Vero细胞批式培养中的氨基酸代谢

用高效液相色谱(HPLC)测定了批式培养的Vero细胞培养液中氨基酸的变化,结果表明Vero细胞在生长过程中需要消耗12种氨基酸,同时生成4种氨基酸, Vero细胞在生长期吸收Leu,Thr,Ile,Lys,Phe和Tyr较多,而在静止期这些氨基酸却消耗较少.     

Amino acids as carbon,energy and nitrogen sources for Penicillium camembertii

Three groups of amino acids were previously characterized on their ability to be assimilated as carbon source by Penicillium camembertii. In view of a deeper understanding of their metabolic behaviour, growth of P. camembertii on glucose, the limiting substrate, and an amino acid was examined in batch culture. Amino acids from the first group (Cys, His, Lys, Met, Trp and Val) are convenient nitrogen sources, but cannot be assimilated as carbon sources. However, they are also dissimilated, namely used for energy supply by oxidation into CO₂, during stationary phase, after glucose depletion, as shown for lysine; and the corresponding nitrogen was released as ammonium. Growth exhibited diauxic behaviour for the second group of amino acids (Arg, Leu), since they can be assimilated as carbon sources, in addition to their assimilation as nitrogen sources, but only after glucose depletion, as shown for arginine. A clear differentiation between the assimilated and the dissimilated carbon was demonstrated for the third group of amino acids (Ala, Asp, Glu, Gly, Pro, Ser, Thr); it was shown that the carbon from glutamic acid was assimilated, while the carbon from glucose was dissimilated. Copyright © 2006 Society of Chemical Industry     

Carbon assimilation and dissimilation during growth of Geotrichum candidum on amino acids and glucose

BACKGROUND: This work examines the metabolic behaviour of amino acids during Geotrichum candidum growth, in the presence of a primary carbon source like glucose. Amino acids were characterized based on their carbon assimilation and dissimilation by G. candidum, in the presence of glucose as the limiting substrate. RESULTS: The first group (Cys, His, Phe, Thr and Trp) was only used as nitrogen sources by G. candidum, with glucose being the carbon and energy source. Glucose repression was shown for the rest of the amino acids, since only after glucose depletion amino acids from the second group (Gly, Lys, Met, Val) were dissimilated for energy supply by oxidation into CO₂, while those from the third group (Ala, Arg, Asp, Glu, Leu, Pro and Ser) were assimilated as carbon sources (and additionally used as nitrogen sources), leading to a diauxic growth. CONCLUSION: This energy‐saving response was not previously shown for the second fungus involved in ripening of soft white cheese—P. camembertii—leading to simultaneous use of some amino acids and glucose as carbon and energy sources, and hence lower growth rates than those recorded during G. candidum growth. Copyright © 2007 Society of Chemical Industry     

Random mutagenesis into the conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes

We analyzed the role played by the conserved Gly154, a constituent of the P1 substrate-binding pocket of Bacillus subtilis subtilisin E, in the catalytic properties of the protease. Using an Escherichia coli expression system, the termination codon at position 154 in subtilisin E was first introduced to abolish the catalytic activity through truncation of the C-terminus from amino acid residues 154-275. We then attempted to obtain revertants with substitutions of various amino acids at position 154 by the polymerase chain reaction using a mixture of oligonucleotides. In addition to the Gly residue (wild-type), six amino acid substitutions (Ala, Arg, Leu, Phe, Pro and Thr) gave caseinolytic activity. When assayed with synthetic peptide substrates, most of the revertants showed a considerable decrease in specific activity and a P1 specificity similar to that of the wild-type enzyme. An Ala154 mutant purified from the periplasmic space in E. coli, however, resulted in an up to 2.3-fold preference for Val rather than Pro as a P2 substrate relative to the wild-type. Further, a significant 2-10-fold increase in the catalytic efficiency occurred in the Gly127Ala plus Gly154Ala combination variant, relative to the single Gly127Ala variant, without any change in the restricted specificity. The kinetic data and molecular modeling analysis demonstrate the important role of position 154 in the catalytic efficiency as well as in the substrate specificity of subtilisin E.      

