Isolation and characterization of tyrosinase produced by marine actinobacteria and its application in the removal of phenol from aqueous environment

The present study was focused on screening and characterization of tyrosinase enzyme produced by marine actinobacteria and its application in phenolic compounds removal from aqueous solution. A total of 20 strains were isolated from marine sediment sample and screened for tyrosinase production by using skimmed milk agar medium. Among 20 isolates, two isolates LK-4 and LK-20 showed zone of hydrolysis and these were taken for secondary screening by using tyrosiue agar medium. Based on the result of secondary screening LK-4 was selected for further analysis, such as tyrosinase assay, protein content and specific activity of the enzyme. The tyrosinase enzyme was produced in a SS medium and was partially purified by ammonium sulfate precipitation, dialysis and SDS PAGE. The isolate （LK-4） was identified as Streptomyces espinosus using 16S rRNA gene sequencing and named as ＂Streptomyces espinosus strain LK4 （KF806735）＂. The tyrosinase enzyme was immobilized in sodium alginate which was applied to remove phenolic compounds from water. The enzyme efficiently removed the phenolic compounds from aqueous solution within few hours which indicated that tyrosinasc enzyme produced by Streptomyces espinosus strain LK-4 can be potently used for the removal of phenol and phenolic compounds from wastewater in industries.     

云南五个不同地区烟草花叶病毒外壳蛋白基因的序列比较

Five Tobacco mosaic virus isolates, obtained from tobacco leaves showing typical symptom in Qujing, Honghe, Dali, Chuxiong and Yuxi in Yunnan province, were selected and studied from 637 TMV samples. Using a pair of primers specific for TMV-U1 strain, a specific fragment of 530bp including the TMV coat protein gene was amplified using IC-PCR. The products of PCR were cloned and sequenced. The nucleotide and amino acid sequences of the coat protein of the five isolates were found to be very similar each other and have more than 90% sequence identity with TMV-U1, TMV-B,TMV-P, TMV-FUJIAN, although minor differences existed among them. The results showed that the five isolates from Yunnan province belong toTMV-U1 strain.     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

酵母菌SH2发酵产物增强干扰素抗病毒活性 及其有效成分的初步研究

The fermentation broth of yeast strain SH-2 (FSH-2) which could enhance the biological potency of human interferon alpha (huIFN-α) was detected. Its enhancing ratio was 1.64～6.86 fold. A group of proteins was seperated and purified from the fermentation supernatant by chromatography on Sephadex G-75 and HPLC. Each protein showed the similar band on PAGE and SDS-PAGE. Their molecular weights ranged from 52 to 72 kD. The proteins were stainded with periodic acid Schiff's agent. Lowry's method and sulfuric acid-phenol method were respectively used to determine the contents of proteins and neutral sugars. The results showed the ratio of protein to sugar was 3∶1. These results indicated that the proteins were extracellular glycoproteins (YEGPs) secreted by yeast strain SH-2. YEGPs could significantly enhance the biological potency of huIFN-α. It was veritied by the experiment of cytopathic effect inhibition in Wish cells. The enhancing ratios were 1.6～2.8 fold and 1.4～4.0 fold, respectively. The enhancing ratio of the elution protein of FSH-2 seperated by Sephadex G-75 was 2.01～5.68 fold. YEGPs alone could not enhance the potency of huIFN-α and had no biological activity as IFNs.     

Meta-analysis of the relationship between Epstein-Barr virus infection and clinicopathological features of patients with gastric carcinoma

