Molecular Biology Reports

Published by Springer Nature

Online ISSN: 1573-4978


Print ISSN: 0301-4851


Prior peritoneal lavage with hot 0.9 % saline induces HSP70 expression and protects against cerulein-induced acute pancreatitis in rats
  • Article

October 2012


71 Reads

Ke Meng


Qingsen Liu


Yan Dou


Qiyang Huang
Recent studies have indicated that pre-induction of heat shock protein 70 (HSP70) expression in the pancreas protects against secretagogue-induced pancreatitis. In those studies, the HSP70 was mostly induced by unfeasible conditions. The aim of this current study was to investigate the effect of peritoneal lavage with hot 0.9 % saline (42 °C) on the pancreatic expression of HSP70 and its protective effect on cerulein-induced acute pancreatitis in rats. Male Wistar rats were peritoneally lavaged with 0.9 % saline at 42 °C for 30 min. HSP70 expression was evaluated by western blotting analysis. Prior peritoneal lavages with hot and warm saline were performed. Acute pancreatitis was induced by administration of intraperitoneal injection of cerulein (20 μg/kg) four times, and its severity was assessed by measuring serum amylase, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and trypsinogen activation peptide (TAP) levels. Pancreatic sections were stained with hematoxylin and eosin for histological evaluation. Peritoneal lavage with hot 0.9 % saline increased intrapancreatic HSP70 expression and ameliorated the cerulein-induced pancreatitis in rats, judged by the significantly reduced serum amylase, TNF-α, and IL-6 concentrations; histopathological scores, and serum TAP levels. Peritoneal lavage with hot 0.9 % saline can induce HSP70 expression and prevent cerulein-induced acute pancreatitis in rats. The results suggest that HSP70 protects against cerulein-induced pancreatitis by preventing proinflammatory cytokine synthesis and trypsinogen activation during acute pancreatitis.

An investigation of hormesis of trichloroethylene in L-02 liver cells by differential proteomic analysis

November 2009


52 Reads

Hai-Yan Huang


Jian-Jun Liu


Ren-Rong Xi




Zhi-Xiong Zhuang
Hormesis is the dose-response pattern of the biological responses to toxic chemicals, characterized by low-dose stimulation and high-dose inhibition. Although it is known that some cell types exhibit an adaptive response to low levels of cytotoxic agents, its molecular mechanism is still unclear and it has yet to be established whether this is a universal phenomenon that occurs in all cell types in response to exposure to every chemical. Trichloroethylene (TCE) is an organic solvent widely used and is released into the atmosphere from industrial degreasing operations. Acute (short-term) and chronic (long-term) inhalation exposure to trichloroethylene can affect the human health. In order to elucidate a cell-survival adaptive response of L-02 liver cells exposed to low dose of TCE, CCK-8 assay was used to assess cytotoxicity, and examined the possible mechanisms of hormesis by proteomics technology. We found that exposure of L-02 liver cells to low level of TCE resulted in adaptation to further exposure to higher level, about 1,000 protein-spots were obtained by two-dimensional electrophoresis (2-DE) and five protein spots were identified by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry sequencing of tryptic peptides. Our results suggest that a relationship may exist between identified proteins and TCE-induced hormesis, which are very useful for further study of the mechanism and risk assessment of TCE.

NAT2 slow acetylation genotypes contribute to asthma risk among Caucasians: Evidence from 946 cases and 1,091 controls

January 2014


42 Reads

NAT2 plays a critical role in external chemical detoxification. Thus, polymorphism of NAT2 has been suggested to associate with several disorders. A number of studies have been devoted to the relationship between NAT2 polymorphism and asthma risk. However, the results were inconclusive. In this study we aimed to derive a more precise estimation of the association. A literature search in the common databases was conducted and then meta-analyses evaluating the association of NAT2 polymorphism and asthma risk were performed. Eligible studies were identified for the period up to May 2013. A total of five case-control studies containing 946 cases and 1,091 controls were lastly included for analysis. The overall data showed that slow acetylators of NAT2 might have an association with increased asthma risk (OR 2.20; 95 % CI 1.31-3.72). The pooled data suggest that slow acetylators of NAT2 might contribute to asthma risk among Caucasians. Future studies are needed to confirm this conclusion.

DNA cleavage in chromatin spacer segments by a non-enzymic probe, 1,10-phenanthroline-copper complex

May 1985


17 Reads

The action of 1,10-phenanthroline-copper complex under aerobic conditions in the presence of 2-mercaptoethanol on chromatin DNA in murine thymocyte nuclei was studied. At limited oxygen supply primarily multiple single-strand breaks of DNA in the spacer segments are observed while the core DNA segments in chromatin remain intact. After the single-strand breaks accumulate in both DNA strands (under conditions of improved oxygen supply), double-strand cleavage of DNA to fragments of nucleosomal size becomes apparent. This regular character of DNA degradation in chromatin is apparently due to the preferential binding of the 1,10-phenanthroline-copper complex to DNA of the spacer segments and to the localized generation of damaging radicals.