A Synthetic MUC1 Glycopeptide Bearing βGalNAc‐Thr as a Tn Antigen Isomer Induces the Production of Antibodies against Tumor Cells

This study presents the synthesis of the novel protected O‐glycosylated amino acid derivatives 1 and 2 , containing βGalNAc‐SerOBn and βGalNAc‐ThrOBn units, respectively, as mimetics of the natural Tn antigen (αGalNAc‐Ser/Thr), along with the solid‐phase assembly of the glycopeptides NHAcSer‐Ala‐Pro‐Asp‐Thr[αGalNAc]‐Arg‐Pro‐Ala‐Pro‐Gly‐BSA ( 3 ‐BSA) and NHAcSer‐Ala‐Pro‐Asp‐Thr[βGalNAc]‐Arg‐Pro‐Ala‐Pro‐Gly‐BSA ( 4 ‐BSA), bearing αGalNAc‐Thr or βGalNAc‐Thr units, respectively, as mimetics of MUC1 tumor mucin glycoproteins. According to ELISA tests, immunizations of mice with βGalNAc‐glycopeptide 4 ‐BSA induced higher sera titers (1:320 000) than immunizations with αGalNAc‐glycopeptide 3 ‐BSA (1:40 000). Likewise, flow cytometry assays showed higher capacity of the obtained anti‐glycopeptide 4 ‐BSA antibodies to recognize MCF‐7 tumor cells. Cross‐recognition between immunopurified anti‐βGalNAc antibodies and αGalNAc‐glycopeptide and vice versa was also verified. Lastly, molecular dynamics simulations and surface plasmon resonance (SPR) showed that βGalNAc‐glycopeptide 4 can interact with a model antitumor monoclonal antibody (SM3). Taken together, these data highlight the improved immunogenicity of the unnatural glycopeptide 4 ‐BSA, bearing βGalNAc‐Thr as Tn antigen isomer.     

Pilot Study on Acute Effects of Pharmacological Intraperitoneal L-Homoarginine on Homeostasis of Lysine and Other Amino Acids in a Rat Model of Isoprenaline-Induced Takotsubo Cardiomyopathy

L-Arginine:glycine amidinotransferase (AGAT) catalyzes the formation of L-homoarginine (hArg) and L-ornithine (Orn) from L-arginine (Arg) and L-lysine (Lys): Arg + Lys ↔ hArg + Orn; equilibrium constant K_hArg. AGAT also catalyzes the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly ↔ GAA + Orn; equilibrium constant K_GAA. In humans, pharmacological hArg is metabolized to Lys. Low circulating and low excretory concentrations of hArg are associated with worse outcomes and mortality in the renal and cardiovascular systems. The metabolism and pharmacology of hArg have been little investigated. In the present study, we investigated the effects of pharmacological hArg (i.p., 0, 20, 220, 440 mg/kg at time point 0 min) on amino acids homeostasis in a rat model of isoprenaline-induced takotsubo cardiomyopathy (i.p., 50 mg/kg at time point 15 min). We measured by gas chromatography-mass spectrometry free and proteinic amino acids, as well as the polyamines putrescine and spermidine in the heart, lung, kidney, and liver of ten rats sacrificed at various time points (range, 0 to 126 min). hArg administration resulted in multiple changes in the tissue contents of several free and proteinic amino acids, as well as in the putrescine-spermidine molar ratio, an indicator of polyamines catabolism. Our results suggest that Lys and Arg are major metabolites of pharmacological hArg. Kidneys and heart seem to play a major metabolic role for hArg. Circulating Lys does not change over time, yet there is a considerable interchange of free Lys between organs, notably kidney and heart, during the presence of isoprenaline in the rats (time range, 15 to 90 min). Antidromic changes were observed for K_hArg and K_GAA, notably in the heart in this time window. Our study shows for the first time that free hArg and sarcosine (N-methylglycine) are positively associated with each other. The acute effects of high-dosed hArg administration and isoprenaline on various amino acids and on AGAT-catalyzed reaction in the heart, lung, kidney, and liver are detailed and discussed.     

