Promoter identification and analysis of key glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Development of indicators for evaluating the appropriate use of triazoles for invasive fungal disease: A Delphi panel survey.

WHAT IS KNOWN AND OBJECTIVES: Triazoles including fluconazole, itraconazole, voriconazole and posaconazole are now widely used, whereas in some countries, they are reportedly used in inappropriate way frequently; thus, it is clearly a matter of urgency to regulate the use of triazole drugs. Several studies have made good attempts to evaluate antifungal use, but they did not cover the entire medication process. This study aimed to establish indicators for the appropriate use of triazoles for invasive fungal disease, so as to produce a reference for evaluating and standardizing the rational application of triazole antifungals. METHODS: A comprehensive analysis and literature review were conducted to construct an evaluation system for indications, therapeutic timing, duration of drug usage, drug dosage, administration method, drug interactions, medication in specific populations and pharmaceutical care. A Delphi questionnaire was designed based on the preliminary evaluation of an indicator system that comprised eight primary indicators, 29 secondary indicators and 54 tertiary indicators. A Delphi panel survey was completed by 28 and 26 experts in the first and second rounds, respectively, via e-mail. Agreement was considered to be reached when the median score was in the top tertile (4 or 5), and 80% of panel scores were in the top tertile. The experts mainly comprised physicians and clinical pharmacists from different regions. Based on expert opinions, the evaluation indicator system was modified and the final version was established. RESULTS: The response rate was high in the first and second rounds, at 100% and 93%, respectively, as was the dependability coefficient (0.83). The expert opinions resulted in four secondary indicators and nine tertiary indicators being deleted, and 10 tertiary indicators being modified. After the two rounds of the Delphi survey, the final established system of indicators comprised eight primary indicators, 25 secondary indicators and 45 tertiary indicators. WHAT IS NEW AND CONCLUSIONS: Contrasted with previous studies that have only focused on a subset of indicators, this research establishes comprehensive indicators for evaluating the use of triazoles for invasive fungal disease and which cover most of the medication process: indications, therapeutic timing, duration of drug usage, drug dosage, administration method, drug interactions, medication in specific populations and pharmaceutical care. The indicators can reflect the characteristic of triazoles throughout the process of clinical administration for an invasive fungal disease, and it will be helpful as references when hospital administrators are regulating the use of antifungals.

Expression of key glycosphingolipid biosynthesis-globo series pathway genes in Escherichia coli F18-resistant and Escherichia coli F18-sensitive piglets.

A pioneering study showed that the glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1 and NAGA) may play an important regulatory role in resistance to Escherichia coli F18 in piglets. Therefore, we analysed differential gene expression in 11 tissues of two populations of piglets sensitive and resistant respectively to E. coli F18 and the correlation of differential gene expression in duodenal and jejunal tissues. We found that the mRNA expression of the seven genes was relatively high in spleen, liver, lung, kidney, stomach and intestinal tract; the levels in thymus and lymph nodes were lower, with the lowest levels in heart and muscle. FUT2 gene expression in the duodenum and jejunum of the resistant population was significantly lower than that in the sensitive group (P < 0.01). ST3GAL1 gene expression was also significantly lower in the duodenum of the resistant population than in the sensitive group (P < 0.05). No significant differences were observed among the remaining genes. The expression level of FUT1 was extremely significantly positively correlated with FUT2 and B3GALNT1 expression (P < 0.01) and also had a significant positive correlation with NAGA expression (P < 0.05). The expression level of FUT2 had extremely significant positive correlations with FUT1, ST3GAL1 and B3GALNT1 (P < 0.01). These results suggest that FUT2 plays an important role in E. coli F18 resistance in piglets. FUT1, ST3GAL1, B3GALNT1 and NAGA may also participate in the mechanism of resistance to E. coli F18.

Characteristic element of matrix attachment region mediates vector attachment and enhances nerve growth factor expression in Chinese hamster ovary cells.