Effects of ovariectomy on microsatellite instability in rat colon tumors induced by 1,2-dimethylhydrazine

April 2009


15 Reads

To study the effects of ovariectomy on tumorigenesis and microsatellite instability (MSI) in rat colon tumors induced by 1,2-dimethylhydrazine, to elucidate the association between postmenopausal ovarian hormones depletion and MSI pathway in colorectal tumorigenesis. Forty female Wistar rats were randomly divided into two groups: Ovariectomized (Ovx) group and Sham-ovariectomized (Sham-Ovx) group. All rats were injected intraperitoneally with 1,2-dimethylhydrazine (DMH) (20 mg/kg b.w) once a week for 20 weeks. Ten weeks after the final DMH injection, all the rats were sacrificed to collect tumors. Microsatellite instability of six microsatellite loci was detected using fluorescent PCR followed by fragment analysis on automatic DNA sequencer with GeneScan 3.7 software. The tumor multiplicity in the OVX group was significantly higher than that in the Sham-OVX group (3.6 +/- 1.4 vs. 2.4 +/- 1.6, P < 0.05). The incidence of MSI-positive tumors in OVX group was higher than that in Sham-OVX group (32.1 vs. 10.8%, P < 0.05).The incidence of tumors showing MSI at multiple loci in OVX group was also higher than that in Sham-OVX group (18.9 vs. 2.7%, P < 0.05). Ovariectomy increased tumor formation and the frequency of MSI in DMH-induced colon tumors. It implied that postmenopausal ovarian hormones depletion might influence colorectal tumorigenesis through MSI pathway.

Expression, purification and functional characterization of a recombinant 2,3-dihydroxybiphenyl-1,2-dioxygenase from Rhodococcus rhodochrous

November 2010


31 Reads

A 2,3-dihydroxybiphenyl (2,3-DHBP) dioxygenase gene from a Rhodococcus sp. strain, named RrbphCI and involved in the degradation of polychlorinated biphenyls (PCBs), was synthesized. RrbphCI was expressed in Escherichia coli and its encoded enzyme was purified. SDS–PAGE analysis indicated that the size of the protein encoded by RrbphCI was about 32 kDa. The activity of the 2,3-DHBP dioxygenase was 82.8 U/mg when the substrate was 2,3-DHBP, with optimum pH 8.0 at 30°C, and optimum temperature was 40°C at pH 8.0. The RrbphCI gene was transformed into Pseudomonas putida strain EG11, to determine the ability of the enzyme to degrade 2,3-DHBP. The wild type EG11 degraded 61.86% of supplied 2,3-DHBP and the transformed EG11 (hosting the RrbphCI gene) utilized 52.68% after 2 min of treatment at 30°C. The overexpressed and purified enzyme was able to degrade 2,3-DHBP. The 2,3-DHBP dioxygenase is a key enzyme in the PCB degradation pathway. RrbphCI and its encoded 2,3-DHBP dioxygenase may have transgenic applications in bioremediation of PCBs.

Fig. 1 Various types of comets observed in rats colonic cells. A Undamaged, B Damaged 
Table 2 Effect of probiotic curd feeding on DMH induced DNA damage 
Inhibition of 1,2 dimethylhydrazine induced genotoxicity in rats by the administration of probiotic curd
  • Article
  • Full-text available

April 2009


180 Reads

Epidemiologic and experimental studies suggest that the probiotic organisms are effective in preventing colon carcinogenesis, which is the major cause of mortality and morbidity in western countries. Keeping this in view, a curd (a common Indian fermented milk product) was prepared by the addition of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and curd culture Lactococcus lactis biovar. diacetylactis. In present study, we have evaluated the anti tumor effect of probiotic curd by monitoring the DNA damage through comet assay. The rats were allocated to four groups, first group was DMH control group, second group was probiotic curd group in which probiotic curd was given along with DMH (1,2-dimethylhydrazine) injection, third group was normal curd group in which normal curd was given along with DMH injection and fourth group was normal control group. Animals received subcutaneous injection of DMH dissolved in normal saline at a dose rate of 20 mg/kg body weight, once weekly for 15 weeks. The rats were dissected at 40th week of experiment and comet assay was done in colonic cells to assess the DNA damage. A significant reduction in DNA damage (54.7%) was observed in probiotic curd group as compared to DMH control group (88.1%). The probiotic curd was effective to significantly reduce the L:W ratio in comparison to DMH control group and normal curd. The results of present study show the protective effects of probiotic curd against DMH induced genotoxicity in colonic cells.