Epstein-Barr virus (EBV) infection has been causally associated with occurrence of many malignant neoplasms. EBV-encoded small RNAs (EBERs) have been detected from about 10% of gastric carcinoma tissue cells, suggesting that EBV infection is associated with the development of gastric carcinoma. The present study pooled the data from the papers concerning EBV-related gastric cancers and performed a meta-analysis of 22 research papers. Among these papers, a total of 5475 cases with gastric cancer were enrolled, of whom 411 cases were found EBV-positive, with the EBV-positive rate being 7.5%. Among the EBV-positive gastric cancer cases, the detection rate was 11.1% in males and 3.0% in females. Compared with EBV-negative gastric cancer, EBV-positive gastric cancer had less lymph node metastasis. Based on the histological typing, of the EBV-positive gastric cancers, the diffuse type was 8.1%, and intestinal type was 8.0%. The examined specimen types included stored paraffin blocks and fresh surgically removed specimens, their EBV positive rates were 7.9% and 6.5% respectively. In terms of geographical distribution, the detection rate of EBV-positive gastric cancer was 9.4% in America, 6.1% in Asia and 9.1% in Europe. Meta-analysis showed that EBV infection occurred only in gastric cancer tissue cells and was significantly associated with the patients’ gender, lymph node metastases, and the location where tumor tissue generated and geographical distribution (P0.05), but was not significantly associated with the patients’ histological types of tumor and the types of specimens (P0.05). These results suggested that EBV-positive gastric cancer has distinct clinicopathological features.     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

“In front of patients,I will always be a pupil.”Dr.Xiaoqian Zhang:the founder of the modern Chinese gastroenterology

Dr.Xiaoqian Zhang(张教骞,Hsiao-Chien Chang)was an outstanding clinician,therapist and medical educator,a member of the Academic Divisions of the Chinese Academy of Sciences,he is considered to be the founder of the modern Chinese gastroenterology(PUMCH,1988).As a doctor,he made a systemic and deep research on human blood volume,gastric secretion function,peptic ulcer disease,gastric ulcer and gastric cancer,celiac tuberculosis,amoebic dysentery,ulcerative colitis and so on.He diagnosed and treated multiple intractable diseases.As an educator,he emphasized the training of clinical basic skills,urged students to grasp the utilization of science in study and work.He trained a large number of talents for Chinese medicine.     

16S rDNA-based phylogeny of non-symbiotic bacteria of Entorno-pathogenic nematodes from infected insect cadavers

Using 16S rDNA gene sequencing technique, three different species of non-symbiotic bacteria of entomopatho-genic nematodes (EPNs) (Steinernema sp.and Heterorhabditis sp.) were isolated and identified from infected insect cadavers(Galleria mellonella larvae) after 48-hour post infections.Sequence similarity analysis revealed that the strains SRK3, SRK4 and SRK5 belong to Ochrobactrum cytisi,Schineria larvae and Ochrobactrum anthropi,respectively.The isolates O.anthropi and S.larvae were found to be associated with Heterorhabditis indica strains BDU-17 and Yer-136,respectively,whereas O.cytisi was associated with Steinernema siamkayai strain BDU-87. Phenotypically, temporal EPN bacteria were fairly related to symbiotic EPN bacteria (Photorhabdus and Xenorhabdus genera). The strains SRK3 and SRK5 were phylogeographically similar to several non-symbionts and contaminated EPN bacteria isolated in Germany(LMG3311T) and China (X-14),while the strain SRK4 was identical to the isolates of S.larvae (L1/57,L1/58, L1/68 and L2/11) from Wohlfahrtia magnifica in Hungary.The result was further confirmed by RNA secondary structure and minimum energy calculations of aligned sequences.This study suggested that the non-symbionts of these nematodes are phylogeographically diverged in some extent due to phase variation.Therefore,these strains are not host-dependent, but environment-specific isolates.     

The variable codons of H5N1 avian influenza A virus haemagglutinin genes

We investigated the selection pressures on the haemagglutinin genes of H5N1 avian influenza viruses using fixed effects likelihood models. We found evidence of positive selection in the sequences from isolates from 1997 to 2007, except viruses from 2000. The haemagglutinin sequences of viruses from southeast Asia, Hong Kong and mainland China were the most polymorphic and had similar nonsyn-onymous profiles. Some sites were positively selected in viruses from most regions and a few of these sites displayed different amino acid patterns. Selection appeared to produce different outcomes in vi-ruses from Europe, Africa and Russia and from different host types. One position was found to be positively selected for human isolates only. Although the functions of some positively selected posi-tions are unknown, our analysis provided evidence of different temporal, spatial and host adaptations for H5N1 avian influenza viruses.     