Modeling of aqueous amino acid and polypeptide solutions with PC-SAFT

Vapor pressures, liquid densities and solubilities of aqueous amino acid and oligopeptide solutions were modeled with an equation of state based on PC-SAFT. The amino acids glycine (Gly), alanine (Ala), serine (Ser), proline (Pro), and valine (Val) as well as the oligopeptides (Gly)_n=1−5, and Ala(Gly)_n=1−4 were considered. Five pure-component model parameters for each amino acid were fitted using experimental densities, vapor pressures, and solubility data. The oligopeptides were treated as co-polymers built up by the respective amino acids. Thus, no additional parameters had to be fitted for the prediction of densities and vapor–liquid behavior of their aqueous solutions besides the segment number. The model is able to excellently reproduce the experimental densities, vapor pressures and solubility data of aqueous amino acid and oligopeptide solutions.     

重组人Tau蛋白的表达、纯化及鉴定

目的 重组人Tau蛋白的诱导表达和纯化。方法 将携带 Tau蛋白基因的工程菌株经IPTG诱导表达后,通过超声破碎、硫酸按盐析、离子交换层析、电洗脱等方法纯化Tau蛋白,以SDS-PAGE、非变性PAGE、Westem blot和N末端氨基酸测定等方法进行鉴定。结果 纯化Tau蛋白的得率为28.5％,在SDS-PAGE和非变性PAGE上呈均一的蛋白条带,其相对分子质量约为59000,N末端5个氨基酸测定结果为NH2-Met-Ala-Glu-Pro-Arg-。结论 本实验采用纯化重组人Tau蛋白方法是可行的。     

Branched-chain Amino Acid Biosensing Using Fluorescent Modified Engineered Leucine/Isoleucine/Valine Binding Protein

A novel fluorescence sensing system for branched-chain amino acids (BCAAs) was developed based on engineered leucine/isoleucine/valine-binding proteins (LIVBPs) conjugated with environmentally sensitive fluorescence probes. LIVBP was cloned from Escherichia coli and Gln149Cys, Gly227Cys, and Gln254Cys mutants were generated by genetic engineering. The mutant LIVBPs were then modified with environmentally sensitive fluorophores. Based on the fluorescence intensity change observed upon the binding of the ligands, the MIANS-conjugated Gln149Cys mutant (Gln149Cys-M) showed the highest and most sensitive response. The BCAAs Leu, Ile, and Val can each be monitored at the sub-micromolar level using Gln149Cys-M. Measurements were also carried out on a mixture of BCAFAs and revealed that Gln149Cys-M-based measurement is not significantly affected by the change in the molar ratio of Leu, Ile and Val in the sample. Its high sensitivity and group-specific molecular recognition ability make the new sensing system ideally suited for the measurement of BCAAs and the determination of the Fischer ratio, an indicator of hepatic disease involving metabolic dysfunction.     

Model polypeptide of mussel adhesive protein. I. Synthesis and adhesive studies of sequential polypeptides (X‐Tyr‐Lys)n and (Y‐Lys)n

The sequential polytripeptides and polydipeptides, (X‐Tyr‐Lys)_n, (XGly, Ala, Pro, Ser, Leu, Ile, Phe), (Y‐Lys)_n, (YGly, Tyr), and (Gly‐Tyr)_n, which imitate a mussel adhesive protein, have been synthesized. The molecular weights of the polypeptides were estimated to be 7,200 ∼ 13,400 (19 ∼ 42 repeating units), and the polypeptides were found to have satisfactory amino acid sequences. The polypeptides were crosslinked by tyrosinase, and the optimal pH in the crosslinking reaction was 7.4 in the case of the polytripeptide, (Gly‐Tyr‐Lys)_n. The optimal tyrosinase amount for the adhesive strength of (Gly‐Tyr‐Lys)_n was 0.34 unit/mg (polypeptide) at pH 7.4. The shear adhesive strength of the polytripeptide increased with an increase in the polytripeptide concentration, and was not influenced by the third amino acid, X. The shear adhesive strengths of polytripeptides (X‐Tyr‐Lys)_n were equal to one of the synthetic polydecapeptides, (Ala‐Lys‐Pro‐Ser‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys)_n and (Gly‐Pro‐Lys‐Thr‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys)_n which were the model polydecapeptides for blue mussel and Californian mussel, respectively. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 929–937, 2000     

