Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA.In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n = 60), healthy controls (n = 40), systemic lupus erythematosus (n = 20), Sjögren’s syndrome (n = 40)) were screened for antibody reactivity.Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity.Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies.     

Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques

Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-Å resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.     

Immature and mature human astrovirus: structure, conformational changes, and similarities to hepatitis e virus

Human astroviruses (HAstVs) are a major cause of gastroenteritis. HAstV assembles from the structural protein VP90 and undergoes a cascade of proteolytic cleavages. Cleavage to VP70 is required for release of immature particles from cells, and subsequent cleavage by trypsin confers infectivity. We used electron cryomicroscopy and icosahedral image analysis to determine the first experimentally derived, three-dimensional structures of an immature VP70 virion and a fully proteolyzed, infectious virion. Both particles display T = 3 icosahedral symmetry and nearly identical solid capsid shells with diameters of ~ 350 Å. Globular spikes emanate from the capsid surface, yielding an overall diameter of ~ 440 Å. While the immature particles display 90 dimeric spikes, the mature capsid only displays 30 spikes, located on the icosahedral 2-fold axes. Loss of the 60 peripentonal spikes likely plays an important role in viral infectivity. In addition, immature HAstV bears a striking resemblance to the structure of hepatitis E virus (HEV)-like particles, as previously predicted from structural similarity of the crystal structure of the astrovirus spike domain with the HEV P-domain [Dong, J., Dong, L., Méndez, E. &; Tao, Y. (2011). Crystal structure of the human astrovirus capsid spike. Proc. Natl. Acad. Sci. USA 108, 12681–12686]. Similarities between their capsid shells and dimeric spikes and between the sequences of their capsid proteins suggest that these viral families are phylogenetically related and may share common assembly and activation mechanisms.     

Thermolabile antifreeze protein produced in Escherichia coli for structural analysis

Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100 mM Tris buffer containing 6 M n-propanol and 2 M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6 M n-propanol-based buffer containing 2 M urea. Existence of native-like secondary structure of r-hGH in 6 M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6 M n-propanol and 2 M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6 M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.     

Purification and crystallization of the ABC-type transport substrate-binding protein OppA from Thermoanaerobacter tengcongensis

Di- and oligopeptide- binding protein OppAs play important roles in solute and nutrient uptake, sporulation, biofilm formation, cell wall muropeptides recycling, peptide-dependent quorum-sensing responses, adherence to host cells, and a variety of other biological processes. Soluble OppA from Thermoanaerobacter tengcongensis was expressed in Escherichia coli. The protein was found to be >95% pure with SDS–PAGE after a series of purification steps and the purity was further verified by mass spectrometry. The protein was crystallized using the sitting-drop vapour-diffusion method with PEG 400 as the precipitant. Crystal diffraction extended to 2.25 Å. The crystal belonged to space group C222₁, with unit-cell parameters of a = 69.395, b = 199.572, c = 131.673 Å, and α = β = γ = 90°.     

Validation of surface plasmon resonance screening of a diverse chemical library for the discovery of protein tyrosine phosphatase 1b binders

We investigated the suitability of surface plasmon resonance (SPR) for providing quantitative binding information from direct screening of a chemical library on protein tyrosine phosphatase 1b (PTP1B). The experimental design was established from simulations to detect binding with K_D < 10^?4 M. The 1120 compounds (cpds) were injected sequentially at concentrations [C(cpd)] of 0.5 or 10 μM over various target surfaces. An optimized evaluation procedure was applied. More than 90% of cpds showed no detectable signal in four screens. The 30 highest responders at C(cpd) = 10 μM, of which 25 were selected in at least one of three screens at C(cpd) = 0.5 μM, contained 22 promiscuous binders and 8 potential PTP1B-specific binders with K_D ～ 10^?5 M. Inhibition of PTP1B activity was assayed and confirmed for 6 of these, including sanguinarine, a known PTP1B inhibitor. C(cpd) dependence studies fully confirmed screening conclusions. The quantitative consistency of SPR data led us to propose a structure–activity relationship (SAR) model for developing selective PTP1B inhibitors based on the ranking of 10 arylbutylpiperidine analogs.     

