Alternative primers are required for pullulan biosynthesis in Aureobasidium melanogenum P16.

Although pullulan has many uses in industry, the detailed mechanisms of its biosynthesis still require clarification. In this study, it was found that a short α-1,4-glucosyl chain (pullulan primer) synthesized by the glycogenins Glg1 and Glg2 for initiation of glycogen biosynthesis was also needed for pullulan synthesis. The primers were also synthesized on sterol glycosides and glucosylceramides by catalysis of sterol glucosyltransferase (Sgt1) and ceramide ß-glucosyltransferase (Gcs1). All the primers might be elongated to be long α-1,4-glucosyl chain (pullulan precursor) by catalysis of the glycogen synthetase domain of the AmAgs2 as previously reported. Then, the amylase domain of the same AmAgs2 was responsible for pullulan biosynthesis. Removal of all the genes encoding Glg1, Glg2, Gcs1 and Sgt1 made all the mutants produce much less pullulan than the strain P16. However, pullulan synthesis could not be stopped totally in these mutants, suggesting that any other unknown alternative pullulan primers may exist in the yeast cells. Complementation of all the genes in the mutants restored pullulan biosynthesis. This is the first time to report that like starch and glycogen biosynthesis, alternative primers are also required for pullulan biosynthesis in Aureobasidium melanogenum P16.

A multidomain α-glucan synthetase 2 (AmAgs2) is the key enzyme for pullulan biosynthesis in Aureobasidium melanogenum P16.

Pullulan, a biological macromolecule, has many applications. However, it is completely unknown how and where it is synthesized. In this study, it was found that the multidomain AmAgs2 (α-glucan synthase 2) encoded by an AmAGS2 gene in Aureobasidium melanogenum P16 contained the amylase domain (Amy_D), the glycogen synthetase domain (Gys_D) and the transmembrane regions in which the exopolysaccharide transporter domain (EPST_D) was embedded. Removal of the AmAGS2 gene in A. melanogenum P16 rendered the disruptants not to synthesize any pullulan and complementation of the AmAGS2 gene in the disruptants restored pullulan synthesis. Overexpression of the gene in Aureobasidium melanogenum CBS105.22, a non-pullulan producer, resulted in the transformants producing pullulan. Therefore, the AmAGS2 gene was the key gene responsible for pullulan biosynthesis in A. melanogenum P16. It was speculated that the short α-1,4-glucosyl chains (pullulan primers) were elongated by the Gys_D of the AmAgs2 to form long α-1,4-glucosyl chains (precursors of pullulan). All the precursors were transported to outside plasma membrane by the EPST_D in the transmembrane regions of the AmAgs2. Then, the Amy_D of the AmAgs2 was responsible for both hydrolysis of the endo-α-1,4-linkages in the precursors to release maltotriose and transfer of the maltotriose to Lph-glucose to form α-1,6 glucosidic bonds between maltotrioses in pullulan molecule. This is the first time to report that the AmAgs2 can play the key role in pullulan biosynthesis.

Macromolecular pullulan produced by Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert and its crucial roles in resistance to the stress treatments.

A novel yeast strain Aureobasidium melanogenum 13-2 isolated from the Taklimakan desert was found to be able to produce a high level of extracellular polysaccharide (EPS). Under the optimal conditions, the yeast strain could yield 73.25 ±â€¯2.3 g/L of EPS within 5 days at a flask level. During a 10-liter fermentation, the yeast strain could produce 78.05 ±â€¯3.5 g/L of EPS within 120 h. The FT-IR spectra of the standard pullulan from Sigma and the purified EPS produced by A. melanogenum 13-2 were almost identical and the purified EPS could be actively hydrolyzed by a pullulanase, demonstrating that the purified EPS was pullulan. The molecular weight (Mw) of the purified pullulan was estimated to be 7.703 × 105 g/moL. Disruption of a pullulan synthase gene (PUL1) made a mutant DAG27 lose the ability to synthesize any pullulan. The mutant DAG27 was more sensitive to radiation of UV light, high NaCl concentration, heat treatment, strong oxidation of H2O2 and desiccation than its wild type strain 13-2, suggesting that the produced pullulan could play an important role in resistance of the yeast cells to various stresses. This was the first time to show that the yeast strain obtained from the desert could produce such high level pullulan and the produced pullulan had an obviously protective effect on its producer.