A Combination of 3D-QSAR, Molecular Docking and Molecular Dynamics Simulation Studies of Benzimidazole-Quinolinone Derivatives as iNOS Inhibitors

Inducible Nitric Oxide Synthase (iNOS) has been involved in a variety of diseases, and thus it is interesting to discover and optimize new iNOS inhibitors. In previous studies, a series of benzimidazole-quinolinone derivatives with high inhibitory activity against human iNOS were discovered. In this work, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking and molecular dynamics (MD) simulation approaches were applied to investigate the functionalities of active molecular interaction between these active ligands and iNOS. A QSAR model with R² of 0.9356, Q² of 0.8373 and Pearson-R value of 0.9406 was constructed, which presents a good predictive ability in both internal and external validation. Furthermore, a combined analysis incorporating the obtained model and the MD results indicates: (1) compounds with the proper-size hydrophobic substituents at position 3 in ring-C (R₃ substituent), hydrophilic substituents near the X₆ of ring-D and hydrophilic or H-bond acceptor groups at position 2 in ring-B show enhanced biological activities; (2) Met368, Trp366, Gly365, Tyr367, Phe363, Pro344, Gln257, Val346, Asn364, Met349, Thr370, Glu371 and Tyr485 are key amino acids in the active pocket, and activities of iNOS inhibitors are consistent with their capability to alter the position of these important residues, especially Glu371 and Thr370. The results provide a set of useful guidelines for the rational design of novel iNOS inhibitors.     

Transport Behavior of Basic Amino Acids through an Organic Liquid Membrane System

Abstract

The transport behavior of basic amino acids (BAA), such as arginine (Arg), histidine (His), and ornithine (Orn), through an organic liquid membrane system (LMS) was investigated. The LMS was composed of two aqueous phases (Phases I and II) separated by an organic phase of chloroform containing sodium di-(2-ethylhexyl) sulfosuccinate (Aerosol OT, AOT). The amount of BAA that moved from Phase I at pH 3 into the organic phase increased with increasing AOT concentration (2–10 mM). The relative amount of extracted BAA was in the following order: Arg > His > Orn. On the other hand, the release of BAA from the organic phase into Phase II at pH 10 did not depend upon their amount in the organic phase. Arg was difficult to release. The relative amount of released BAA was in the following order: Arg = His > Orn. BAA were extracted from Phase I at pH 5 into the organic phase containing 4 mM AOT because they exist as cationic species. Other amino acids possessing nonionic residues were untransportable under these conditions except leucine, tryptophan, and phenylalanin, which have highly hydrophobic residues. However, they were transportable in their cationic forms at pH 1. These transport phenomena are essentially controlled by the interaction of the anionic group of AOT and a cationic form. These results suggested that BAA can be separated from most amino acids under an appropriate pH by using AOT.     

1,6‐hexanediol diacrylate‐crosslinked polystyrene: Preparation,characterization, and application in peptide synthesis

A copolymer of 1,6‐hexanediol diacrylate (HDODA) and styrene was prepared by a suspension polymerization method. The resin was characterized by infrared and carbon‐13 cross‐polarization magic‐angle spin (¹³C CP‐MAS) spectroscopy. The topology of the resin was examined by scanning electron microscopy (SEM). The polymer swells extensively in common solvents used for peptide synthesis. The resin exhibited chemical stability even in neat trifluoroacetic acid. The applicability of the new resin was demonstrated by synthesis of Val‐Ala‐Val‐Ala‐Ala‐Gly, Gln‐Val‐Gly‐Gln‐Val‐Glu‐Leu‐Gly, and Val‐Gln‐Ala‐Ala‐Ile‐Asp‐Tyr‐Ile‐Asn‐Gly. Comparative synthetic studies showed that the new resin is superior to divinylbenzene (DVB)‐based resin in the case of the synthesis of hydrophobic peptide sequences. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 87: 1290–1296, 2003     