Purification and characterization of chaperonins 60 and 10 from Methylobacillus glycogenes

Two proteins belonging to the group I chaperonin family were isolated from an obligate methanotroph, Methylobacillus glycogenes. The two proteins, one a GroEL homologue (cpn60: M. glycogenes 60 kDa chaperonin) and the other a GroES homologue (cpn10: M. glycogenes 10 kDa chaperonin), composed a heteropolymeric complex in the presence of ATP. Both proteins were purified from crude extracts of M. glycogenes by anion-exchange (DEAE-Toyopearl) and gel-filtration (Sephacryl S-400) chromatography. The native molecular weights of each chaperonin protein as determined by high-performance liquid chromatography (HPLC) gel-filtration were 820 000 for cpn60 and 65 000 for cpnl0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit molecular weights of cpn60 and cpnl0 were 58 000 and 10 000, respectively. Both cpn60 and cpnl0 possessed amino acid sequences which were highly homologous to other group I chaperonins. M. glycogenes cpn60 displayed an ATPase activity which was inhibited in the presence of cpn10. The chaperonins also displayed an ability to interact with and facilitate the refolding of Thermus malate dehydrogenase and yeast enolase in a manner similar to that of GroEL/ES. The similarities between the Escherichia coli GroE proteins are discussed.     

Myocardial improvement with human embryonic stem cell-derived cardiomyocytes enriched by p38MAPK inhibition

Background aimsWe have shown previously that inhibition of the p38 mitogen-activated protein kinase (p38MAPK) directs the differentiation of human embryonic stem cell (hESC)-derived cardiomyocytes (hCM). We investigated the therapeutic benefits of intramyocardial injection of hCM differentiated from hESC by p38MAPK inhibition using closed-chest ultrasound-guided injection at a clinically relevant time post-myocardial infarction (MI) in a mouse model.MethodsMI was induced in mice and the animals treated at day 3 with: (a) hCM, (b) human fetal fibroblasts (hFF) as cell control, or (c) medium control (n = 10 animals/group). Left ventricular ejection fraction (LVEF) was evaluated post-MI prior to therapy, and at days 28 and 60 post-cell therapy. Hearts were analyzed at day 60 for infarct size, angiogenesis, cell fate and teratoma formation.ResultsLVEF was improved in the hCM-treated animals compared with both hFF and medium control-treated animals at day 28 (39.03 ± 1.79% versus 27.89 ± 1.27%, P < 0.05, versus 32.90 ± 1.46%, P < 0.05, respectively), with sustained benefit until day 60. hCM therapy resulted in significantly smaller scar size, increased capillary bed area, increased number of arterioles, less native cardiomyocyte (CM) apoptosis, and increased CM proliferation compared with the other two groups. These benefits were achieved despite a very low retention rate of the injected cells at day 60, as assessed by immunohistochemistry and quantitative real-time polymerase chain reaction (qPCR). Therapy with hCM did not result in intramyocardial teratoma formation at day 60.ConclusionsThis study demonstrates that hCM derived from p38MAPK-treated hESC have encouraging therapeutic potential.     

Effects of starvation on growth, survival, and body biochemical composition among different sizes of Manila clam Ruditapes philippinarum

A study was conducted to investigate the impact of starvation on different sizes of Manila clam Ruditapes philippinarum (0.66 ± 0.11, 2.12 ± 0.38, and 11.65 ± 0.84 mm in shell length, respectively) in the summer of 2008. Different size clams were starved for 7, 15, 30, 45, and 60 d, respectively, and followed by refeeding for 30 d. During the study, the water temperature ranged 26.2–28.4 °C, salinity 22–24‰, and pH 7.80–8.12. Compensatory growth occurred in the smallest size-group after 7 and 15 d of starvation, respectively. The point-of-no-return (PNR₅₀) was determined to be 18.7 d. However, no compensatory growth was noted in the medium size-group, and the PNR₅₀ for this group was 25.2 d. The complete compensatory growth was observed for the largest size-group following food depravation for 7 and 30 d, respectively. In the same group, over-compensatory growth occurred 15 d post-starvation. The PNR₅₀ for the largest size-group was 46.3 d. The survival rate of different groups decreased as the starvation time prolonged. To discuss the change in body biochemical composition of individuals in the process of starvation and refeeding, the biochemical composition of the largest group individuals at different stages was determined. There were no significant differences in moisture and ash concentrations of the largest size-group during starvation and refeeding (P > 0.05). The relative body protein content increased as the starvation period prolonged and the level returned to normal after refeeding. The lipid content of the clam at the end of starvation was significantly lower than the initial level (P < 0.05), and remained below the initial level at end of the refeeding period.     