Genome sequencing of Aureobasidium pullulans P25 and overexpression of a glucose oxidase gene for hyper-production of Ca2+-gluconic acid.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97 Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GA g/l and 2480 U/mg of Gox activity. The productivity of Ca2+-GA was 2.78 g/l/h and the yield was 1.1 g/g.

α-Amylase, glucoamylase and isopullulanase determine molecular weight of pullulan produced by Aureobasidium melanogenum P16.

A high molecular weight (Mw) pullulan has many potential applications in various fields. α-Amylase, glucoamylase and pullulanase were thought to play an important role in high Mw pullulan biosynthesis. However, there is no genetic evidence for this role. In this study, the genes encoding α-amylase, glucoamylase and pullulanase were cloned from Aureobasidium melanogenum P16, a high pullulan producing yeast and characterized. The proteins deduced from the cloned α-amylase gene, the glucoamylase gene and the isopullulanase gene, not a pullululanse gene had their corresponding conserved amino acid sequences, respectively. After the single gene of them was deleted, the Mw of the pullulan produced by the single disruptants greatly increased and the pullulan concentration decreased. It was found that the triple mutant DT15 grown at the flask level could produce 46.2 g/L of pullulan with a Mw of 3.02 × 106 Da and grown in the 10-L fermentor could yield 58.14 g/L of pullulan with the same Mw while its wild type strain P16 produced 65.5 ±â€¯3.5 g/L of pullulan with a Mw of 0.35 × 106 Da. After the genes were complemented, pullulan production, Mw of the produced pullulan and others were restored. All the results demonstrated that the α-amylase, glucoamylase and isopullulanase indeed could determine the Mw of the produced pullulan.

Cell wall integrity is required for pullulan biosynthesis and glycogen accumulation in Aureobasidium melanogenum P16.

BACKGROUND: Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect pullulan and glycogen biosynthesis. METHODS: The genes related to cell wall integrity were cloned, characterized, deleted and complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and gene expression were examined. RESULTS: In this study, the GT6 and GT7 genes encoding different α1,2 mannosyltransferases in Aureobasidium melanogenum P16 were cloned and characterized. The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family. The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphate glucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. GENERAL SIGNIFICANCE: This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis in the pullulan producing yeast.

Optimized construction of MUC1-VNTRn DNA vaccine and its anti-pancreatic cancer efficacy.

Considering mucin 1-variable number tandem repeat (MUC1-VNTRn) as a novel target for pancreatic cancer immunotherapy, the present study aimed to screen and identify the pVAX1-MUC1-VNTRn DNA vaccine with the strongest immunogenicity. Following construction of a pVAX1-MUC1-VNTRn plasmid, immature dendritic cells (DCs) were subjected to transfection, and mature DCs were then co-cultured with autologous T-cells. The numbers of cytotoxic T lymphocytes (CTLs) secreting interferon (IFN)-γ were determined using an enzyme-linked immunospot assay, and CytoTox® was also used to examine the MUC1-VNTRn-specific Lethal effect of CTLs on Capan2 cells. Additional in vivo experiments in mice were performed to confirm the antitumor effect of the DNA vaccine candidate. The present study successfully constructed the pVAX1-MUC1-VNTRn plasmid, which expresses the target protein in eukaryotic cells. Additionally, upon uptake of the pVAX1-MUC1-VNTRn plasmid, the immature DCs differentiated into mature DCs. The levels of the DC surface molecules cluster of differentiation (CD) 80, CD86, human leukocyte antigen-antigen D related, interleukin (IL)-12, IL-17 and IFN-γ were significantly higher, while the levels of IL-10 and IL-14 were lower, in mature DCs of the stimulated groups compared with the immature DCs of the non-stimulated groups (all P<0.01). In addition, the MUC1-VNTR6 and MUC1-VNTR9 groups, in which DCs were capable of activating autologous T-cells, showed increased IFN-γ-producing T-cells compared with the other groups (strong MUC1-VNTR1, weak VNTR1, VNTR3, VNTR4 and MUC1-cDNA groups; all P<0.001). In addition, the Lethal effect of CTLs on Capan2 cells in these two groups was stronger compared with the other groups (all P<0.001). Furthermore, the induced protective and therapeutic immune responses in mouse experiments showed that the pVAX1-MUC1-VNTR6DNA vaccine likely possessed the strongest immunogenicity, and its ability to inhibit panc02-MUC1 tumor growth was superior to other DNA vaccines (P<0.01). The present study provides compelling evidence that pVAX1-MUC1-VNTRn has the potential to express the target protein in eukaryotic cells, and thatpVAX1-MUC1-VNTR6 was characterized by the strongest Lethal effect in both in vivo and in vitro experiments.