Computational Prediction of O-linked Glycosylation Sites That Preferentially Map on Intrinsically Disordered Regions of Extracellular Proteins

O-glycosylation of mammalian proteins is one of the important posttranslational modifications. We applied a support vector machine (SVM) to predict whether Ser or Thr is glycosylated, in order to elucidate the O-glycosylation mechanism. O-glycosylated sites were often found clustered along the sequence, whereas other sites were located sporadically. Therefore, we developed two types of SVMs for predicting clustered and isolated sites separately. We found that the amino acid composition was effective for predicting the clustered type, whereas the site-specific algorithm was effective for the isolated type. The highest prediction accuracy for the clustered type was 74%, while that for the isolated type was 79%. The existence frequency of amino acids around the O-glycosylation sites was different in the two types: namely, Pro, Val and Ala had high existence probabilities at each specific position relative to a glycosylation site, especially for the isolated type. Independent component analyses for the amino acid sequences around O-glycosylation sites showed the position-specific existences of the identified amino acids as independent components. The O-glycosylation sites were preferentially located within intrinsically disordered regions of extracellular proteins: particularly, more than 90% of the clustered O-GalNAc glycosylation sites were observed in intrinsically disordered regions. This feature could be the key for understanding the non-conservation property of O-glycosylation, and its role in functional diversity and structural stability.     

钙盐和氨基酸对味精等电母液模拟料双极膜电渗析过程膜污染的影响

采用恒压分批操作方式考察了味精等电母液模拟料液中钙盐和氨基酸对双极膜电渗析过程膜污染的影响.结果表明,在处理含氨基酸和Ca2+的盐溶液时,碱室溶液中检测到各氨基酸含量为Glu＞Ala＞Gly＞Thr＞Lys,它们与料液中NH4+同步迁移至碱室,迁移量与料液中氨基酸浓度成正比;料液中Ca2+是引起碱室侧阳膜面出现固形污染...     

Activity of linked HIV-1 proteinase dimers containing mutations in the active site region

Mutations were introduced into the active site triplet (Asp–Thr–Gly)of one or both subunits of a linked dimer of human immunodeficiencyvirus type 1 proteinase. Mutation of Thr to Ser in one or bothsubunits did not alter the activity of the enzyme substantially,whereas its mutation to Asn in one subunit caused a dramaticdecrease in catalytic efficiency. Mutation of Gly to Val inone subunit also yielded an enzyme with very low activity. Theenzymes containing Thr Asn and Gly Val mutations in both subunitsresulted in inactive enzymes, based on their inability to self-processand on assay with an oligopeptide substrate. The dramatic decreasein enzyme efficiency of the mutants was interpreted using molecularmodels of the enzymes.     

Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis

Xylose isomerases (XIs) from Thermoanaerobacterium thermosulfurigenes(TTXI) and Thermotoga neapolitana (TNXI) are 70.4% identicalin their amino acid sequences and have a nearly superimposablecrystal structure. Nonetheless, TNXI is much more thermostablethan TTXI. Except for a few additional prolines and fewer Asnand Gln residues in TNXI, no other obvious differences in theenzyme structures can explain the differences in their stabilities.TNXI has two additional prolines in the Phe59 loop (Pro58 andPro62). Mutations Gln58Pro, Ala62Pro and Gln58Pro/Ala62Pro inTTXI and their reverse counterpart mutations in TNXI were constructedby site-directed mutagenesis. Surprisingly, only the Gln58Promutation stabilized TTXI. The Ala62Pro and Gln58Pro/Ala62Promutations both dramatically destabilized TTXI. Analysis of thethree-dimensional (3D) structures of TTXI and its Ala62Pro mutantderivative showed a close van der Waal's contact between Pro62-C     

Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae)

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H‐Val‐Asn‐Trp‐Lys‐Lys‐Val‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH₂ (LL‐I), H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Ala‐Lys‐NH₂ (LL‐II) and H‐Val‐Asn‐Trp‐Lys‐Lys‐Ile‐Leu‐Gly‐Lys‐Ile‐Ile‐Lys‐Val‐Val‐Lys‐NH₂ (LL‐III). These lasioglossins exhibited potent antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of α‐helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved α‐helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N‐terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL‐III peptide. C‐terminal deamidation of LL‐III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL‐III and the observed NOE contacts indicated the possible formation of a bifurcated H‐bond between hydrogen from the Lys15 CONH peptide bond and one H of the C‐terminal CONH₂ to the Ile11 oxygen atom. Such interactions cannot form with C‐terminal esterification.     

Grafting of oligopeptide on poly(aminostyrene)s and characterization of the polymers

Graft copolymers that have oligopeptides (OP) as graft molecules were prepared through the coupling reaction of the carboxylic acids of OP with the amino groups of poly(aminostyrene)s (PAS). The three OPs are Boc‐Gly, Z‐Gly‐Pro, and Z‐Gly‐Pro‐Leu‐Gly‐Pro, where Boc and Z are, respectively, the t‐butyloxycarbonyl group and benzyloxycarbonyl group as protective groups on nitrogen. The two PASs are poly(4‐vinylphenylamine) and poly(N‐isopropyl‐4‐vinylbenzylamine). These polymers that have narrow molecular weight distributions were prepared via anionic living polymerization. The coupling reaction to form the peptide bonds was mediated by dicyclohexylcarbodiimide in a mixed solvent of N,N′‐dimethylformamide and methyl chloride. The degree of grafting (DOG) on PAS was determined from the ¹H‐NMR spectrum. The dependence of the reaction time (0–8 h) on the DOG, the dependence of the reaction temperature (0–45°C) on the DOG, and the dependence of the molar ratio of OP to the amino group of PAS (1–4 times) on the DOG were studied. By alternating the reaction time and the molar ratio, the DOG values of PAS could be controlled in the range from 0 to 100%. DOG seems to be independent of the molecular weight of OP and the degree of basicity of PAS. The contact angle of the resultant graft copolymers was measured and the preliminary nonthrombogenic test was also performed. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 1558–1567, 2000     

CARD15/NOD2, CD14 and Toll-like 4 Receptor Gene Polymorphisms in Saudi Patients with Crohn's Disease

Crohn's disease (CD) is a multifactorial disease with a genetic component and an observed association with genes related to the innate immune response. Polymorphisms in the CARD15/NOD2 gene, in addition to functional variants of the toll-like receptor-4 (TLR4) and CD14 genes, have been associated with the development of Crohn's disease. There is no information about the frequency of these polymorphisms in the Saudi population. We examined the frequency of the three major CARD15/NOD2 risk alleles (Leu1007fsinsC, Arg702Trp, and Gly908Arg) and the TLR4 (Thr399Il) polymorphism as well as a functional polymorphism in the promoter of the CD14-159C/T in 46 Saudi CD patients and 50 matched controls. Genotyping was performed by allele-specific PCR or by restriction fragment length polymorphism (PCR-RFLP) analysis. The mutant genotype frequencies of the Leu1007fsinsC, Arg702Trp and Gly908Arg in the patient group were 6.5, 21.7 and 6.5%, respectively, compared with frequencies of 0, 4 and 2%, respectively, in the control group. There were 15 patients who carried the mutant alleles for all three CARD15/NOD2 variants, Leu1007fsinsC, Arg702Trp and Gly908Arg, while none of the control candidates carried the three alleles. This genetic study provides evidence that the three major CARD15/NOD2 variant alleles and the CD14 -159C/T polymorphism are associated with Crohn's disease (CD) susceptibility in the Saudi population; however, there is no evidence that the TLR4 (Thr399Il) or CARD15/NOD2 polymorphisms can be considered risk factors for Crohn's disease.