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.     

Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and se- quenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indi- cated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and vali- dated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a pro- tein. The superposition of the Cα traces of cpn60 structure and predicted structure of leptospiral GroEL indi- cates good agreement of secondary structure elements with an RMSD value of 1.5 . Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.     

Molecular cloning,structure, and reactivity of the second bromoperoxidase from Ascophyllum nodosum

The sequence of bromoperoxidase II from the brown alga Ascophyllum nodosum was determined from a full length cloned cDNA, obtained from a tandem mass spectrometry RT-PCR-approach. The clone encodes a protein composed of 641 amino-acids, which provides a mature 67.4 kDa-bromoperoxidase II-protein (620 amino-acids). Based on 43% sequence homology with the previously characterized bromoperoxidase I from A. nodosum, a tertiary structure was modeled for the bromoperoxidase II. The structural model was refined on the basis of results from gel filtration and vanadate-binding studies, showing that the bromoperoxidase II is a hexameric metalloprotein, which binds 0.5 equivalents of vanadate as cofactor per 67.4 kDa-subunit, for catalyzing oxidation of bromide by hydrogen peroxide in a bi-bi-ping-pong mechanism (k_cat = 153 s⁻¹, 22 °C, pH 5.9). Bromide thereby is converted into a bromoelectrophile of reactivity similar to molecular bromine, based on competition kinetic data on phenol bromination and correlation analysis. Reactivity provided by the bromoperoxidase II mimics biosynthesis of methyl 4-bromopyrrole-2-carboxylate, a natural product isolated from the marine sponge Axinella tenuidigitata.     

Association between interleukin 15 receptor, alpha (IL15RA) polymorphism and Korean patients with ossification of the posterior longitudinal ligament

ObjectivesRecently, a number of evidences have been reported concerning the genetic factor involved in the development of ossification of the posterior longitudinal ligament (OPLL). The purpose of this study was to investigate single nucleotide polymorphisms (SNPs) of the interleukin 15 receptor, alpha (IL15RA) gene as a risk factor in Korean patients with OPLL.DesignTo investigate the genetic association, two coding SNPs (rs2296139, Thr73Thr; rs2228059, Asn182Thr) in IL15RA were genotyped in 166 OPLL patients and 230 control subjects. SNPStats, SNPAnalyzer, and Helixtree programs were used for association analysis.ResultsIn the present study, we found the association between a missense SNP (rs2228059) and the risk of OPLL in codominant (p = 0.0028, OR = 1.58, 95% CI = 1.17–2.14), dominant (p = 0.0071, OR = 1.82, 95% CI = 1.17–2.82), and recessive models (p = 0.036, OR = 1.79, 95% CI = 1.04–3.09). The frequency of rs2228059 allele was significantly associated with the susceptibility of OPLL (p = 0.0043, OR = 1.52, 95% CI = 1.14–2.02). After Bonferroni correction, the missense SNP (rs2228059, Asn182Thr) still had significant correlations (p = 0.0056 in codominant model; p = 0.0142 in dominant model; p = 0.0086 in allele analysis). Haplotype variation in IL15RA was associated with OPLL (global haplotype test, p = 0.025).ConclusionsThese results suggest that IL15RA polymorphism may be associated with the susceptibility of OPLL in Korean population.     