Subtractive hybridization analysis of gastric diseases-associated Helicobacter pylori identifies peptidyl-prolyl isomerase as a potential marker for gastric cancer

Helicobacter pylori, a microaerophilic Gram-negative bacterium, is known to cause chronic gastritis, peptic ulcer and gastric cancer. Genes that are present in certain isolates may determine strain-specific traits such as disease association and drug resistance. In order to understand the pathogenic mechanisms of gastric diseases, identify molecular markers of the diseases associated with H. pylori strains and provide clues for target treatment of H. pylori-related diseases, a subtracted DNA library was constructed from a gastric cancer-associated H. pylori strain and a superficial gastritis-associated H. pylori strain by suppression subtractive hybridization. The presence of gastric cancer-specific genes was identified by dot blot hybridization, DNA sequencing and PCR-based screening. Twelve gastric cancer-specific high-copy genes and nine low-copy genes were found in gastric cancer compared with the superficial gastritis strain. These genes were confirmed by PCR analysis of H. pylori isolates. Notably, peptidyl-prolyl cis-trans isomerase (PPIase) was detected positively in 11 out of 22 (50%) gastric cancer-associated H. pylori strains. In contrast, <24% of the H. pylori strains from superficial gastritis showed positive results. Given the potential role of PPIases in cell growth, apoptosis and oncogenic transformation, our results suggest that PPIase may represent a novel marker and potential therapeutic target for gastric cancer.     

No correlation of babA2 with vacA and cagA genotypes of Helicobacter pylori and grading of gastritis from peptic ulcer disease patients in Brazil

BACKGROUND: The babA2 gene, which encodes a blood-group antigen-binding adhesin that mediates attachment of Helicobacter pylori to human Lewis(b) antigens on gastric epithelial cells, has been associated with a higher risk of peptic ulcer and gastric cancer. The purpose of this study was to ascertain the frequency of babA2 genotype in H. pylori strains of patients with peptic ulcer and to correlate with other virulence factors. MATERIALS AND METHODS: vacA, cagA, and babA2 genotypes of H. pylori were determined by using polymerase chain reaction (PCR). DNA was extracted from positive urease test gastric samples of 150 patients with peptic ulcer. Antrum and corpus biopsies were taken for histologic examination according to the updated Sydney system classification. RESULTS: babA2 genotype was present in 104 (69.3%) and cagA in 113 (75.3%) gastric samples. No significant correlation was observed between babA2 and vacAs1 genotype or between babA2 and cagA status. The correlation of vacAs1 genotype with positive cagA was statistically significant ( p < .001). The babA2-positive strain was more frequently found from the gastric samples of men, than of women (p = .01). Strains harboring cagA, vacAs1, and babA2 genotypes had no association to the grading of gastritis, presence of glandular atrophy, or intestinal metaplasia. The simultaneous presence of cagA, vacAs1, and babA2 was found in 32.6% of the H. pylori strains. CONCLUSIONS: babA2 genotype is frequently found in H. pylori strains from peptic ulcer disease in Brazil, although it has no significant correlation to the worsening of the gastritis and to other virulence markers such as vacAs1 and cagA.     

Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules,the selectins--a preliminary report

The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer. We are exploring the possibility that carbohydrate components on H. pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. Lipopolysaccharides of most H. pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens. Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes. Here we investigate H. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins. Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.     

