Effect of UV-B radiation on growth, flavonoid and podophyllotoxin accumulation, and related gene expression in Sinopodophyllum hexandrum.

Sinopodophyllum hexandrum is an alpine medicinal plant that produces the anticancer compound podophyllotoxin (PPT). Although a positive relationship between PPT content and altitude has been proved and low temperature enhances plant growth and PPT accumulation has also been revealed, the role of UV radiation in regulating growth and PPT accumulation is still unclear In this study, morphophysiological traits, metabolites content and related genes expression were investigated by exposing S. hexandrum seedlings to treatment with UV-B radiation. The results showed that the contents of soluble sugars and flavonoids, and the expression levels of genes involved in glycometabolism (XET and ß-1,3-glucanase) and flavonoid biosynthesis (PAL,C4H,4CL,CHS1 and DTX41) were enhanced in response to UV-B compared to CK. Moreover, genes involved in stress tolerance (MYB, WRKY,APX3 and EX2) were also upregulated in response to UV-B radiation. Although the whole plant biomass exhibited slightly increased values that depended largely on root development, the contents of chlorophyll and PPT and the expression levels of genes involved in photosynthesis (matK, ndhF,rbcL and ycf5) and PPT biosynthesis (C3H,CCoAMT,CCR,CAD, DPO, PLR,SDH, CPY719A23,OMT3,CYP71CU1,OMT1and 2-ODD) were significantly decreased in response to UV-B compared to CK. It can be concluded that UV-B radiation promotes soluble sugars and flavonoids accumulation, but inhibits PPT biosynthesis in S. hexandrum.

[Influence of Xuebijing injection and its component paeoniflorin on immune function and survival rate of septic rats].