Upregulation of interleukin-1β production by 1,25-Dihydroxyvitamin D3 in activated human macrophages

March 2011


25 Reads

1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) plays important roles in the immune system. In contrast to its well known function in the adaptive immune system, much less is known about the immunoregulatory effects of 1,25(OH)(2)D(3) in the innate immune system, especially on activated human macrophages. Here we found that 1,25(OH)(2)D(3) strongly stimulated the production of interleukin-1β (IL-1β) in PMA-differentiated U937 cells and human monocyte-derived macrophages treated with lipopolysaccharide (LPS) or PMA. In this study, Erk1/2 appeared to mediate 1,25(OH)(2)D(3)-induced expression of IL-1β. Parallel to the increased production of IL-1β, 1,25(OH)(2)D(3) increased the expression and phosphorylation of the CCAAT enhancer-binding protein β (C/EBPβ), which is one of the key transcriptional regulatory factors for IL-1β transcription. These results suggest that 1,25(OH)(2)D(3) may function as a proinflammatory molecule in inflammatory macrophages.

Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein from oriental river prawn, Macrobrachium nipponense

March 2014


46 Reads

The lipopolysaccharide and β-1,3-glucan binding protein (LGBP), one of the pattern recognition proteins, plays an important role in the innate immune response of invertebrates. A 1,506 bp full-length cDNA of a LGBP gene was cloned and characterized from the oriental river prawn Macrobrachium nipponense (named as MnLGBP). Analysis of the nucleotide sequence revealed that the cDNA clone has an open reading frame of 1,119 bp, encoding a protein of 372 amino acids including a 21-aa signal peptide. The calculated molecular mass of the mature protein (351 aa) was 39.9 kDa with an estimated pI of 4.63. The MnLGBP sequence contains: (1) two putative integrin-binding motifs, (2) a glucanase motif, (3) two putative N-glycosylation sites, (4) one protein kinase C phosphorylation site, and (5) a putative recognition motif for β-1,3-linkage of polysaccharides. Sequence comparison based on the deduced amino acid sequence of MnLGBP showed varied identity of 89, 76 and 74 % with those of Macrobrachium rosenbergii LGBP, Marsupenaeus japonicus β-1,3-glucan binding proteins, and Fenneropenaeus chinensis LGBP, respectively. Quantitative RT-PCR results showed that MnLGBP was expressed in nerve, intestine, muscle, gill, heart, haemocytes and at the highest level in hepatopancreas. After challenge with the pathogen, Aeromonas hydrophila and Vibrio parahaemolyticus, the expression of MnLGBP mRNA was significantly upregulated in the hepatopancreas compared to the control group. At the same time, the mRNA level of MnproPO increased dramatically at 48 h after injection of bacteria. These data should be helpful to better understand the function of MnLGBP in the prawn immune system.

Fig. 1 Different concentration of b3Gn-T8 siRNA was transfected into AGS cells. a 1 untreated cells, 2 cells transfected with negative control siRNA, 3 cells transfected with 10 nM siRNA, 4 cells transfected with 20 nM siRNA, 5 cells transfected with 40 nM siRNA. b Densitometry measurements of the bands 
Figure 2 of 2
Regulation of MMP-2 expression and activity by β-1,3-N-acetylglucosaminyltransferase-8 in AGS gastric cancer cells

October 2010


68 Reads

β-1,3-N-acetylglucosaminyltransferase-8(β3Gn-T8) catalyzes the transfer of GlcNAc to the non-reducing terminus of the Galβ1-4GlcNAc of tetraantennary N-glycan in vitro. It has been reported to be involved in malignant tumors, but a comprehensive understanding of how the glycolsyltransferase correlates with the invasive potential of human gastric cancer is not currently available. Therefore, we investigated the ability and possible mechanism involved with β3Gn-T8 in modulating matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in AGS gastric cancer cells. Here, we found out that siRNA-mediated suppression of the β3Gn-T8 could directly reduce the MMP-2 expression and activity as observed in RT-PCR, western blot and gelatin zymography analysis. Meanwhile, TIMP-2 expression had been increased. Cell invasion assay using matrigel matrix-coated transwell inserts showed that the invasive property was greatly suppressed in β3Gn-T8 siRNA transfected cells. Furthermore, cells overexpressing β3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene, but TIMP-2 expression had been inhibited. The invasive ability of these cells was also enhanced. Protein-protein interaction analysis using STRING database showed that β3Gn-T8 and MMP-2 may have related signal pathway. In summary, our results reveal a new mechanism by which β3Gn-T8 can regulate MMP-2 and TIMP-2. We suggest that β3Gn-T8 can be used as a novel therapeutic target for human gastric treatment.

