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PubMed1995

Ostrich trypsinogen: purification, kinetic properties and characterization of the pancreatic enzyme.

Trypsinogen is a serine protease zymogen (EC.3.4.21.4) which has proved to be of key significance in a family of about 20 structurally and functionally related pancreatic digestive enzymes. This study was an endeavour to isolate, purify and characterize a stable form of ostrich trypsinogen, which has thus far not yet been accomplished. Trypsinogen (anionic) was isolated and purified by alkaline extraction of pancreatic acetone powder, followed by Toyopearl DEAE 650M, hydroxylapatite and LBTI-Sepharose affinity chromatography. The enzyme was chemically physically and kinetically characterized, using amidase and esterase activity and spectrofluorometric determinations. Effects of CaCl2 and pH, among others, were examined. Purification of homogeneous anionic ostrich trypsinogen was achieved. Immunochemical analysis and spectrofluorometric reaction with sulphonyl-Ala-Ala-Pro-Arg-7-amino-4-methylcoumarin indicated trypsin-free ostrich trypsinogen, with an average Mr of 23,016 and a pI of 4.93. N-terminal sequence data revealed an unique activation peptide sequence, VPGDADDDK. Certain concentrations of Ca2+ enhanced trypsinogen activation, whilst others appeared to have the opposite effect. The kcat/Km values obtained at different pHs, using N alpha-benzoyl-DL-arginine-p-nitroanilide, p-toluenesulphonyl-arginine-methylester and p-toluenesulphonyl-lysine-methylester, followed the pH profile activity trend closely, with maximum catalytic activity at about pH 8 for both ostrich and bovine activated trypsinogen. Ostrich trypsin has significantly higher amidase activity than bovine trypsin, while esterase activities of the two enzymes have an inverse ratio. Kinetic pKa values were 7.2 and 7.4 for ostrich and bovine activated trypsinogens, respectively. The existence of ostrich trypsinogen in a now homogeneous stable form, free of autocatalytic inducing impurities, together with its characterization scenario will hopefully make a significant contribution to the field of comparative biochemistry. This study also confirms that ostrich trypsinogen is closely related to its serine protease counterparts.

Amino Acid Sequence↗

PubMed2025

Experimental strategy for characterization of novel TnpB orthologs.

TnpB proteins encoded in IS200/IS605 and IS607 mobile genetic elements are among the most widespread proteins in the microbial world. They function as RNA-guided DNA nucleases that play a critical role in transposon proliferation and are the predecessors of CRISPR-Cas12 effector proteins of the type V CRISPR-Cas family. Small size of TnpB nucleases makes them an attractive alternative for larger Cas9 and Cas12 proteins in genome editing applications. However, only a small fraction of TnpB nucleases characterized to date are active in human cells, highlighting the need to identify new TnpB variants that can function as genome editors. Here, we present an experimental pipeline for the characterization of TnpB proteins by combining in silico analysis with in vitro assays. To validate it we determined guide RNA and identified TAM for a set of TnpB orthologs. The proposed workflow can be employed for rapid screening and characterization of the huge TnpB protein family to identify novel TnpB variants that might expand the genome editing toolbox.

Humans↗

PubMed2004

Characterizing high-affinity antigen/antibody complexes by kinetic- and equilibrium-based methods.

Two biophysical methods, Biacore and KinExA, were used to kinetically and thermodynamically characterize high-affinity antigen/antibody complexes. Three to five independent experiments were performed on each platform with three different antigen/antibody complexes possessing nanomolar to picomolar equilibrium dissociation constants. By monitoring the dissociation phase on Biacore for 4 h, we were able to measure dissociation rate constants (kd) on the order of 1 x 10(-5)s(-1). To characterize high-affinity interactions by KinExA, samples needed to be equilibrated for up to 35 h to reach equilibrium. In the end, we show that similar kinetic rate constants and affinities were determined with both solution-phase and solid-phase methodologies. These results help further validate both interaction technologies and illustrate their suitability for characterizing extremely high-affinity interactions.

Animals↗

PubMed2006

High-resolution characterization of antibody fragment/antigen interactions using Biacore T100.

A Biacore T100 optical biosensor was used to characterize the binding kinetics of a panel of antigen binding fragments (Fabs) directed against the PcrV protein from Pseudomonas aeruginosa. PcrV protein forms part of the type III secretion system complex of this opportunistic pathogen. We demonstrate that the biosensor response data for each Fab collected from three different surface densities of the antigen could be fit globally to a simple 1:1 interaction model. Importantly, we found that the Fabs with the slowest dissociation rate provided the best protection in cell cytotoxicity studies. To further characterize the Fab interactions, binding data were automatically acquired at different temperatures and under different buffer conditions. The comprehensive characterization of these Fabs shows how Biacore T100 can be used to complement protein therapeutic discovery programs from basic research to the selection of therapeutic candidates.