Objective: To explore the influence of Xuebijing injection (hereinafter referred to as Xuebijing) and its component paeoniflorin on immune function of regulatory T cells (Tregs) of spleen and survival rate of septic rats. Methods: (1) CD4(+) CD25(+) Tregs and CD4(+) T cells were isolated and purified from spleens of three 9 to 12 weeks old Sprague-Dawley male rats (the same age, breed, and gender below) by immunomagnetic beads. According to the random number table (the same grouping method below), CD4(+) CD25(+) Tregs were divided into blank control group, simple CD3/CD28 group, simple endotoxin/lipopolysaccharide (LPS) group, LPS+ Xuebijing group, and LPS+ paeoniflorin group, with 6 wells in each group. The cells in simple CD3/CD28 group, simple LPS group, LPS+ Xuebijing group, and LPS+ paeoniflorin group were cultured in RPMI 1640 medium containing fetal bovine serum in volume fraction of 10%, 1.25 µg CD3, and 2.5 µg CD28 for 24 hours. Then 1 µg/mL LPS in the volume of 1 µL was added to the cells in simple LPS group, LPS+ Xuebijing group, and LPS+ paeoniflorin group. Moreover, 5 mg/mL Xuebijing in the volume of 1 µL and 80 µmol/L paeoniflorin in the volume of 1 µL were added to the cells in LPS+ Xuebijing group and LPS+ paeoniflorin group, respectively, which were cultured for another 72 hours. Cells in blank control group were routinely cultured in RPMI 1640 medium containing fetal bovine serum in volume fraction of 10% for 96 hours. The expressions of cytotoxic T lymphocyte antigen 4 (CTLA-4) and forkhead wing-link transcription factor 3 (Foxp3) and apoptosis of CD4(+) CD25(+) Tregs were measured by flow cytometry. The interleukin-10 (IL-10) level from culture supernatant of CD4(+) CD25(+) Tregs was determined by enzyme-linked immunosorbent assay (ELISA). CD4(+) T cells were divided into blank control' group, simple CD3/CD28' group, simple LPS' group, LPS+ Xuebijing' group, and LPS+ paeoniflorin' group, with 6 wells in each group. After being cocultured with the corresponding CD4(+) CD25(+) Tregs treated as before for 72 hours, the proliferative activity of CD4(+) T cells was measured by flow cytometry, and IL-4 level from culture supernatant of CD4(+) T cells was determined by ELISA. (2) One hundred and twenty rats were divided into sham surgery group, simple sepsis group, sepsis+ Xuebijing group, and sepsis+ paeoniflorin group, with 30 rats in each group. The septic rat model was reproduced by cecal ligation and puncture surgery in simple sepsis group, sepsis+ Xuebijing group, and sepsis+ paeoniflorin group. In sham surgery group, the rats were only performed with laparotomy to simulate surgery. In sepsis+ Xuebijing group, the rats were given post-surgical injection of 4 mL/kg Xuebijing through tail vein, twice a day. In sepsis+ paeoniflorin group, the rats received 978 µg paeoniflorin via tail vein, twice a day. The survival rates of rats in the four groups on post surgery day 1, 2, 3, 4, 5, 6, and 7 were observed and recorded. The surviving cure of Kaplan-Meier was drawn. Data were statistically analyzed with one-way analysis of variance, least significant difference t test. The surviving curve was analyzed by Log-rank (Mantel-Cox) test. Results: (1) Compared with those in blank control group, the expressions of CTLA-4 and Foxp3 of CD4(+) CD25(+) Tregs (t=27.19, 17.00, P<0.01) and IL-10 level from culture supernatant (t=40.76, P<0.01) were significantly increased in rats in simple LPS group. Compared with those in simple LPS group, the expressions of CTLA-4 and Foxp3 of CD4(+) CD25(+) Tregs (t(LPS+ Xuebijing group)=31.03, 11.27, t(LPS+ paeoniflorin group)=5.79, 5.64, P<0.01) and IL-10 level from culture supernatant (t=15.49, 4.20, P<0.01) was significantly decreased in LPS+ Xuebijing group and LPS+ paeoniflorin group. Compared with that in blank control group, the apoptosis rate of CD4(+) CD25(+) Tregs in simple LPS group was significantly declined (t=6.02, P<0.01). Compared with the rate in simple LPS group, the apoptosis rates of CD4(+) CD25(+) Tregs in LPS+ Xuebijing group and LPS+ paeoniflorin group were significantly increased (t=20.32, 8.60, P<0.01). (2) Compared with those in simple CD3/CD28' group, the proliferative rate of CD4(+) T cells was significantly decreased in simple LPS' group (t=22.47, P<0.01), while IL-4 level from culture supernatant was significantly elevated (t=3.51, P<0.01). Compared with those in simple LPS' group, the proliferative rates of CD4(+) T cells in LPS+ Xuebijing' group and LPS+ paeoniflorin' group were significantly increased (t=16.31, 11.48, P<0.01), while IL-4 level from culture supernatant showed no obvious change. (3) The post-operative 7-day survival rates of rats in sham surgery group, simple sepsis group, sepsis+ Xuebijing group, sepsis+ paeoniflorin group were 100% (30/30), 30% (9/30), 57% (17/30), and 47% (14/30), respectively. Compared with that in simple sepsis group, the survival rate within post-operative 7-day of rats in sepsis+ Xuebijing group was significantly higher (χ(2)=4.34, P<0.05), while the survival rate within post-operative 7-day of rats in sepsis+ paeoniflorin group showed no obvious change. Conclusions: Both Xuebijing and its component paeoniflorin are capable of reversing sepsis-induced inhibitory immune function and apoptotic resistant of Tregs in rats, and further improving the proliferative activity of T cells. In addition, the effect of paeoniflorin on improvement of survival rate of rats with sepsis is weaker than Xuebijing.

First Report of Subanguina moxae Infecting Mugwort in Yunnan, China.