MiR-27a rs895819 is involved in increased atrophic gastritis risk, improved gastric cancer prognosis and negative interaction with Helicobacter pylori.

MiR-27a rs895819 is a loop-stem structure single nucleotide polymorphism affecting mature miR-27a function. In this study, we performed a comprehensive analysis about the association of rs895819 with gastric cancer risk and prognosis, atrophic gastritis risk, as well as the interactions with environmental factors. A total of 939 gastric cancer patients, 1,067 atrophic gastritis patients and 1,166 healthy controls were screened by direct sequencing and MALDI-TOF-MS. The association of rs895819 with clinical pathological parameters and prognostic survival in 357 gastric cancer patients was also been analyzed. The rs895819 variant genotype increased the risk for atrophic gastritis (1.58-fold) and gastric cancer (1.24-fold). While in stratified analysis, the risk effect was demonstrated more significantly in the female, age >60y, Helicobacter pylori (H. pylori) negative and non-drinker subgroups. Rs895819 and H. pylori showed an interaction effect for atrophic gastritis risk. In the survival analysis, the rs895819 AG heterozygosis was associated with better survival than the AA wild-type in the TNM stage I-II subgroup. In vitro study by overexpressing miR-27a, cells carrying polymorphic-type G allele expressed lower miR-27a than wild-type A allele. In conclusion, miR-27a rs895819 is implicated as a biomarker for gastric cancer and atrophic gastritis risk, and interacts with H. pylori in gastric carcinogenesis.

[Correlation between CYP2C19 Gene Polymorphism with Clopidogrel Resistance and Distribution of Chinese Medicine Syndrome in 229 Acute Coronary Syndrome Patients].