Animals↗

PubMed2006

Characterization and development of a peptide substrate-based phosphate transfer assay for the human vascular endothelial growth factor receptor-2 tyrosine kinase.

Vascular endothelial growth factor (VEGF), a critical regulator in angiogenesis, exerts its angiogenic effect via binding to its receptor, VEGF receptor-2 tyrosine kinase (VEGFR2) or kinase insert domain-containing receptor (Kdr), on the surface of endothelial cells. Kdr-mediated signaling plays an important role in the proliferation, migration, differentiation, and survival of endothelial cells. Therefore, the inhibition of this signaling pathway represents a promising therapeutic approach for the discovery of novel anticancer agents by destabilizing the progression of solid tumors via abrogating tumor-induced angiogenesis. To explore Kdr as an anticancer target and further characterize the enzyme, we purified a cytoplasmic domain of human Kdr (Kdr-CD) and characterized its autophosphorylation activity. We also designed and synthesized peptides containing amino acid sequences corresponding to the autophosphorylation sites of Kdr and developed a simple, robust, high-throughput assay for measuring the phosphate transfer activity of the enzyme. This assay was validated by the experiments showing that the phosphate transfer activity of the purified Kdr-CD required Mg2+ or Mn2+ and preactivation by adenosine 5'-triphosphate (ATP) and was inhibited by known Kdr inhibitors. Using this assay, we examined effects of Mg2+ and Mn2+ on the enzyme activity; optimized the concentrations of Kdr-CD, peptide and ATP substrates, and metal ions in the assay; and determined the kinetic properties of the enzyme for the peptide and ATP as well as IC50 values of two known Kdr inhibitors. Thus, the results of these studies have validated the utilities of this assay for biochemical characterizations of the enzyme and its inhibitors. This approach of designing peptides corresponding to the autophosphorylation sites of Kdr as substrates for the enzyme has general practical implications to other kinases.

Adenosine Diphosphate↗

PubMed2005

Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase.

A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.

Aminohydrolases↗

PubMed2005

Microarray expression profiling and functional characterization of AtTPS genes: duplicated Arabidopsis thaliana sesquiterpene synthase genes At4g13280 and At4g13300 encode root-specific and wound-inducible (Z)-gamma-bisabolene synthases.

The Arabidopsis thaliana genome contains at least 32 terpenoid synthase (AtTPS) genes [Aubourg et al., Mol. Genet. Genom. 267 (2002) 730] a few of which have recently been characterized. Based on hierarchical cluster analysis of AtTPS gene expression, measured by microarray profiling and validated with published expression data, we identified two groups of predominantly root expressed AtTPS genes containing five members with previously unknown biochemical functions (At4g13280, At4g13300, At5g48110, At1g33750, and At3g29410). Among the root expressed AtTPS genes, a pair of tandem-organized genes, At4g13280 (AtTPS12) and At4g13300 (AtTPS13), shares 91% predicted amino acid identity indicating recent gene duplication. Bacterial expression of cDNAs and enzyme assays showed that both At4g13280 and At4g13300 encode sesquiterpene synthases catalyzing the conversion of farnesyl diphosphate to (Z)-gamma-bisabolene and the additional minor products E-nerolidol and alpha-bisabolol. Expression of beta-glucuronidase (GUS) reporter gene fused to upstream genomic regions of At4g13280 or At4g13300 showed constitutive promoter activities in the cortex and sub-epidermal layers of Arabidopsis roots. In addition, highly localized promoter activities were found in leaf hydathodes and flower stigmata. Mechanical wounding of Arabidopsis leaves induced local expression of At4g13280 and At4g13300. The functional characterization of At4g13280 gene product AtTPS12 and At4g13230 gene product AtTPS13 as (Z)-gamma-bisabolene synthases, together with the recent characterization of two flower-specific AtTPS [At5g23960 and At5g44630; Tholl et al., Plant J. 42 (2005) 757], concludes the biochemical functional annotation of all four predicted Arabidopsis sesquiterpene synthase genes. Our data suggest biological functions for At4g13280 and At4g13300 in the rhizosphere with additional roles in aerial plant tissues.

Alkyl and Aryl Transferases↗

PubMed2005

Lack of herbal supplement characterization in published randomized controlled trials.