Mugwort is a perennial in the Compositae family distributed throughout Asia and Europe. The leaves are reported to have various pharmaceutical properties, e.g., antibacterial, antiviral, antitussive, and hemostatic properties, and have been used in traditional Chinese medicine for more than 2,000 years. In August 2011, a field of mugwort in Kunming, Yunnan Province, China, exhibited more than 90% incidence of whitish and rounded galls on the leaves. There were approximately 10 galls on each leaf, impacting the quality of the leaves for medicinal use. Parasitic nematodes were found upon dissection of the galls, then eggs, second-stage juveniles (J2), and mature males and females were observed. Through the morphologic observation of juveniles and female and male adults, the parasitic nematode was identified as Subanguina moxae (Yokoo and Choi, 1968) Brzeski, 1981 (3). Key morphological features are as follows: eggs (n = 20) measured 54.0 to 71.4 × 24.1 to 30.0 µm; J2 (n = 20) had the following characteristics: body length 689.3 to 873.2 µm (x = 775.5 µm); stylet length 8.2 to 9.8 µm (x = 8.8 µm); tail length 49.5 to 74.5 µm (x = 60.1 µm); a (total body length/maximum body width) ranged from 28.6 to 38.6 µm (x = 34.1 µm); and c (total body length/the length of the tail) ranged from 11.2 to 16.0 µm (x = 13.0 µm). Females (n = 20) had the following characteristics: body length 1,252.8 to 1,665.2 µm (x = 1,475.7 µm); stylet length 7.2 to 9.2 µm (x = 8.2 µm); V of 88.0 to 92.3 µm (x = 89.6 µm); a ranged from 17.6 to 24.5 µm (x = 21.3 µm); and c ranged from 20.2 to 28.9 µm (x = 22.8 µm). Males (n = 20) had the following characteristics: body length 994.2 to 1,453.6 µm (x = 1,253.2 µm); stylet length 7.5 to 9.9 µm (x= 9.1 µm); tail length 69.2 to 88.1 µm (x = 78.0 µm); spicule length 22.2 to 33.4 µm (x = 29.4 µm); gubernaculum length 10.4 to 14.2 µm (x = 12.2 µm); a ranged from 23.1 to 37.2 µm (x = 29.9 µm); and c ranged from 13.9 to 18.7 µm (x = 16.1 µm). Amplification of the rDNA-internal transcribed spacer (ITS) region and the D2/D3 (1) fragments of the 28S RNA with universal primers rDNA1/rDNA2 and D2A/D3B yielded PCR fragments of 934 bp and 754 bp, respectively. The ITS sequence (JN865234) and D2D3 sequence (JN885540) were submitted to GenBank. The ITS sequence (JN865234) exhibited 99.4% similarity with Mesoanguina moxae (AF396314) (synonym of S. moxae) (4). S. moxae has been identified from the common mugwort in Japan (2) and in China, was reported to infect wheat in Guizhou Province, but to our knowledge, this is the first report of this nematode affecting mugwort in Yunnan, China. References: (1) S. Amiri et al. Eur. J. Plant Pathol. 108:497, 2002. (2) K. Daigo et al. Bull. School Agric. Meiji University. 56:237, 2007. (3) M. R. Siddiqi. Tylenchida: Parasites of Plants and Insects. CABI Publishing, New York, 2000. (4) S. A. Subbotin et al. Mol. Phylogenet. Evol. 30:226, 2004.

Cornel iridoid glycoside promotes neurogenesis and angiogenesis and improves neurological function after focal cerebral ischemia in rats.

The aim of this study was to investigate the effects of cornel iridoid glycoside (CIG), an ingredient extracted from a traditional Chinese herb Cornus officinalis, on neurological function and neurogenesis after ischemic stroke. CIG was intragastrically administered to rats in doses of 20, 60 and 180 mg/kg/day, starting 3 h after the onset of middle cerebral artery occlusion (MCAO). The behavioral test was performed by using the modified neurological severity score (mNSS). Rats were sacrificed 7, 14, or 28 days after ischemia occurred. Neurogenesis and angiogenesis were detected by using immunofluorescence staining. The messenger ribonucleic acid (mRNA) expression of vascular endothelial growth factor (VEGF) and its receptor Flk-1 was measured by RT-PCR, and the protein expression of VEGF was determined by Western blotting analysis. The treatment with CIG at the doses of 60 and 180 mg/kg/day significantly improved neurological function, and increased the number of bromodeoxyuridine (BrdU)-positive cells and nestin-positive cells in the subventricular zone of rats 7, 14 and 28 days after ischemia. The number of newly mature neurons and blood vessels in striatum, as indicated by BrdU/NeuN and vWF immunoreactivity, respectively, was also increased in CIG-treated rats 28 days after stroke. CIG treatment obviously enhanced the mRNA expression of VEGF and its receptor Flk-1 and the protein expression of VEGF 7 and 28 days after ischemia. The results indicated that CIG promoted neurogenesis and angiogenesis and improved neurological function after ischemia in rats, and the mechanism might be related to CIG's increasing VEGF and Flk-1 in the brain.

Extract from Arca granosa L. inhibits proliferation of human tumour cell lines with kidney and lung origin.

Marine organisms are being considered increasingly as sources of anti-tumour agents. The extract from Arca granosa L. has been shown to decrease the growth of tumours and this study was undertaken to determine its ability and mechanism of inhibition. The extract inhibited the proliferation of six human tumour cell lines from different origins with varying sensitivity. The cell lines Ketr-3, A549 and NCl-H460 with kidney or lung origins were more sensitive to the extract than those of the HepG-2, MCF-7 and MGC-803 cells from other origins. In the three sensitive cell lines (Ketr-3, A549 and NCI-H460) the extract was shown to block different phases of the cell cycle progression and inhibit DNA synthesis in a concentration-dependent manner. It was concluded that the extract from A. granosa is potentially a novel anti-tumour agent, especially on kidney and lung tumour cell lines.