Cloning and characterization of a β-1,3-glucan-binding protein from shrimp Fenneropenaeus chinensis

December 2010


47 Reads

The β-1,3-D: -glucan binding protein (βGBP), one kind of the pattern recognition proteins (PRPs), was cloned and characterized from the Chinese Shrimp Fenneropenaeus chinensis, and named as FcβGBP-HDL. The results indicated that the full length cDNA of 6713 bp had an open reading frame encoded a polypeptide of 2139 amino acids with two glucanase-like motifs and one RGD motif, while without signal peptide. The calculated molecular mass of mature protein was 240.7 kDa and theoretical pI was 5.95. Sequence comparison of the deduced amino acid sequence of FcβGBP-HDL showed varied identity of 88, 54 and 53% with those of Litopenaeus vannamei βGBP-HDL, Pacifastacus leniusculus βGBP, and Pontastacus leptodactylus βGBP, respectively. qRT-PCR analysis indicated that FcβGBP-HDL was expressed in intestines, hepatopancreas, muscle, gill and hemocytes, and its profile was modified post WSSV challenge. The differential expressions of FcβGBP-HDL in different tissues post WSSV challenge suggested that FcβGBP-HDL might play an important role in shrimp immune and perform differently in different tissues. These data would be helpful to better understand the WSSV-resistance mechanism of farming shrimp.

Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and 3-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis

January 2008


49 Reads

A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp.

Molecular characterization and expression analysis of lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) from pearl oyster Pinctada fucata

November 2009


36 Reads

The lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) plays an important function in the innate immune response of invertebrates as a pattern recognition receptor (PRR). Herein, we described the isolation and characterization of pearl oyster Pinctada fucata LGBP (designated as poLGBP). The poLGBP cDNA was 2,075 bp long and consisted of a 5'-untranslated region (UTR) of 18 bp, a 3'-UTR of 299 bp with one cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1,758 bp encoding a polypeptide of 585 amino acids with an estimated molecular mass of 65.1 kDa and a theoretical isoelectric point of 5.80. Homology analysis of the deduced amino acid sequence of the poLGBP with other known LGBP sequences by MatGAT software revealed that the poLGBP shared 26.3-56.7% identity and 40.5-70.9% similarity to the other known LGBP sequences. SMART and alignment analysis revealed that the poLGBP possessed a potential polysaccharide-binding motif, a glucanase motif, a LPS-binding site, a β-1,3-linkage of polysaccharide, a glycine-rich region, a threonine-rich region and two N-glycosylation sites. In healthy pearl oyster, the poLGBP mRNA was specifically expressed in digestive gland, and not detected in gill, adductor muscle, gonad, intestine, mantle and hemocytes. However, after bacteria stimulation, the expression of the poLGBP mRNA was significantly up-regulated in digestive gland and also weakly detected in haemocytes, gonad and intestine. After LPS stimulation, the poLGBP mRNA expression was significantly up-regulated at 8 and 12 h in digestive gland, and the expression level was 10.7-fold higher than the PBS group at 12 h. After bacteria stimulation, the expression level of the poLGBP mRNA was also significantly up-regulated in digestive gland and was 12.9-fold higher than the PBS group at 8 h. However, during the experiment, the poLGBP mRNA expression was not detected in gill after LPS or bacteria stimulation. The tissue-specific expression and the expression up-regulation after LPS or bacteria stimulation in digestive gland suggested that the poLGBP was an inducible acute-phase protein and might play an important function in digestion as digestive enzyme and pattern recognition receptor.

Cloning and characterization of a wheat b-1,3-glucanase gene induced by the stripe rust pathogen Puccinia striiformis f. sp. tritici

September 2009


82 Reads

b-1,3-Glucanases are a group of pathogenesis related proteins that have been reported to be involved in plant defense against pathogens in many other plant pathogen systems. However, it was not clear if these genes play similar role in wheat (Triticum aestivum L.) against Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen. To investigate the role of b-1,3-glucanase (EC3.2.1.39) in the resistance response of wheat (cv. Suwon11) to stripe rust, a wheat b-1,3-glucanase gene induced by Pst, designated as TaGlu, was cloned and characterized.TaGlu was predicted to encode a basic protein of 334 amino acids. Quantitative real-time PCR analyses revealed that the transcription of TaGlu was induced during both compatible and incompatible interactions with Pst, but the transcription level was much higher in the incompatible interaction than that in the compatible interaction. TaGlu also showed noticeable induction of gene expression in young green leaf tissues treated with salicylic acid, methyl jasmonate or ethylene. Immunogold labeling assays showed that the enzyme were localized mainly in the host cell wall and over the extra haustorial matrix, and the labeling densities were found significantly higher in the incompatible interaction than those in the compatible interaction.