Preliminary studies have suggested that a characteristic element of the matrix attachment region (MAR) in human interferon-ß mediates the adhesion of vectors to Chinese hamster ovary (CHO) cells. In this study, we investigated if vector adhesion increased nerve growth factor (NGF) expression in CHO cells. The MAR characteristic element sequence of human interferon-ß was inserted into the multiple-cloning site of the pEGFP-C1 vector. The target NGF gene was inserted upstream of the MAR characteristic element sequence to construct the MAR/NGF expression vector. The recombinant plasmid was transfected into CHO cells and stable monoclonal cells were selected using G418. NGF mRNA and protein expression was detected by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Plasmid reduction experiments were used to determine the state of transfected plasmid in mammalian cells. The insertion of MAR into the vector increased NGF expression levels in CHO cells (1.93- fold) compared to the control. The recombinant plasmid expressing the MAR sequence was digested into a linear space vector. The inserted MAR and NGF sequences were consistent with those inserted into the plasmid before recombination. Therefore, we concluded that the MAR characteristic element mediates vector adhesion to CHO cells and enhances the stability and efficiency of the target gene expression.

Regulating effects of insertion direction of matrix attachment regions on transgenic expression in stably transformed Chinese hamster ovary cells.

We investigated the effects of different directions of insertion of matrix attachment region (MAR) sequences on transgenic expression in stably transformed Chinese hamster ovary (CHO) cells. The MAR sequences were inserted in forward or reverse directions into the expression vectors, and transfected into CHO cells. The expression of the chloramphenicol acetyltransferase (CAT) reporter gene and the relative copy numbers of the CAT gene were analyzed. The CAT gene expression levels in the vector with the MAR sequence inserted in the forward or reverse directions increased compared with expression without the MAR sequence. The relative copy numbers of the CAT gene with MAR sequenced vectors inserted in the reverse and forward directions were lower, than in the control group. The direction of insertion of MAR sequences had no significant effect on expression levels. The expression levels were not proportional to the copy numbers of the gene.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Dynamic changes in TAP1 expression levels in newborn to weaning piglets, and its association with Escherichia coli F18 resistance.

The transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. This study showed the dynamic changes in the TAP1 expression level in newborn to weaning piglets. Tissue expression profiles revealed that the TAP1 gene was expressed at low levels in all tissues, and the expression levels were relatively higher in the lung, spleen, lymph, and thymus; further, no significant difference was observed in the expression in each tissue among the 3 unweaned stages (8, 18, and 30 days). Nevertheless, the postweaning (35 days) expression levels in tissues, including the spleen, lung, lymph, duodenum, and jejunum were significantly higher than those in the unweaned stages. Furthermore, gene ontology and pathway analysis showed that TAP1 took part in 38 biological functions and 5 pathway processes, including ABC transporters and antigen processing and presentation. These analyses showed that the TAP1 gene, which was related to MHC I immune regulation, had a stable and low expression level in unweaned stages; however, its expression increased in the postweaning stages. The high expression level of TAP1 indicated that the gene might play an important role in Escherichia coli F18 resistance.

Stipe Canker Caused by Trichothecium roseum on the Edible Shaggy Mane Coprinus comatus in China.

Coprinus comatus, the shaggy mane, is one of the most popular and widely cultivated edible mushrooms in China. Its young fruiting body has good nutritional and medicinal value as well as a special flavor. In July 2010, an unusual stipe rot symptom was observed in cultivation tunnels in Pingyin county of Shandong Province. The lower part of the stipe was infected and water soaked scab occurred. The scab then expanded, a few mycelia and pink spores emerged on the scab surface, and finally, the stipe decayed and the fruiting body became wilted. The pathogen was isolated from infected tissues of C. comatus and the colonies on CYM were whitish at first, then pink sorus emerged, later forming concentric rings of sporulation. Mycelia were floccose, colorless, slender, and septate. Conidiophores bore upright, nonbranched, and colorless sporogenous cells, and slightly rounded spores were borne on the top and aggregated in pink cephaloid. Conidia were obovoid or obpyriform. Almost all spores were two-celled and one septate, while one was nearly round and the other bore an apex (1). The size of spores ranged from 10 to 21 µm long and 5 to 7 µm wide, which was consistent with the characteristics of Trichothecium roseum (1). The species identification was confirmed by sequencing the ribosomal ITS sequences. The ribosomal ITS1-5.8S-ITS2 region was amplified from the isolated strain using primers ITS1 and ITS4. A BLAST search in GenBank revealed the highest similarity (99%) to T. roseum (JQ434580). Pathogenicity was tested on different parts of 20 fruiting bodies of C. comatus with or without the wound treatment. One inoculum was prepared by flooding the agar surface with sterilized double distilled water for spore suspension (6.5 × 103 conidia/ml), and the other was by 0.2 × 0.2 cm mycelial plugs without spore production on CYM at 25°C for 5 days. After 1 to 2 days, only inoculated stipes showed water soaked and slight decay on the injured surface of all 20 fruiting bodies, while control fruiting bodies remained healthy. The symptoms were similar to those observed in the cultivation tunnels. No symptom was observed on the pileus, either with or without wound treatment. Pathogens reisolated from the inoculated stipes were confirmed to be T. roseum based on morphological characteristics. Because T. roseum is generally regarded as a postharvest disease of fruits and vegetables such as apple, pear, and muskmelon (2), apples and pears were inoculated with this fungus as well using the same methods. The parts inoculated were sunken, wettish, and decayed with brown stain at 25°C and 90% relative humidity after 5 days. Thus, it was confirmed that the T. roseum from the C. comatus stipe canker could infect the fruits of apple and pear. To our knowledge, this is the first report that T. roseum can cause disease on agaric. References: (1) G. Dal Bello. Australas. Plant Dis. Notes 3:103, 2008. (2) J.-H. Kwon et al. Plant Pathol. J. 26:296, 2010.