Co-localization of NK(1)-receptor mRNA with glutamate immunopositivity in cat hypothalamic neurons by the combination of in situ hybridization and immunohistochemistry.

Previous studies had demonstrated that, in the cat, aggression is mediated by glutamatergic neurons in the anterior medial hypothalamus which project to the periaqueductal gray. Additionally, NK(1) receptor activation in the medial hypothalamus plays a role in the regulation of aggressive behavior by the medial amygdala. In the present study, in situ hybridization and immunohistochemistry were combined in order to provide neurochemical characterization of medial hypothalamic neurons containing NK(1)-receptor mRNA. In order to identify NK(1) receptors in cat brain, a 650-bp fragment of the cat NK(1) cDNA was cloned. This fragment was used to synthesize a riboprobe for in situ hybridization. Partial DNA sequence analysis of the fragment indicated a 90% homology with human cDNA. In situ hybridization revealed the presence of NK(1)-receptor mRNA in cat hypothalamic neurons. Tissue used to localize NK(1) receptors was also processed for glutamate immunopositivity. The results demonstrated that NK(1)-receptor mRNA is present in glutamate-immunopositive neurons in the anterior medial hypothalamus of cat, thus reinforcing the hypothesis that NK(1) receptors play an important role in this neural circuit.

Tea as a potential chemopreventive agent in PhIP carcinogenesis: effects of green tea and black tea on PhIP-DNA adduct formation in female F-344 rats.

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is formed during the cooking of proteinaceous animal foods (meat, chicken, and fish). PhIP is a carcinogen in the Fischer 344 (F-344) rat; it induces mammary tumors in female rats and lymphomas and colon and prostate tumors in male rats. In F-344 rats, PhIP forms DNA adducts in various organs, including the target organs. Inhibition of PhIP-DNA adduct formation is likely to lead to inhibition of PhIP tumorigenicity. We have examined the chemopreventive properties of green tea and black tea in PhIP carcinogenesis by evaluating their effects on PhIP-DNA adduct formation in the female F-344 rat. Young adult animals were maintained on powdered AIN-76A diet while receiving regular drinking water or 2% (wt/vol) infusions of green tea or black tea for a total of six weeks. During Weeks 3, 4, and 5, all animals received PhIP by gavage (1 mg/kg/day). Three rats per group were euthanized on Days 1 and 8 after termination of PhIP exposure. DNA was isolated from a number of organs and analyzed for PhIP-DNA adducts by 32P-postlabeling assays. Compared with animals on regular drinking water, PhIP-DNA adduct formation was inhibited in small intestine, colon, liver, and mammary epithelial cells (MECs) of animals receiving green tea or black tea as the sole source of drinking fluid. Green tea inhibited adduct formation in colon, liver, and MECs (33.3-80.0%) on both days, but only on Day 8 (54.4%) in small intestine. Black tea inhibited adduct formation on both days in liver (71.4-80.0%), on Day 1 in colon (40.0%), and on Day 8 in small intestine (81.8%); it had no effect on MEC adducts. Neither green tea nor black tea had an effect on adduct levels in pancreas, lungs, white blood cells, heart, kidneys, spleen, cecum, or stomach. Similarly, these teas did not affect the rate of adduct removal (percent change from Day 1 to Day 8) in any organ. It is concluded that green tea and black tea are potential chemopreventive agents in PhIP-induced tumorigenesis in the F-344 rat.

Glutamyl hydrolase. pharmacological role and enzymatic characterization.

gamma-Glutamyl hydrolase (GH, EC 3.4.19.9) is a lysosomal and secreted glycoprotein that hydrolyzes the gamma-glutamyl tail of antifolate and folate polyglutamates. Tumor cells that have high levels of GH are inherently resistant to classical antifolates, and further resistance can be acquired by elevations in GH following exposure to this class of antitumor agents. The highest level of expression in normal tissues occurs in the liver and kidney in humans. When panels of tumors are compared with normal tissues, GH expression is elevated in cancerous hepatic and breast tissue. A second poly-gamma-glutamate hydrolyzing enzyme, glutamate carboxypeptidase II, is a transmembrane protein whose active site is on the outside of the cell, occurring in the prostate gland, small intestine, brain, kidney, and tumor neovasculature. It is a high-affinity (nanomolar), low-turnover, zinc co-catalytic enzyme. In contrast, GH is a low-affinity (micromolar), high-turnover enzyme that has a cysteine at the active site. Data are presented suggesting that Cys110 is the nucleophile that attacks the gamma-amide linkage and causes hydrolysis. GH is being evaluated as an intracellular target for inhibition in order to enhance the therapeutic activity of antifolates and fluorouracil.

