responses of mice to pig paratyphoid vaccine].

Cationic PLG nanoparticles and liposome were prepared and used as package molecules to pack up pUC18-CpG. The effects of the packed pUC18-CpG on the cellular and humoral immune responses were detected in the mice that were inoculated with pig paratyphoid vaccine. The results showed that compared with the control, the amount of IgG and the titre of specific antibody were significantly increased in the sera of mice immunized with the CpG plasmid entrapped by cationic PLG nanoparticles; the proliferation and induced IL-2 bioactivity of lymphocytes were significantly enhanced in the spleen of the immunized mice; the stimulatory effect of cationic PLG nanoparticles was similar to or stronger than that of cationic liposome. These indicated that cationic PLG nanoparticle could be employed as an effective package molecule to promote the immunostimulatory effect of pUC18-CpG.

Constructing an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing.

An amylolytic brewing yeast Saccharomyces pastorianus, free of vector sequences and drug-resistance genes, was constructed by disrupting the alpha-acetolactate synthase gene and introducing the alpha-amylase gene as a selective marker. The resulting recombinant strain was able to utilize starch as the sole carbon source and its alpha-acetolactate synthase activity was lowered by 30%. Fermentation tests confirmed that the diacetyl concentration and the residual oligosaccharide were reduced by 70% and 25%, respectively, in fermented wort by the recombinant strain, while the brewing performance of the recombinant strain was retained.

Cloning and expression of the AtGRP9 gene related to salt stress tolerance in Arabidopsis thaliana.

The fission yeast Schizosaccharomyces pombe is a single cell eukaryotic organism. Taking advantage of the genetic simplicity of S.pombe and of the functional conservation of some salt tolerance-related proteins, this organism was used as a simple system to identify Arabidopsis thaliana salt-tolerance proteins by screening for cDNA clones that confer salt resistance when overexpressed. By this "cross-phylogenetic" screen, an A.thaliana gene was isolated encoding a glycine-rich protein, which was named AtGRP9. Overexpression of AtGRP9 in yeast cells led to significant increase in salt tolerance of the yeast strain. Northern blotting analysis revealed that AtGRP9 was expressed specifically in the roots of A.thaliana and was induced by NaCl. The growth of some transgenic plants was easily controlled by using the medium with different NaCl concentrations. Taken together, these results show that AtGRP9 may be involved in the salt stress response in A.thaliana.