Perioperative inflammatory response and protein S-100ß concentrations - relationship with post-operative cognitive dysfunction in elderly patients.

BACKGROUND: One major concern in about one third of elder patients after total hip-replacement surgery is post-operative cognitive dysfunction (POCD). Previous studies have suggested that cognitive impairment is accompanied with changes in serum S-100ß protein (S-100ß) and inflammatory markers. Thus, the aim of the current study was to investigate the value of serum S-100ß and interleukin(IL)-1ß, IL-6, tumour necrosis factor-α (TNF-α), and C-reactive protein (CRP) in reflecting POCD after total hip-replacement surgery. METHODS: Forty-two elderly patients were enrolled, and 37 patients completed the follow-up. Serum S-100ß protein and IL-1ß, IL-6, TNF-α, and CRP were determined pre-operatively, as well as 1 h and 6 h post-operatively. Neuropsychological tests were performed pre-operatively, as well as on day 1, 3, and 7 post-operatively. RESULTS: Seventeen (45.9%, 17/37) patients developed POCD 1 day after surgery, and three (8.1%, 3/37) developed POCD 7 days after surgery. [Correction added after publication 7 February 2012: in the preceding sentence (54.1%, 17/37) was corrected to (45.9%, 17/37)]. Patients with POCD 1 day after surgery had significantly higher serum levels of IL-6 at 6 h (135 ± 32 pg/ml vs. 91 ± 29 pg/ml, P < 0.05) and S-100ß at 1 h (1872 ± 385 pg/ml vs. 1289 ± 143 pg/ml, P < 0.05. No significant post-operative change was detected in levels of TNF-α, IL-1, or CRP. CONCLUSION: The serum levels of pro-inflammatory marker IL-6 and S-100ß protein increased after total hip-replacement in elderly patients, and such increase may serve as predicting parameters for the occurrence of POCD.

A simplified universal genomic DNA extraction protocol suitable for PCR.

Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 µg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.

An assessment of teicoplanin use and monitoring serum levels in a Chinese teaching hospital.

OBJECTIVE: To validate a high performance liquid chromatography (HPLC) method for serum teicoplanin measurement and use the method for clinical monitoring of teicoplanin levels to analyze the clinical application of teicoplanin. METHODS: 55 patient profiles were collected and analyzed for the clinical teicoplanin application. 10 critically ill patients of the 55 cases were monitored for teicoplanin trough concentration using the HPLC method. RESULTS: The modified HPLC method exhibited excellent linearity, with correlation coefficient r = 0.9995. The intra-day and inter-day coefficients of variation were less than 10%. The lower limit of detection of teicoplanin was 5.63 mg/l. The recovery of teicoplanin was above 90%. Of the 55 patients in this study, there were 42 patients without load-dosing. There were only 29 patients treated with teicoplanin documented Gram-positive infections by etiological diagnoses. In the 10 patients with teicoplanin serum trough concentration monitoring, all cases received a loading dose of 400 mg every 12 h for 3 doses, and the mean trough concentration of teicoplanin was 10.82 ± 4.51 mg/l. The mean trough levels were 13.04 ± 6.23 mg/l in 4 patients with microbiological eradication and improvement of symptoms of diseases and 9.34 ± 2.61 mg/l in 6 patients with persistence of previous clinical infectious symptoms, respectively. CONCLUSION: The modified HPLC method is robust, highly reproducible and suited to monitor the concentration of teicoplanin. In critically ill Chinese patients, we should consider more appropriate loading doses and evaluate the relationship between teicoplanin trough concentration and the efficacy using microbiological and clinical parameters.