Fig. 5 Effect of TcGlu2 on Moniliophthora perniciosa growth. PB phosphate buffer; CP control proteins corresponding to purified protein extract from E. coli expressing pET28a without insert  
Fig. 6 Membrane permeability of Moniliophthora perniciosa hyphae. TcPR-10 was incubated with M. perniciosa hyphae at 25 or 4 °C with or without NaN 3 , and membrane integrity was checked by using PI uptake assay. Hyphae were observed under fluorescence microscope at 30 min and 1 h and images were taken under phase contrast and PI filter  
Recombinant β-1,3-1,4-glucanase from Theobroma cacao impairs Moniliophthora perniciosa mycelial growth

May 2013


139 Reads

In this work, we identified a gene from Theobroma cacao L. genome and cDNA libraries, named TcGlu2, that encodes a β-1,3-1,4-glucanase. The TcGlu2 ORF was 720 bp in length and encoded a polypeptide of 239 amino acids with a molecular mass of 25.58 kDa. TcGlu2 contains a conserved domain characteristic of β-1,3-1,4-glucanases and presented high protein identity with β-1,3-1,4-glucanases from other plant species. Molecular modeling of TcGlu2 showed an active site of 13 amino acids typical of glucanase with β-1,3 and 1,4 action mode. The recombinant cDNA TcGlu2 obtained by heterologous expression in Escherichia coli and whose sequence was confirmed by mass spectrometry, has a molecular mass of about 22 kDa (with His-Tag) and showed antifungal activity against the fungus Moniliophthora perniciosa, causal agent of the witches' broom disease in cacao. The integrity of the hyphae membranes of M. perniciosa, incubated with protein TcGlu2, was analyzed with propidium iodide. After 1 h of incubation, a strong fluorescence emitted by the hyphae indicating the hydrolysis of the membrane by TcGlu2, was observed. To our knowledge, this is the first study of a cacao β-1,3-1,4-glucanase expression in heterologous system and the first analysis showing the antifungal activity of a β-1,3-1,4-glucanase, in particular against M. perniciosa.

Associations between the FAS -670 A/G and -1,377 G/A polymorphisms and susceptibility to autoimmune rheumatic diseases: a meta-analysis

October 2012


17 Reads

The aim of this study was to explore whether FAS -670 A/G and -1,377 G/A polymorphisms confer susceptibility to autoimmune rheumatic diseases. A meta-analysis was conducted on the associations between the FAS -670 A/G and -1,377 G/A polymorphisms and autoimmune rheumatic diseases using allele contrast, a recessive model, a dominant model, and an additive model. Thirteen articles with 21 comparison studies (16 on FAS -670 A/G and 5 on -1,377 G/A polymorphisms) including systemic lupus erythematosus (SLE), four systemic sclerosis, four Sjogren's syndrome, three rheumatoid arthritis (RA), one juvenile idiopathic arthritis, and one spondyloarthropathy were available for the meta-analysis. Meta-analysis revealed an association between rheumatic diseases and the FAS -670 A/G polymorphism in the dominant model (odds ratio [OR] = 0.761, 95 % confidence interval [CI] = 0.621-0.932, p = 0.008]. Stratification by ethnicity indicated an association between the FAS -670 G allele carrier and rheumatic diseases in Asian (OR = 0.569, 95 % CI = 0.409-0.791, p = 0.001). Furthermore, stratification by disease indicated an association between the FAS -670 G allele carrier and SLE and RA (OR = 0.578, 95 % CI = 0.358-0.934, p = 0.025; OR = 0.609, 95 % CI = 0.398-0.934, p = 0.023, respectively). The FAS -670 G allele was negatively associated with SLE susceptibility. Meta-analysis of the FAS -1,377 G/A polymorphism stratified by disease showed an association between the FAS -1,377 A allele and SLE (OR = 0.783, 95 % CI = 0.613-0.997, p = 0.047). Meta-analyses using the dominant model also showed a significant association in SLE (OR = 0.712, 95 % CI = 0.528-0.961, p = 0.027). This meta-analysis demonstrates that the FAS -670 A/G polymorphism confers susceptibility to rheumatic diseases in Asians and SLE and RA, and the FAS -1,377 G/A polymorphism is associated with SLE susceptibility.

Pre and post cloning characterization of a β-1,4-endoglucanase from Bacillus sp

July 2009


23 Reads

Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over a pH range of 6.0–8.0. The K m and V max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min−1, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.

Kinetic and thermodynamic study of cloned thermostable endo-1,4-??-xylanase from Thermotoga petrophila in mesophilic host