PURPOSE: Herbal supplements in the United States and abroad have poor quality control and high content variability. We assessed the extent to which recently published randomized controlled trials of herbal supplements characterized and verified the content of the supplement under study. METHODS: We identified all MEDLINE-indexed English language randomized controlled trials evaluating single-herb preparations of echinacea, garlic, ginkgo, saw palmetto, or St. John's wort that were published between January 1, 2000, and February 9, 2004. From each article we extracted information characterizing the herbal supplement studied. RESULTS: Of 81 randomized controlled trials meeting inclusion criteria, 12 (15%) reported performing tests to quantify actual contents, and 3 (4%) provided adequate data to compare actual with expected content values of at least one chemical constituent. In those 3 studies, actual content varied between 80% and 113% of expected values. Studies of higher overall quality (Jadad score > or =3) performed testing somewhat less frequently (5/54; 9%) than those with lower Jadad scores (7/27; 26%) (P = .09). CONCLUSION: Documented characterization of herbal supplements in published randomized controlled trials is inadequate. Investigators may be unaware of the extent to which herbal quality-control issues may detract from the value of otherwise well-designed clinical trials. The scientific and clinical utility of future herbal randomized controlled trials would be enhanced if authors provided evidence that the herbal products studied were of high quality.

Drug Contamination↗

PubMed2004

Characterization of endoleaks by dynamic computed tomographic angiography.

BACKGROUND: Current imaging modalities may not be able to detect endoleaks, differentiate between type II and type III, or localize inflow and outflow sources. We describe a new technique that can characterize endoleaks to guide secondary intervention. METHODS: One hundred four patients with Zenith (Cook, Inc.) endograft repair of abdominal aortic aneurysms (AAAs) were monitored by serial computed tomographic angiography (CTA). Endoleaks were evaluated with a dynamic CTA using a stationary table position, 24-mm beam collimation, and continuous scanning over 30 to 40 seconds to create a cine. RESULTS: Twelve patients (12%) had endoleaks that persisted or appeared more than 30 days post-deployment. Five patients in whom the standard CT surveillance protocol could not differentiate type II versus type III endoleaks underwent dynamic CTA. This technique accurately characterized the endoleaks and localized inflow and outflow branches to guide the subsequent successful secondary interventions. CONCLUSIONS: Dynamic CTA is a useful technique to evaluate endoleaks for characterization and precise localization to guide secondary interventional therapy.

Aged↗

PubMed2006

Characterization of hepatic glutathione S-transferases in coho salmon (Oncorhynchus kisutch).

The glutathione S-transferases (GSTs) are a family of phase II detoxification enzymes which protect against chemical injury. In contrast to mammals, GST expression in fish has not been extensively characterized, especially in the context of detoxifying waterborne pollutants. In the Northwestern United States, coho salmon (Oncorhynchus kisutch) are an important species of Pacific salmon with complex life histories that can include exposure to a variety of compounds including GST substrates. In the present study we characterized the expression of coho hepatic GST to better understand the ability of coho to detoxify chemicals of environmental relevance. Western blotting of coho hepatic GST revealed the presence of multiple GST-like proteins of approximately 24-26kDa. Reverse phase HPLC subunit analysis of GSH affinity-purified hepatic GST demonstrated six major and at least two minor potential GST isoforms which were characterized by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI MS-MS) and Fourier transform-ion cyclotron resonance (FT-ICR) MS analyses. The major hepatic coho GST isoforms consisted of a pi and a rho-class GST, whereas GSTs representing the alpha and mu classes constituted minor isoforms. Catalytic studies demonstrated that coho cytosolic GSTs were active towards the prototypical GST substrate 1-chloro-2,4-dinitrobenzene, as well as towards ethacrynic acid and nitrobutyl chloride. However, there was no observable cytosolic GST activity towards the pesticides methyl parathion or atrazine, or products of oxidative stress, such as cumene hydroperoxide and 4-hydroxynonenal. Interestingly, coho hepatic cytosolic fractions had a limited ability to bind bilirubin, reflecting a potential role in the sequestering of metabolic by-products. In summary, coho salmon exhibit a complex hepatic GST isoform expression profile consisting of several GST classes, but may have a limited a capacity to conjugate substrates of toxicological significance such as pesticides and endogenous compounds associated with cellular oxidative stress.

Amino Acid Sequence↗

PubMed2006

Purification and characterization of two protein kinases acting on the aquaporin SoPIP2;1.