Objective To observe correlation between CYP2C19 *2/CYP2C19 *3 gene polymorphism with clopidogrel resistance and distribution of Chinese medicine ( CM) syndrome in acute coronary syndrome (ACS) population. Methods Peripheral blood was collected from 229 ACS patients from June 2014 to March 2015. DNAs were extracted, amplified, and sequenced. Correlations between CYP2C19 *2/CYP2Cl9 *3 gene polymorphisms and clopidogrel resistance/distribution of CM syndrome were analyzed. Gene frequency and allele frequency were tested using gene counting and one-sample K-S test. Correlation between gene types and distribution of CM syndrome was tested by Pearson corre- lation test. Results (1) The CYP2C19 *2 polymorphism distribution: CYP2C19 *2(A/A) (mutant homozygous) 12 cases (5. 2%) ; CYP2C19 * 2 ( G/A ) ( mutant heterozygote ) 93 cases (40. 6%), and CYP2C19 *2 (G/G) (normal homozygous) 124 cases (54. 2%). The mutant allele frequency was 0. 255. (2) The CYP2C19 *3 polymorphism distribution: CYP2C19 *3 (A/A) 0 case (0) ; CYP2C19 *3 (G/A) 26 cases (11. 4%), and CYP2C19 *3 (G/G) 203 cases (88. 6%). The mutant allele frequency was 0. 056. (3) Correlation between CYP2C19 gene polymorphism and clopidogrel resistance: Clopidogrel resistance was more liable to occur in mutant homozygous than in mutant heterozygote and normal homozygous (R =0. 30, P <0. 01). Clopidogrel resistance was more liable to occur in mutant heterozygote than in normal homozygous (R =0. 34, P <0. 01). (4) Among the 229 patients, the CM syndrome distribution were distributed as follows. Blockage of Xin vessels syndrome (BXVS, 33 cases, 14. 41%) ; qi deficiency blood stasis syndrome (QDBSS, 51 cases, 22. 27%) ; qi stagnation blood stasis syndrome (QSBSS, 92 cases, 40.18%) ; phlegm obstructing Xin vessel syndrome (POXVS, 17 cases, 7. 42%) ; yin-cold coag- ulation syndrome (YCCS, 8 cases, 3. 49%) ; qi-yin deficiency syndrome (QYDS, 13 cases, 5.68%) ; Xin-Shen yin deficiency syndrome (XSYDS, 5 cases, 2.18%), yang and qi deficiency syndrome (YQDS, 10 cases, 4. 37%). (5) CYP2C19 *2 gene type was significantly correlated with syndrome typing of CM (R =0. 26, P <0. 01). Mutant homozygous and most mutant heterozygote patients were syndrome typed as QDBSS. Conclusions The polymorphism of CYP2C19 was closely correlated with clopidogrel resist- ance in 229 ACS patients. Its occurrence rate was correlated with CYP2C19 *2/CYP2C19 *3 gene muta- tion frequency. Blood stasis syndrome ( QSBSS, QDBSS, BXVS) were main syndromes of ACS. Be- sides, QSBSS was obviously higher than the rest syndrome types. The polymorphism of CYP2C19 * 2 was correlated with syndrome typing of CM. CYP2C19 *2 gene defect mostly existed in QSBSS.

The complete mitochondrial genome of malbrouck monkey, Chlorocebus cynosuros (Chlorocebus, Cercopithecinae).

In this study, the complete mitochondrial genome sequence of malbrouck monkey, Chlorocebus cynosuros, with the total length of 16,334 bp, is reported for the first time. Similar to other monkeys, it contains a typically conserved structure including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region (D-loop). The base composition was A (32.1%), G (12.4%), C (30.1%), and T (25.4%), so the percentage of A and T (57.5%) was higher than that of G and C. Most of the genes are encoded on H-strand, except for the ND6 subunit gene and 8 tRNA genes. The complete mitochondrial genome sequence provided here would be useful for further phylogenetic analysis and population genetic studies in C. cynosuros.

[Detection of Helicobacter pylori genotypes from human gastric mucosa and its association with gastric diseases].

OBJECTIVE: To investigate the feasibility of detecting Helicobacter pylori (Hp) directly from gastric mucosa and the relationship of HP genotypes to gastric diseases. METHODS: Specimens of gastric mucosa were collected by biopsy from 217 patients, 90 with superficial gastritis (GS), 70 with atrophic gastritis (GA), 28 with gastroesophageal erosions and ulcers (GEU), and 29 with gastric cancer (GC), to undergo pathological examination, culture of Hp, and DNA isolation from the gastric mucosa respectively. Routine phenol/chloroform method was used to isolate the DNA in the cultured Hp. PCR was conducted to detect the ureB, cagA, vacAs1, vacAm1, vacAm2, iceA1, iceA2, and baba2 genotypes in both the gastric mucosa-originated Hp-DNA and cultured Hp-DNA. RESULTS: The concordance rates of ureB, cagA, vacAs1, vacAm1, vacAm2, iceA1, iceA2, and baba2 genotypes between the gastric mucosa-originated Hp-DNA and cultured Hp-DNA were 74.23%, 73.39%, 93.69%, 62.16%, 78.16%, 89.13%, 88.37%, and 75% respectively (all P > 0.05). There were no significant differences in the distribution rates of ureB, cagA, vacAs1, vacAm1b, iceA1, iceA2, and baba2 genotypes among different gastric diseases (all P > 0.05). However, the distribution frequency of vacAs1m1 strain in the GA patients was 22.22%, significantly higher than that in the GA patients (5.71%, chi2 = 8.416, P = 0.004), and the distribution frequency of vacAs1m2 strains in the GA patients was 47.14%, significantly higher than that in GS patients (18.89%, chi2 = 13.336, P = 0.000). CONCLUSION: Rapid detection of Hp can be achieved by using DNA isolated directly from infected gastric mucosa. VacAs1 m1 is associated with GA and vacAs1m2 is associated with GA.