February 2012


138 Reads

The 1,044 bp endo-1,4-β-xylanase gene of a hyperthermophilic Eubacterium, "Thermotoga petrophila RKU 1" (T. petrophila) was amplified, from the genomic DNA of donor bacterium, cloned and expressed in mesophilic host E. coli strain BL21 Codon plus. The extracellular target protein was purified by heat treatment followed by anion and cation exchange column chromatography. The purified enzyme appeared as a single band, corresponding to molecular mass of 40 kDa, upon SDS-PAGE. The pH and temperature profile showed that enzyme was maximally active at 6.0 and 95 °C, respectively against birchwood xylan as a substrate (2,600 U/mg). The enzyme also exhibited marked activity towards beech wood xylan (1,655 U/mg). However minor activity against CMC (61 U/mg) and β-Glucan barley (21 U/mg) was observed. No activity against Avicel, Starch, Laminarin and Whatman filter paper 42 was observed. The K(m), V(max) and K (cat) of the recombinant enzyme were found to be 3.5 mg ml(-1), 2778 μmol mg(-1)min(-1) and 2,137,346.15 s(-1), respectively against birchwood xylan as a substrate. The recombinant enzyme was found very stable and exhibited half life (t(½)) of 54.5 min even at temperature as high as 96 °C, with enthalpy of denaturation (ΔH*(D)), free energy of denaturation (ΔG*(D)) and entropy of denaturation (ΔS*(D)) of 513.23 kJ mol(-1), 104.42 kJ mol(-1) and 1.10 kJ mol(-1)K(-1), respectively at 96 °C. Further the enthalpy (ΔH*), Gibbs free energy (ΔG*) and entropy (ΔS*) for birchwood xylan hydrolysis by recombinant endo-1,4-β-xylanase were calculated at 95 °C as 62.45 kJ mol(-1), 46.18 kJ mol(-1) and 44.2 J mol(-1) K(-1), respectively.

Fig. 1 Complete nucleotide and deduced amino acid sequence of the AcRbcS cDNA. An open reading frame of 498 bp from 29 to 526 encoding 165 amino acids is shown below the nucleotide sequence. The conserved N-myristoylation site, tyrosine kinase phosphorylation active site and protein kinase C phosphorylation site are underlined,  
Fig. 2 Bioinformatics analysis of the amino acid sequences of AcRbcS. a Conserved domains of AcRbcS obtained via NCBI searches. b Comparison of the deduced amino acid sequences of RbcS from A. convolutus with those of other green algae: Acetabularia mediterranea (GenBank accession no. CAA36108), Acetabularia peniculus (CAA36106), Batophora oerstedii (CAA40538), Chlorella pyrenoidosa (ACU32662), Chlamydomonas reinhardtii (CAA28160), Dunaliella salina (AAU93597), Dunaliella tertiolecta (AAS48504), Nannochloris bacillaris (BAD42340), Ulva pertusa (BAI47769) and Volvox carteri (AAO46872). The conserved residues are indicated by black and similar residues are indicated by gray. Putative transit sequences and mature peptide sequences of RbcS are shown separately with a vertical line. Dashed lines are gaps introduced to maximize alignments. The highly conserved residues are indicated with asterisks, and the similar residues with colons or dots. c Secondary structure prediction of AcRbcS. The helices, strands, turns and coils are indicated in order from the longest to the shortest vertical lines  
Fig. 3 Phylogenetic relationship of RbcS among green algae. One thousand bootstrap trials were run using the neighbour-joining algorithm by MEGA 4.1. Branch lengths were proportional to estimates of evolutionary change. The number associated with each internal branch was the local bootstrap probability, which was an indicator of confidence  
Fig. 4 Southern hybridization analysis of AcRbcS. A. convolutus genomic DNA (20 lg) was digested with MspI (lane 1), NcoI (lane 2) and PstI (lane 3) restriction enzymes, and hybridized with the partial cDNA of AcRbcS. M presents the molecular weight marker  
Fig. 5 The expression of AcRbcS was analysed by RT-PCR using total RNA with or without (-ve) reverse transcriptase. Total RNA was isolated from cells grown under four conditions. Condition 1: cells were grown in continuous light for 3 days after 1-day subculture. Condition 2: cells were grown under Condition 1 and then for 12 h in dark. Condition 3: cells were grown under Condition 2 and then for 24 h in light. Condition 4: cells were grown under Condition 2 and then for 48 h in light. 18S rRNA gene was used as the control to show the equality of the templates in PCR reactions  
Cloning and characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) cDNA from green microalga Ankistrodesmus convolutus

February 2011


213 Reads

An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5′ untranslated region (UTR), 225 bp of 3′ non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.

Table 1 Characteristics of studies included to the meta-analysis
Meta-analysis of association between TT genotype of MTHFR gene and pediatric ischemic stroke. OR and 95 % CI of the TT genotype versus CC + CT were calculated from data presented in individual studies. A random effects model with the method of Dersimonian Laird was used to calculate pooled weighted OR. One study was excluded from the calculation because of the lack of TT genotype, both in cases and controls [14]
Meta-analysis of association between carriers of T allele (individuals with CT + TT genotypes) of MTHFR gene and pediatric ischemic stroke. OR and 95 % CI of the CT + TT genotypes versus CC were calculated from data presented in individual studies. A random effects model with the method of Dersimonian Laird was used to calculate pooled weighted OR. Four of the studies were excluded from the calculation due to lack of accurate data of CT genotype, both in cases and controls [10, 19, 21, 22]
The TT genotype of methylenetetrahydrofolate reductase 677C>T polymorphism increases the susceptibility to pediatric ischemic stroke: Meta-analysis of the 822 cases and 1,552 controls