Aquaporins are water channel proteins that facilitate the movement of water and other small solutes across biological membranes. Plants usually have large aquaporin families, providing them with many ways to regulate the water transport. Some aquaporins are regulated post-translationally by phosphorylation. We have previously shown that the water channel activity of SoPIP2;1, an aquaporin in the plasma membrane of spinach leaves, was enhanced by phosphorylation at Ser115 and Ser274. These two serine residues are highly conserved in all plasma membrane aquaporins of the PIP2 subgroup. In this study we have purified and characterized two protein kinases phosphorylating Ser115 and Ser274 in SoPIP2;1. By anion exchange chromatography, the Ser115 kinase was purified from the soluble protein fraction isolated from spinach leaves. The Ca2+-dependent Ser274 kinase was purified by peptide affinity chromatography using plasma membranes isolated from spinach leaves. When characterized, the Ser115 kinase was Mg2+-dependent, Ca2+-independent and had a pH-optimum at 6.5. In accordance with previous studies using the oocyte expression system, site-directed mutagenesis and kinase and phosphatase inhibitors, the phosphorylation of Ser274, but not of Ser115, was increased in the presence of phosphatase inhibitors while kinase inhibitors decreased the phosphorylation of both Ser274 and Ser115. The molecular weight of the Ser274 kinase was approximately 50 kDa. The identification and characterization of these two protein kinases is an important step towards elucidating the signal transduction pathway for gating of the aquaporin SoPIP2;1.

Amino Acid Sequence↗

PubMed2004

Kinetic and equilibrium characterization of the interaction between bovine trypsin and I-ovalbumin.

Serpins are a superfamily of structurally linked proteins with interesting functional properties. Most serpins act as proteinase suicide inhibitors and play a key role in a number of physiological processes. Structural flexibility properties make serpins extremely available to conformational transitions, often causing changes in protein function. Ovalbumin is a member of the serpin family that is not able to inhibit serine proteinases in its native form. In contrast, I-ovalbumin, the product of a heating transition, is a potent reversible serine proteinase inhibitor. In this paper, a detailed equilibrium and kinetic characterization of the interaction between the serpin ovalbumin and bovine trypsin, using a resonant mirror technique, is reported. This methodology revealed that the high affinity interaction between the two binding partners is characterized by high kinetic association constants and low kinetic dissociation constants. The modulation exerted by protons in solution, examined taking into account structural motifs characterizing the binding interface between the two macromolecules, suggests an interaction reminiscent of that observed for the binding of other serine proteases to their ligands.

Animals↗

PubMed2006

Characterization of protein aggregation: the case of a therapeutic immunoglobulin.

In this paper, a therapeutic immunoglobulin (Antibody A) has been characterized in two solutions: (1) 0.1% acetic acid containing 50 mM magnesium chloride, a solution in which the immunoglobulin is stable, and (2) 10 mM sodium phosphate buffer pH approximately 7. The protein solutions were characterized by microscopy, asymmetrical flow field-flow fractionation (FFF), light scattering, circular dichroism, fluorescence and fluorescence lifetime spectroscopy. The results show that Antibody A dissolved in 0.1% acetic acid containing 50 mM magnesium chloride exists as 88% monomer, 2% low molecular weight aggregates and 10% high molecular weight aggregates (>1 million Dalton). In phosphate buffer, Antibody A formed micrometre-sized aggregates that were best characterized by fluorescence microscopy. The aggregation of Antibody A in phosphate buffer was shown to be concomitant with conformational changes in amino acid residue side chains. The aggregates formed in phosphate buffer were easily disrupted during FFF analysis, indicating that they are formed by weak interactions. The combination of microscopy, asymmetrical flow field-flow fractionation (FFF) and spectroscopy allowed a reliable assessment of protein self association and aggregation.

Acetic Acid↗

PubMed2003

Molecular characterization of human eye and heart fatty acid ethyl ester synthase/carboxylesterase by site-directed mutagenesis.

The FAEES/carboxylesterase enzyme nonoxidatively metabolizes alcohol into FAEEs, potentially toxic molecules linked with alcohol-induced end-organ damage. Our laboratory has isolated and characterized a FAEES/carboxylesterase cDNA from human eye and heart (G12). In an effort to further characterize the G12 enzyme, this laboratory has analyzed the FAEES activity, as well as performed mutagenic studies on a proposed active site of G12. We have mutated Ser 204 and His 451 in the G12 enzyme in an attempt to characterize the active site. We found that both mutations cause almost total loss of carboxylesterase enzyme activity with the native enzyme remaining active for carboxylesterase activity (560 nmol/mg protein/h for G12). With oleic acid as a substrate, the FAEES activity was 170 nmol/mg protein/h for G12, 10.8 nmol/mg protein/h for M204, and 8.5 nmol/mg protein/h for M451. When ethanol was used as substrate, the FAEES activity was 240 nmol/mg protein/h for G12, 15.1 nmol/mg protein/h for M204, and 6.2 nmol/mg protein/h for M451. Since both carboxylesterase and FAEES enzyme activities are significantly lowered by mutating Ser 204 and His 451 of the G12 enzyme, this study indicates that these residues may be important for the key enzymatic activities of the enzyme.