Root health evaluation of three alfalfa varieties in Kerquin sandy land

Without a robust and healthy root system, establishment, productivity, and persistence are compromised. Consequently, research on alfalfa root morphology and health is very important in development of technology for efficient improvement and production of alfalfa. The objectives of this study were to evaluate the root morphology and health of three alfalfa varieties, Algonquin, Golden Queen, and Yellow Flower and to determine relationships among root morphology traits and root health. Yields from these varieties ranged from 5.83 to 43.93 t/ha, total root length ranged from 215.17 to 708.89 mm, root surface area from 124.95 to 468.37 cm², volume from 3.24 to 57.72 cm³, and forks from 1.25 × 10³ to 10.54 × 10³, and tips from 0.65 × 10³ to 3.17 × 10³. Root infestation score was negatively correlated with yield (r = ?0.997, P < 0.01), and was positively correlated with all root morphology traits (r = 0.466–0.997, P < 0.01), and yield was negatively associated with root morphology traits (r = ?0.755 to ?0.998, p < 0.01) with the exception of root tips (r = 0.448, P < 0.01). Results from these analyses indicated that root infestation score was the lowest averaged over age of alfalfa stand in Algonquin. Yield in 2-year old stands was greater in Golden Queen compared to the other two cultivars.     

Involvement of endogenous hydrogen sulfide in cigarette smoke-induced changes in airway responsiveness and inflammation of rat lung

Hydrogen sulfide (H₂S), recently considered the third endogenous gaseous transmitter, may have an important role in systemic inflammation. We investigated whether endogenous H₂S may be a crucial mediator in airway responsiveness and airway inflammation in a rat model of chronic exposure to cigarette smoke (CS). Rats randomly divided into control and CS-exposed groups were treated with or without sodium hydrosulfide (NaHS, donor of H₂S) or propargylglycine (PPG, inhibitor of cystathionine-γ-lyase [CSE], an H₂S-synthesizing enzyme) for 4-month exposure. Serum H₂S level and CSE protein expression in lung tissue were higher, by 2.04- and 2.33-fold, respectively, in CS-exposed rats than in controls (P < 0.05). Exogenous administration of NaHS to CS-exposed rats alleviated airway reactivity induced by acetylcholine (Ach) or potassium chloride (KCl) by 17.4% and 13.8%, respectively, decreased lung pathology score by 32.7%, inhibited IL-8 and TNF- α concentrations in lung tissue by 34.2% and 31.4%, respectively, as compared with CS-exposed rats (all P < 0.05). However, blocking endogenous CSE with PPG in CS-exposed rats increased airway reactivity induced by Ach or KCl, by 24.1% and 24.5%, respectively, and aggravated lung pathology score, by 44.8%, as compared with CS-exposed rats (all P < 0.01). Incubation in vitro with NaHS, 1–3 mmol/L, relaxed rat tracheal smooth muscle precontracted by Ach or KCl. However, the NaHS-induced relaxation was not blocked by glibenclamide (10^?4 mol/L), L-NAME (10^?4 mol/L), or ODQ (1 μmol/L) or denudation of epithelium. Endogenous H₂S may have a protective role of anti-inflammation and bronchodilation in chronic CS-induced pulmonary injury.     

Circulating antibodies directed against “polycyclic aromatic hydrocarbon-like” structures in the sera of cancer patients

Background: An increase in immunoglobulin (Ig) A isotype directed against benzo(a)pyrene (BP) structure has previously been described in sera of cancer patients. In this study, new polycyclic aromatic hydrocarbon (PAH) conjugates were synthesized in order to more closely mimic the endogenous ligands of the cytosolic aryl hydrocarbon receptor (AhR). PAH [benzo(a)pyrene; 1,2-benzanthracene; dibenz[a,c]anthracene; 7,12-dimethylbenza[a]anthracene; benzo(ghi)perylene] were bound to protein carriers such as bovine serum albumin (BSA) via N-acetyl-cysteine (NAC). Methods: The levels of circulating antibodies (Abs) directed against PAH–NAC conjugates in the sera of cancer patients were evaluated using an Enzyme-Linked Immunosorbent Assay (ELISA) with these new conjugates. The avidity (IC₅₀) and specificity of these circulating Abs were assessed via competition experiments. Results: An increase in Ig directed against these PAH–NAC conjugates was found in the sera of cancer patients, irrespective of the state and stage of the tumors. These Ig were principally of the A isotype. Sera from cancer patients had significantly higher optical density (OD) ranges than the controls, p < 0.0001. The ELISA test for breast cancer (n = 155) and ovarian cancer (n = 62) identified 82% and 92% of positive patients, respectively. The percentage positive in the control group (n = 60) was around 5%. Moreover, competition experiments with the different PAH–NAC conjugates and NAC–BSA revealed an estimated avidity of 10^?6 M for the circulating IgA antibodies. Conclusions: The Abs discriminated between the different PAH–NAC conjugates and NAC–BSA. Therefore, these Abs recognize a carcinogenic PAH–NAC structure and not only a BP structure. These markers may be useful in the future for monitoring cancer evolution and recurrence.     