[Construction of cDNA library of Mortierella and screening of delta 9 fatty acid desaturase cDNA sequence].

Mortierella species have potential for fermentative production of polyunsaturated fatty acids including gamma-linolenic acid, Arachidonic acid and EPA, etc. In order to clone genes encoding enzymes in the unsaturated fatty acid biosynthetic pathway, cDNA library of Mortierella was constructed using lambda gt 10 vector. Using cDNA encoding conserved region of delta 9 fatty acid desaturase gene as probe, Mortierella cDNA library was screened. After two rounds of screening one positive clone was identified which has insert length of larger than 1.6 kb.

Structural organization of the human gamma-glutamyl hydrolase gene.

Gamma-glutamyl hydrolase (GH) plays an important role in the metabolism of folic acid and the pharmacology of antifolates such as methotrexate. We have previously cloned and characterized the human GH cDNA. In this report, the complete organization and structure of the human GH gene was determined. The human GH gene spans 24 kb in the human genome, with nine exons sized from 51 to 371 bp. All of exon-intron splice junctions follow the GT-AG rule. The sequence upstream of exon 1 consists of a promoter-like, GC-rich region and a number of putative cis active elements including Sp1, AP1, and MZF1 sites. A TATA sequence in the 5' region of human GH gene was not observed, similar to housekeeping genes known to be tissue-specific and differentially expressed. S1 nuclease protection analysis with human liver, prostate, brain, and mammary gland revealed a major transcription start point at nucleotide -125 relative to the ATG start codon and several minor transcription start points. Analysis of GH cDNA isolated from human liver indicated a nucleotide change, T-->C, in the leader sequence of GH, which suggested a polymorphism. Studies of cDNA from different human tissue sources provided evidence that there is a single spliced cDNA species in human.

Neurokinin-1 expression and co-localization with glutamate and GABA in the hypothalamus of the cat.

Recent behavioral studies using pharmacological techniques have demonstrated that the high affinity substance P (SP) receptor, neurokinin-1 receptor (NK-1), in the medial hypothalamus could be important in mediating defensive rage behavior in the cat. These observations prompted us to use molecular techniques to determine the distribution of NK-1 in the hypothalamus and in other regions of the forebrain relevant to the control of rage behavior. We cloned a 650 bp fragment of the cat NK-1 cDNA. Partial DNA sequence analyses of this fragment indicate 90% homology with the human cDNA. By in situ hybridization (ISH), we showed that NK-1 mRNA was localized in the cytoplasm but not nuclei of cat forebrain neurons. Furthermore, NK-1 mRNA was co-localized in neurons that displayed positive immunolabeling for glutamate or GABA. Moderate labeling was visualized in the anterior medial hypothalamus which receives significant SP input via the stria terminalis from the medial amygdala. Strong labeling was also observed in the basal amygdaloid complex. The functional significance of this labeling pattern is suggested from the observation that both the medial and basal complex of amygdala serve as powerful modulators of defensive rage behavior. Weaker labeling was seen over the posterior medial and lateral hypothalamus. The distribution of NK-1 in the hypothalamus was matched by that of SP-immunoreactive axons and pre-terminals that were observed in the hypothalamus. The overall findings provide anatomical evidence to show that the high affinity SP receptor, NK-1, is linked to glutamate and GABA neurons in the anterior medial hypothalamus and further suggests its likely role in the regulation of feline aggression.

[Effect of nei-yi recipe on plasma beta-endorphin levels during menstrual cycle in women with endometriosis].

In order to investigate the mechanism of Nei-Yi Recipe (NYR) in treatment of endometriosis, we observe the changes of plasma beta-endorphin concentrations in women with endometriosis treated by NYR. It was found that in luteal phase beta-endorphin concentrations in plasma were significantly reduced in moderate and severe dysmenorrhea groups compared with mild and control groups. Moreover, beta-endorphin level in severe dysmenorrhea group was lower in the luteal phase than that in the follicular phase, and it was lower in patients with pelvic pain than that without pelvic pain. In normal women, the contents of beta-endorphin in plasma were not changed during menstrual cycle. It was also found that plasma beta-endorphin levels were significantly increased in luteal phase and follicular phase after treatment by NYR. The therapeutical mechanism of NYR on dysmenorrhea and pelvic pain and enhancement of immune function might be mediated by increase of plasma beta-endorphin levels.