[Investigation of the role of hepatic oval cell in primary hepatocarcinogenesis using specific Y chromosome].

OBJECTIVE: To determine the role of hepatic oval cell in primary hepatocarcinogenesis using specific Y chromosome. METHODS: The model of hepatic oval cell proliferation was established by feeding 30 male Wistar rats with 0.06% 3'3-diaminobenzidine (DAB) for 4 weeks. Another 40 female Wistar rats were equally randomized into two groups: control group and experimental group. Experimental group was inoculated with oval cells suspension from the prepared male rats under the hepatic amicula and fed with DAB for 14 weeks continuously to promote hepatocarcinogenesis. Control group was fed with DAB for 14 weeks. After primer fragments were worked out on the male rats' SRY genes using the software of primer design, DNAs were extracted from the hepatic carcinomas of the female rats of the two groups and then amplified with PCR. Electrophoretic analysis was performed on the products. RESULTS: After 14 weeks, primary hepatocarcinogenesis was found in the livers of all the 40 female rats. Electrophoresis showed the positive straps from the experimental group with the same length to the designed segments, whereas no positive straps were seen in the control group. CONCLUSIONS: The hepatic oval cells can differentiate into hepatic cancer cells, which provides the evidence that the hepatocellular carcinoma has its source from the hepatic oval cells.

[Rosiglitazone inhibits atherosclerosis in apolipoprotein E-knockout mice].

OBJECTIVE: To study the effect of rosiglitazone on atherosclerosis and potential mechanism in ApoE-knockout mice. METHODS: Thirty-two 6-week-old ApoE-knockout mice were used as atherosclerosis model in two groups: rosiglitazone group (n = 18) and control group (n = 14). Each group contained equal numbers of male and female mice. All mice were fed with normal chow diet. In addition to normal diet, rosiglitazone group received rosiglitazone 17 mg/kg of body weight/day. Venous bloods were collected for plasma glucose and lipid analysis, and aorta were prepared for morphologic and immunohistochemical analysis after 14 weeks. Aortic root (1 cm) was cut and prepared for paraffin slice. The histomorphometric analysis of atherosclerotic lesion was performed by means of HE; positive percentage of macrophage cell and tumor necrosis factor-alpha were measured by means of immunohistochemistry in cross section. The ratio of lesion/aortic wall surface in the rest aorta was measured by means of Sudan IV staining in longitudinal section. RESULTS: The amount of fatty streak in rosiglitazone group was significantly greater than that of control group; the gross number of lesions and the number of fibrous plaque and atheromatous plaque were similar in two groups. There were no differences in percentage of lesions in cross section in two groups. Rosiglitazone could significantly reduce the extend of atherosclerosis of longitudinal section, decrease the amount of macrophage cell and the level of tumor necrosis factor-alpha in lesions. The plasma glucose was normal and similar in two groups, and total cholesterol, LDL-cholesterol and triglyceride were significantly higher in rosiglitazone group. CONCLUSION: Rosiglitazone suppresses the expression of tumor necrosis factor-alpha, reduces the number of macrophage cell in lesion, and inhibits the development of atherosclerosis.