Acyltransferases↗

PubMed2005

Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme.

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 degrees C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.

Bacterial Proteins↗

PubMed2005

Cloning and pharmacological characterization of the cat urotensin-II receptor (UT).

Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [(125)I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity (K(D) 288+/-13pM) and high density (B(max) 747+/-66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT (K(i) 1.8-5.3nM). Cat UT was coupled to intracellular [Ca(2+)] release (EC(50) 0.6+/-0.2nM) and total inositol phosphate (IP) formation (EC(50) 0.4+/-0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla (K(D) 815+/-34) and cortex and (K(D) 316+/-39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.

Amino Acid Sequence↗

PubMed2006

Identification and characterization of the Mustang promoter: regulation by AP-1 during myogenic differentiation.

We previously identified Mustang (musculoskeletal temporally activated novel gene) with expression exclusively in the musculoskeletal system. Although its expression is almost undetectable in intact bone, it is robustly upregulated during bone regeneration. It is also abundantly expressed in adult skeletal muscle and tendon. As such, Mustang represents a marker for these cells and thus identifying its promoter would enable us to characterize its transcriptional regulation. To this end, we have isolated and characterized a 1512-bp mouse genomic clone representing the Mustang 5'-flanking region and identified a transcription start site, a TATA box, and multiple putative transcription factor binding sites (including AP-1 and AP-2). The activity of this promoter was detected in musculoskeletal cells and embryonic fibroblasts, even exceeding levels (145%) of the control SV40 promoter (in C2C12 cells). Further, the contribution of specific AP-1 and AP-2 sites was determined with serially deleted and mutated promoter constructs. Results indicate that one of the four AP-1 sites is required for substantial transcriptional activation, as its specific deletion or mutation decreases promoter activity by 32% and 40%, respectively. In contrast, deletion of both identified AP-2 sites results in only a 12% decrease in promoter activity. We further characterized the key AP-1 site by EMSA and determined that in both proliferating and differentiating C2C12 cells, only c-Fos, Fra-2 and JunD were required for transcriptional activation. Mustang's restricted tissue specificity and strong promoter makes this gene an ideal candidate for utilization in cell lineage studies that could unveil cellular/molecular mechanisms responsible for musculoskeletal development and regeneration.

Animals↗

PubMed2004

Multiple epithelial and nonepithelial tumors in hereditary nonpolyposis colorectal cancer: characterization of germline and somatic mutations of the MSH2 gene and heterogeneity of replication error phenotypes.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal inherited cancer syndrome characterized by germline plus somatic mutations of DNA mismatch repair genes and familial clustering of cancers of colorectum and other visceral organs. So far, to our knowledge, there has been no proof of nonepithelial tumors in association with HNPCC. Here we report on a MSH2 frameshift HNPCC family with a carrier found to have multiple primary tumors, including endometrial hyperplasia, ovarian adenocarcinoma, skin cavernous hemangioma, and skin dermatofibrosarcoma protuberans (DFSP). We studied the replication error (RER) phenotype in noncoding (Bat-26, Bat-25, D2S123, D5S346, and D17S250) and coding (MSH3, MSH6, BAX, and TGFBR2 genes) DNA sequences, and characterized the germline and somatic mutations of the MSH2 gene in the tumors described above and in endometrial carcinomas from two of her affected siblings. RER was observed in an order of hyperplasic endometrium (6/10 markers), ovarian carcinoma (5/10 markers), endometrial carcinomas (4/9 and 3/10), DFSP (2/9 markers), and cavernous hemangioma (2/10 markers). All the tumors showed the same germline mutation of G5-->G6 frameshift at 183-187 and polymorphism of C1168T in a heterozygous pattern. In an endometrial carcinoma, deletion of the second allele of MSH2 was evident. Heterogeneous RER patterns were noted in multiple primary tumors of the same individual and in premalignant and malignant endometrial tumors from different individuals. The study demonstrated the two hits of the hMSH(2) gene as well as intra- and interindividual variations of RER phenotypes in HNPCC. The first characterized nonepithelial tumors in HNPCC seem to carry a limited panel of RER, including a framesift at the (A)(10) tract of TGFBR2.

Adult↗