Structural insights into the dual substrate specificities of mammalian and Dictyostelium dihydropteridine reductases toward two stereoisomers of quinonoid dihydrobiopterin

Up to now, d-threo-tetrahydrobiopterin (DH₄, dictyopterin) was detected only in Dictyostelium discoideum, while the isomer l-erythro-tetrahydrobioterin (BH₄) is common in mammals. To elucidate the mechanism of DH₄ regeneration by D. discoideum dihydropteridine reductase (DicDHPR), we have determined the crystal structure of DicDHPR complexed with NAD⁺ at 2.16 Å resolution. Significant structural differences from mammalian DHPRs are found around the coenzyme binding site, resulting in a higher K_m value for NADH (K_m = 46.51 ± 0.4 μM) than mammals. In addition, we have found that rat DHPR as well as DicDHPR could bind to both substrates quinonoid-BH₂ and quinonoid-DH₂ by docking calculations and have confirmed their catalytic activity by in vitro assay.Structured summary of protein interactionsDHPR binds to DHPR by X-ray crystallography (View interaction)     

Polymorphisms in MSH2 gene and risk of gastric cancer, and interactions with lifestyle factors in a Chinese population

Background: Although polymorphisms in DNA mismatch repair (MMR) gene MSH2 have been associated with risks of many cancers, little is known about their etiology role in gastric cancer (GC) and the potential interacting role with lifestyle factors known to damage DNA. Methods: A population-based study was conducted in 3 counties (Jintan, Taixing and Huaian) of Jiangsu Province, the high-risk areas of GC in China. We investigated the association of polymorphisms IVS12?6T>C and IVS10+12G>A in MSH2 gene with the risk of GC and the potential gene–lifestyle interaction. Results: The risk of GC was found to be associated with the IVS12?6C allele (CC vs TT, OR = 2.34, 95% CI: 1.17–4.71) and IVS10+12A allele (GA or AA vs GG, OR = 1.55, 95% CI: 1.14–2.21; and GA vs GG, OR = 1.51, 95% CI: 1.04–2.17). Stratified analysis indicated that an increased risk of GC also was observed in: suspected familial subjects carrying the IVS12?6T>C (OR = 1.68, 95% CI: 1.27–2.66) or IVS10+12G>A (OR = 2.57, 95% CI: 1.53–4.10); or younger subjects carrying the IVS12?6T>C (OR = 2.15, 95% CI: 1.24–3.91) or IVS10+12G>A (OR = 2.23, 95% CI: 1.20–4.33); or male subjects carrying the IVS10+12G>A (OR = 1.64; 95% CI: 1.10–2.54). Furthermore, the combined IVS12?6CC and IVS10+12AA genotypes also significantly increased the risk of GC (OR = 2.12, 95% CI: 1.22–3.66). Statistically significant interactions were observed between: IVS10+12G>A and drinking, high pickled food or fried food intake (OR = 2.32; 95% CI: 1.43–3.78, OR = 2.55; 95% CI: 1.48–4.21 and OR = 2.88; 95% CI: 1.70–4.94, respectively); and IVS12?6T>C and high pickled food intake or fried food intake (OR = 2.65; 95% CI: 1.62–4.47 and OR = 2.48; 95% CI: 1.42–4.13, respectively). Conclusion: The IVS10+12G>A and IVS12?6T>C polymorphisms in MSH2 gene appear to be associated with risk of GC in this Chinese population. Risk for GC, stratified by related genotypes, was further modified by drinking, high pickled food or fried food intake. Larger prospective studies are needed to confirm these findings.