Helicobacter pylori stool antigen test in patients with bleeding peptic ulcers

BACKGROUND: Helicobacter pylori has been linked to chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. Invasive tests are less sensitive than noninvasive tests in diagnosing H. pylori infection in patients with bleeding peptic ulcers. The H. pylori stool antigen test has been useful in diagnosing H. pylori in patients with peptic ulcers before and after eradication of H. pylori. The aim of this study was to evaluate the H. pylori stool antigen test in patients with bleeding peptic ulcers. METHODS: Patients with bleeding and nonbleeding peptic ulcers underwent a rapid urease test, histology, bacterial culture and H. pylori stool antigen test. Positive H. pylori infection was defined as a positive culture or both a positive histology and a positive rapid urease test. Helicobacter pylori stool antigen was assessed with a commercial kit (Diagnostec H. pylori antigen EIA Kit, Hong Kong). RESULTS: Between October 2000 and April 2002, 93 patients with bleeding peptic ulcers (men/women: 78/15, gastric ulcer/duodenal ulcer: 58/35) and 59 patients with nonbleeding peptic ulcers (men/women: 47/12, gastric ulcer/duodenal ulcer: 30/29) were enrolled in this study. Forty-seven (50.5%) patients with bleeding peptic ulcers and 30 (50.8%) patients with nonbleeding peptic ulcers, were found to be infected with H. pylori (p > .1). Helicobacter pylori stool antigen tests were positive in 54 (58.1%) and 30 (50.8%) patients with bleeding peptic ulcers and nonbleeding peptic ulcers, respectively (p > .1). The sensitivity (82% vs. 93%), specificity (68% vs. 93%), positive predictive value (74% vs. 93%), negative predictive value (77% vs. 93%) and diagnostic accuracy (75% vs. 93%) were all lower in patients with bleeding vs. nonbleeding peptic ulcers. The specificity, positive predictive value, and diagnostic accuracy of the H. pylori stool antigen test in patients with bleeding peptic ulcers were significantly lower than those in patients with nonbleeding peptic ulcers (p = .01, p = .02 and p = .003, respectively). CONCLUSION: The H. pylori stool antigen test is not reliable for diagnosing H. pylori infection in patients with bleeding peptic ulcers.     

Helicobacter pylori, T cells and cytokines: the "dangerous liaisons"

Helicobacter pylori infection is the major cause of gastroduodenal pathologies, but only a minority of infected patients develop chronic and life threatening diseases, as peptic ulcer, gastric cancer, B-cell lymphoma, or autoimmune gastritis. The type of host immune response against H. pylori is crucial for the outcome of the infection. A predominant H. pylori-specific Th1 response, characterized by high IFN-gamma, TNF-alpha, and IL-12 production associates with peptic ulcer, whereas combined secretion of both Th1 and Th2 cytokines are present in uncomplicated gastritis. Gastric T cells from MALT lymphoma exhibit abnormal help for autologous B-cell proliferation and reduced perforin- and Fas-Fas ligand-mediated killing of B cells. In H. pylori-infected patients with autoimmune gastritis cytolytic T cells infiltrating the gastric mucosa cross-recognize different epitopes of H. pylori proteins and H+K+ ATPase autoantigen. These data suggest that peptic ulcer can be regarded as a Th1-driven immunopathological response to some H. pylori antigens, whereas deregulated and exhaustive H. pylori-induced T cell-dependent B-cell activation can support the onset of low-grade B-cell lymphoma. Alternatively, H. pylori infection may lead in some individuals to gastric autoimmunity via molecular mimicry.     

The prevalence of peptic ulcer not related to Helicobacter pylori or non-steroidal anti-inflammatory drug use is negligible in southern Europe

BACKGROUND AND AIM: Helicobacter pylori is the major cause of peptic ulcer disease, but the proportion of H. pylori-negative peptic ulcers seems to be increasing in developed countries. We investigated the frequency of H. pylori-negative peptic ulcer without intake of nonsteroidal anti-inflammatory drugs (NSAIDs) in a Mediterranean European country. MATERIALS AND METHODS: We prospectively collected consecutive patients with an endoscopically verified active peptic ulcer over 6 months from different areas of Spain. Helicobacter pylori infection was assessed by rapid urease test and histologic examination (corpus and antral biopsies). A (13)C-urea breath test was performed if H. pylori was not detected with the invasive test. Patients were considered H. pylori-negative if all three tests were negative. NSAID use was determined by structured data collection. RESULTS: Of 754 consecutive peptic ulcer patients, 16 (2.1%) were H. pylori-negative and had not used NSAIDs before the diagnosis. Of the 472 patients who had duodenal ulcers, 95.7% (n = 452) were H. pylori-positive and only 1.69% (n = 8) were negative for both H. pylori infection and NSAID use; 193 patients had benign gastric ulcers and 87% (n = 168) of them were infected by H. pylori (p <.001 vs. duodenal ulcers). NSAID intake was more frequent in gastric ulcer patients (52.8%) than in duodenal ulcer patients (25.4%; p <.001). Consequently, the frequency of H. pylori-negative gastric ulcer in patients not using NSAID was 4.1% (n = 8). CONCLUSION: Peptic ulcer disease is still highly associated with H. pylori infection in southern Europe, and only 1.6% of all duodenal ulcers and 4.1% of all gastric ulcers were not associated with either H. pylori infection or NSAID use.     

Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance

Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer. It has been recently reported that metronidazole resistance from H. pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA). We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations. To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H. pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504. All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole. The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H. pylori 2600. In addition, the frxA gene cloned from the H. pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E. coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates.     

Effect of Helicobacter pylori eradication on peptic ulcer disease complicated with outlet obstruction

Background. At present, the prevalence of Helicobacter pylori ( H. pylori ) in complicated peptic ulcer and the effect of H. pylori eradication on complicated peptic ulcer have not been fully established. In this study, we report the prevalence of H. pylori in peptic ulcer patients complicated with gastric outlet obstruction, effectiveness of oral eradication therapy on these patients, and their long-term follow up.
Patients and Methods. Ten consecutive patients presenting with clinically and endoscopically significant obstructed peptic ulcers were included in this study. During each endoscopy, seven gastric biopsy specimens were obtained and analyzed for H. pylori colonization.
Results. The antral mucosal biopsy specimens were positive for H. pylori in nine patients. H. pylori infection was eradicated and complete ulcer healing was observed in all patients. The mean follow-up period was 14 (7–24) months. One patient had duodenal perforation and underwent surgical intervention following medical treatment, despite the eradication of H. pylori. Ulcer recurrence was noted in two (22.2%) of nine patients, and in one of them the recurrent ulcer was complicated with obstruction (11.1%). The mean time to ulcer recurrence was 17 months (range, 10–24 months). The biopsies and CLOtests were H. pylori negative at the time of ulcer or erosion recurrence in two patients.
Conclusion. We suggest that H. pylori eradication may improve the resolution in obstructive ulcer cases with colonization.     

Persistence factors of Helicobacter pylori

Isolates of H. pylori from patients with chronic gastritis and peptic ulcer, were found to be capable of inactivating lysozyme and intercide (the bactericidal component of human leukocytic interferon). The expression and penetration capacity of their antilysozyme activity (ALA) and antiintercide activity was determined. The wide spread of ALA among H. pylori clinical isolates associated with inflammatory changes in bioptic specimens, confirmed the leading role of this microorganism in the pathogenesis of the gastric mucosal lesions. The retrospective analysis of clinical cases made it possible to recommend the use of the ALA sign as one of the criteria for choosing the scheme of eradication therapy even at the stage of the initial diagnostics of H. pylori infection.     

The status of the cagA gene does not predict Helicobacter pylori-associated peptic ulcer disease in Singapore

Discrepancies among reports from different geographical regions worldwide on the association between the presence of cagA and peptic ulcer disease prompted this study on the predictive value of the cagA gene in Helicobacter pylori-associated gastroduodenal diseases in the Singapore population. H. pylori strains were obtained from 169 patients with a peptic ulcer, 83 with non-ulcer dyspepsia, and nine with gastric cancer. The presence of the cagA gene was evaluated by polymerase chain reaction (PCR). The expected 400 bp PCR product coding for the cagA gene was present in 232/261 (89%) H. pylori isolates. Of these, 151/169 (89%) strains from patients with peptic ulcer, 73/83 (88%) strains from patients with non-ulcer dyspepsia and 8/9 (89%) strains from cancer patients were positive for the cagA gene. There was no statistically significant difference between the prevalence of cagA-positive strains from patients with distinct clinical outcomes (p > 0.05). The prevalence of cagA-positive strains in the Singapore population is high regardless of clinical disease status. The results suggest that the cagA gene is not a universal virulence marker of H. pylori.