[Effect of anticancer polypeptide from Buthus Martensii venom on immune function in the H22-bearing mice].

OBJECTIVE: To observe the effect of anticancer polypeptide from Buthus Martensii Venom (APBMV) on Immune function in the H22-bearing mice. METHODS: The MTT colorimetric method, homolysin assay, lymphocyte transformation test, delayed hypersensitivity assay and WBC-count of peripheral blood were used in this study. RESULTS: APBMV could obviously augment NK activity, promote proliferation of lymphocytes induced by Con A, potentiate the response of DTH induced by DNCB, antagonize the decrease of WBC in peripheral blood induced by 5-Fu in the H22-bearing mice. CONCLUSION: APBMV can obviously increase immune function in the H22-bearing mice and antagonize hypoimmunity immunodeficiency or immunodeficiency induced by chemotherapy or the tumor.

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa).

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed.

Etiological role of Alternaria alternata in human esophageal cancer.

In this paper, the mutagenicity and carcinogenicity of alternariol monomethyl ether (AME), alternariol (AOH), and their relevance to the etiology of human esophageal cancer were studied. These mycotoxins were produced by Alternaria alternata which was the main contaminating fungi isolated from the grain in Linxian County, an area with high incidence of esophageal cancer. This study demonstrated that: 1. AME and AOH might cause cell mutagenicity and transformation; 2. AME and AOH could combine with the DNA isolated from human fetal esophageal epithelium, activate the oncogens, c-H-ras and c-mys in it, and promote proliferation of human fetal esophageal epithelium in vitro; 3. squamous cell carcinoma of the fetal esophagus could be induced by AOH. According to the results of the studies of AME and AOH mentioned above, we consider that Alternaria alternata plays an important role in the etiology of human esophageal cancer.

The effect of trithiomolybdate and some selenium compounds upon 109Cd labeled rat metallothionein in vitro: the possibilities for cadmium chelation therapy.

The main binding protein for 109Cd was metallothionein after in vitro incubation of various tissue cytosol preparations obtained from rats supplemented with zinc. The exception was heart cytosol where the label was associated with higher molecular weight proteins. The metallothionein-bound 109Cd was sensitive to trithiomolybdate and moved too higher molecular weight proteins, presumably because of the creation of new stronger ligands by the association of thiomolybdate with these proteins. The 109Cd binding was affected by selenate, selenite, and selenide while molybdate, sulphate, and thiosulphate were ineffective. It is proposed that thiomolybdates should be investigated for use in the therapy of in vivo cadmium toxicity because they can remove the accumulated metal from metallothionein.

Regulation of rat ileal water absorption by central alpha 2 adrenergic receptors.

This study examined the role of central adrenergic receptors in the regulation of water absorption by the rat ileum. Clonidine, an alpha 2-adrenergic receptor agonist, increased water absorption in vivo following intracerebroventricular but not intravenous injection. Intracerebroventricular injection of prazosin, an alpha 1-adrenergic receptor antagonist, did not alter basal water absorption. Prazosin did not prevent the clonidine effect. In contrast, the alpha 2-adrenergic receptor antagonist, yohimbine, reduced basal absorption and prevented the intracerebroventricular clonidine effect. Treatment with reserpine reduced ileal norepinephrine content by 98%, reduced basal water absorption consistent with a loss of sympathetic outflow to the mucosa, but did not prevent the increase in water absorption due to intracerebroventricular clonidine. These results suggest that central alpha 2 receptors regulate water absorption by the rat ileum. The clonidine-induced increase in water absorption is not mediated by the sympathetic nerves innervating the intestine.