May 2012


76 Reads

The 677C>T polymorphism within methylenetetrahydrofolate reductase (MTHFR) gene is related to an elevated level of homocysteine. Thus it may be considered as a genetic risk factor in ischemic stroke. Apparently studies of this type of polymorphism in childhood stroke have shown conflicting results. We performed meta-analysis of all the data that are available in relation with MTHFR polymorphism and the risk of ischemic stroke in children. We searched PubMed (last search dated December 2010) using "MTHFR polymorphism", "ischemic stroke" "child", "children", "pediatric stroke" as keywords and reference lists of studies and reviews on the topic. Finally, 15 case-control studies corresponded to the inclusion criteria for meta-analysis. These studies involved the total number of 822 children and adolescents after ischemic stroke and 1,552 control subjects. Fixed or random effects models were used depending on the heterogeneity between the studies. The association between ischemic stroke and 677C>T polymorphism within MTHFR gene was observed in three of the studies. The pooled analysis showed that TT genotype of MTHFR gene is more common in stroke patients than in controls (p = 0.0402, odds ratio = 1.57, 95 % confidence interval 1.02-2.41). The Egger's test did not reveal presence of a publication bias. The results based on a sizeable group of cases and controls have proved that the 677C>T polymorphism in MTHFR gene is associated with the development of ischemic stroke in children.

Fig. 1 Relative quantity of MmeFBA mRNA in different tissues of M. meretrix Values are normalized to b-actin (mean ± SE, n = 4). Different letters indicate the significant difference among tissues (P \ 0.05)  
Fig. 2 The part of MmeFBA genomic DNA sequence containing three discovered exon SNPs Shading characters indicate exon sequence. None shading characters indicate intron sequence. The underlined bold letters indicate SNPs. The characters with border represent the reverse primer used for sequencing  
Fig. 3 Different genotype peak patterns of seven SNPs on the sequence map Black frames indicate the SNP sites. A, B, C, D, E, F, G indicate mmfbai-1, mmfbai-2, mmfbai-3, mmfbai-4, mmfbae-1, mmfbae-2 and mmfbae-3, respectively  
Fig. 4 The position of the amino acid affected by the nonsynonymous SNP, named mmfbae-2, in the three dimensional structure of MmeFBA. a The nSNP-affected methionine (MET166) in there dimensional structure of MmeFBA. LYS231 is the active site of MmeFBA. b The nSNP-affected leucine (LEU166) in partial structure of MmeFBA (the van der Waals surfaces of the leucine side chain is exhibited). c The nSNP-affected methionine (MET166) in partial structure of MmeFBA (the van der Waals surfaces of the methionine side chain is exhibited)  
Identification of a fructose-1,6-bisphosphate aldolase gene and association of the single nucleotide polymorphisms with growth traits in the clam Meretrix meretrix

December 2011


112 Reads

This study investigated whether there were single nucleotide polymorphisms (SNPs) in fructose-1,6-bisphosphate aldolase (FBA) gene associated with growth traits of the clam Meretrix meretrix. A FBA gene was identified in M. meretrix and its deduced amino acid residues shared high identity with type I aldolase. The FBA (MmeFBA) mRNA expression profile was examined by real-time PCR in different tissues and the significantly high expression level in foot and adduct muscle suggests that MmeFBA is a muscle type aldolase which functions in glycolytic pathway. In the MmeFBA gene, we identified four intron SNPs and three exon SNPs including a nonsynonymous SNP (mmfbae-2). These SNPs were genotyped in 205 clams from two clam populations with significantly different growth performance. Results showed that allele frequencies of three SNPs (mmfbai-1, mmfbai-3 and mmfbae-2) and the genotype frequency of mmfbai-1 were all significantly different between the two populations. The haplotype analysis further supported the three SNPs distributed differently between the two populations. This study successively characterized three growth-related SNPs in a gene involved in energy metabolism of M. meretrix. These findings could contribute the development of phenotype-selective breeding program in M. meretrix.

Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis

May 2013


134 Reads

Fructose-1,6-bisphosphatase (FBPase), a key regulatory enzyme of gluconeogenesis, plays an essential role in metabolism and development of most organisms. To the wealth of available knowledge about FBPase from Clonorchis sinensis (CsFBPase), in this study, the characteristics of CsFBPase and its potential role in pathogenesis of clonorchiasis were investigated. The Km value of CsFBPase was calculated to be 41.9 uM. The optimal temperature and pH of CsFBPase were 37 °C and pH 7.5-8.0, respectively. In addition, Mg(2+) or K(+) played a regulatory role in enzyme activity of CsFBPase. Both transcriptional and translational level of CsFBPase were higher in metacercariae (one of larva stages) than those in adult worm (P < 0.05). CsFBPase were observed to extensively express in the intestine, vitellaria and tegument of adult worms and ubiquitously in metacercariae. Moreover, CsFBPase was confirmed as a component of excretory/secretory products. Consequently, the translocation of CsFBPase could be detected on epithelial cells of bile duct in liver of C. sinensis infected rat. Recombinant CsFBPase can specifically bind to the membrane of human hepatic stellate cell line LX-2 by immunofluorescence analysis and stimulated proliferation and activation of LX-2 which demonstrated by Cell Counting Kit-8 and upregulation of key fibrosis-related factors, such as α-smooth muscle actin, collagen I and collagen III using qRT-PCR. Thus, we predicated that CsFBPase might be a multifunctional enzyme which played as both regulatory enzyme and virulence factor in pathogenesis of C. sinensis infection.

Table 1 6His-tagged Dkk1 protein purification table
Fig. 4. Oligomerization studies of 6His-tagged Dkk1 protein. (A) Chromatogram of Superdex 200 HR10-30 (24 ml) gel filtration (UV 280nm ) performed on 6His-tagged Dkk1 protein eluted from nickel chelate column. Injection: 0.5 ml. Flow rate: 0.4 ml/min. (B) Elutions fractions from P2 and P3 gel filtration peaks were subjected to 10-20% SDSPAGE followed by Coomassie blue staining. Ten microliters of each fraction was loaded. (C) Fibroblast L-cells were transiently transfected with TOPflash reporter and grown in the absence (Ctrl) or presence of (others) Wnt3a-CM. Increasing amounts (25-250 ng/ml) of 6His-tagged Dkk1 protein from fractions ''start, P1, P2 and P3" were added to the culture medium. Luciferase activity was measured as described in Materials and methods. 
Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris

May 2011


172 Reads

Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59-97 above kDa glycoprotein, 52-57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 μg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 μg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 μg hyper-glycosylated and non-glycosylated NA. In the 0.2 μg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.

CD14 -159C/T polymorphism contributes to the susceptibility to tuberculosis: Evidence from pooled 1,700 cases and 1, 816 controls

February 2014


18 Reads

CD14 is a receptor for lipopolysaccharide and plays an important role in innate immune against infections induced by microorganisms. A functional polymorphism in promoter region of CD14 gene, -159C/T, was extensively investigated with tuberculosis (TB) risk, but the association results were inconclusive. We performed a meta-analysis to synthesize association results of CD14 -159C/T polymorphism with TB risk from 8 studies including 1,700 TB cases and 1,816 controls. Based on the heterogeneity between studies evaluated by χ(2)-based Q test, a fixed- or random-effect model was applied to estimate the pooled odds ratio (OR) and 95 % confidence interval (CI). Potential publication bias was evaluated with the funnel plot as well as the linear regression asymmetry test proposed by Egger et al. We found that the -159T allele was significantly associated with an increased risk of TB (OR 1.27, 95 % CI 1.01-1.61) as compared with -159C allele. Individuals with -159TT genotype showed a significantly increased risk of TB than those with -159CT/CC genotype (OR 1.52, 95 % CI 1.11-2.08). These associations were not attributed to potential publication bias (P > 0.05 for Egger's test). The results from this meta-analysis indicate that CD14 -159C/T polymorphism is associated with TB predisposition and may serve as a candidate of susceptibility biomarker for TB.

Functional polymorphism rs4072037 in MUC1 gene contributes to the susceptibility to gastric cancer: Evidence from pooled 6,580 cases and 10,324 controls

September 2013


57 Reads

Genome-wide association studies have reported a promising association of rs4072037 with gastric cancer (GC). This variant was associated with altered physiological function of MUC1 possibly by modulating promoter activity and alternative splicing of MUC1. However, the association results were inconclusive and estimate of the effect of this variant was not well evaluated. A meta-analysis by systematically reviewing relevant reports may facilitate to address these concerns. Association studies involving MUC1 rs4072037 polymorphism and GC risk were identified up to June 30, 2012. Odds ratio (OR) and 95 % confidence interval (CI) in additive model were estimated or extracted from each study. The pooled effect size was quantitatively synthesized using meta-analysis. Heterogeneity between studies was measured by the Q test and I 2 statistic, and publication bias was evaluated by a funnel plot and the Egger’s test. A total of 10 independent case–control studies including 6,580 GC cases and 10,324 controls were included in this meta-analysis. Eight of the ten studies were Asian ethnicity and two European. The G allele of MUC1 rs4072037 was significantly associated with a decreased risk of GC (OR = 0.72, 95 % CI 0.68–0.77; P = 7.82 × 10−25), as compared with A allele. Stratification for different ethnicity, tumor localization or type showed similar results. These findings represent important evidence for association of MUC1 rs4072037 variant with GC risk, and also provide a relatively reliable estimate of effect size. MUC1 is a strong candidate as a susceptibility gene of GC.

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