A simple fluorescent assay for the discovery of protein-protein interaction inhibitors.

Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors, called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example, and the FluorIA protocol design, optimization and successful application to HTS of large chemical libraries are described. Here enhanced green fluorescent protein (EGFP)-tagged RAD52 detected the PPI using full-length RPA heterotrimer coated, black microtiter plates and loss in fluorescence intensity identified small molecule inhibitors (SMIs) that displaced the EGFP-tagged RAD52. The FluorIA design and protocol can be adapted and applied to detect PPIs for other protein systems. This should push forward efforts to develop targeted therapeutics against protein complexes in pathological processes.

Smoking, variation in N-acetyltransferase 1 (NAT1) and 2 (NAT2), and risk of non-Hodgkin lymphoma: a pooled analysis within the InterLymph consortium.

PURPOSE: Studies of smoking and risk of non-Hodgkin lymphoma (NHL) have yielded inconsistent results, possibly due to subtype heterogeneity and/or genetic variation impacting the metabolism of tobacco-derived carcinogens, including substrates of the N-acetyltransferase enzymes NAT1 and NAT2. METHODS: We conducted a pooled analysis of 5,026 NHL cases and 4,630 controls from seven case-control studies in the international lymphoma epidemiology consortium to examine associations between smoking, variation in the N-acetyltransferase genes NAT1 and NAT2, and risk of NHL subtypes. Smoking data were harmonized across studies, and genetic variants in NAT1 and NAT2 were used to infer acetylation phenotype of the NAT1 and NAT2 enzymes, respectively. Pooled odds ratios (ORs) and 95 % confidence intervals (95 % CIs) for risk of NHL and subtypes were calculated using joint fixed effects unconditional logistic regression models. RESULTS: Current smoking was associated with a significant 30 % increased risk of follicular lymphoma (n = 1,176) but not NHL overall or other NHL subtypes. The association was similar among NAT2 slow (OR 1.36; 95 % CI 1.07-1.75) and intermediate/rapid (OR 1.27; 95 % CI 0.95-1.69) acetylators (p (interaction) = 0.82) and also did not differ by NAT1*10 allelotype. Neither NAT2 phenotype nor NAT1*10 allelotype was associated with risk of NHL overall or NHL subtypes. CONCLUSION: The current findings provide further evidence for a modest association between current smoking and follicular lymphoma risk and suggest that this association may not be influenced by variation in the N-acetyltransferase enzymes.

Perfect Crystals: microgravity capillary counterdiffusion crystallization of human manganese superoxide dismutase for neutron crystallography.

The NASA mission Perfect Crystals used the microgravity environment on the International Space Station (ISS) to grow crystals of human manganese superoxide dismutase (MnSOD)-an oxidoreductase critical for mitochondrial vitality and human health. The mission's overarching aim is to perform neutron protein crystallography (NPC) on MnSOD to directly visualize proton positions and derive a chemical understanding of the concerted proton electron transfers performed by the enzyme. Large crystals that are perfect enough to diffract neutrons to sufficient resolution are essential for NPC. This combination, large and perfect, is hard to achieve on Earth due to gravity-induced convective mixing. Capillary counterdiffusion methods were developed that provided a gradient of conditions for crystal growth along with a built-in time delay that prevented premature crystallization before stowage on the ISS. Here, we report a highly successful and versatile crystallization system to grow a plethora of crystals for high-resolution NPC.

Meat intake and risk of non-Hodgkin lymphoma.

We conducted a population-based, case-control study to test the hypothesis that consumption of meat and meat-related mutagens increases the risk of non-Hodgkin lymphoma (NHL), and whether the associations are modified by N-acetyltransferase (NAT) 1 and 2. Participants (336 cases and 460 controls) completed a 117-item food frequency questionnaire. The risk of NHL was associated with a higher intake of red meat (OR = 1.5; CI, 1.1-2.2), total fat (OR = 1.4; CI, 1.0-2.1), and oleic acid (OR = 1.5; CI, 1.0-2.2). NHL risk was also associated with a higher intake of very well-done pork (OR = 2.5; 95 % CI, 1.4-4.3) and the meat-related mutagen MeIQx (OR = 1.6; 95 % CI, 1.1-2.3). Analyses of the major NHL histologic subtypes showed a positive association between diffuse large B cell lymphoma (DLBCL) and higher intake of red meat (OR = 2.1; 95 % CI, 1.1-3.9) and the association was largely due to meat-related mutagens as a positive association was observed for higher intakes of both MeIQx (OR = 2.4; 95 % CI, 1.2-4.6) and DiMeIQx (OR = 1.9; 95 % CI, 1.0-3.5). Although the OR for follicular lymphoma (FL) was also increased with a higher red meat intake (OR = 1.9; 95 % CI, 1.1-3.3), the association appeared to be due to increased oleic acid (OR = 1.7; 95 % CI: 0.9-3.1). We found no evidence that polymorphisms in NAT1 or NAT2 modify the association between NHL and meat-related mutagens. Our results provide further evidence that red meat consumption is associated with an increase in NHL risk, and new evidence that the specific components of meat, namely fat and meat-related mutagens, may be impacting NHL subtype risk differently.

Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction.

Synthetic lethality is a successful strategy employed to develop selective chemotherapeutics against cancer cells. Inactivation of RAD52 is synthetically lethal to homologous recombination (HR) deficient cancer cell lines. Replication protein A (RPA) recruits RAD52 to repair sites, and the formation of this protein-protein complex is critical for RAD52 activity. To discover small molecules that inhibit the RPA:RAD52 protein-protein interaction (PPI), we screened chemical libraries with our newly developed Fluorescence-based protein-protein Interaction Assay (FluorIA). Eleven compounds were identified, including FDA-approved drugs (quinacrine, mitoxantrone, and doxorubicin). The FluorIA was used to rank the compounds by their ability to inhibit the RPA:RAD52 PPI and showed mitoxantrone and doxorubicin to be the most effective. Initial studies using the three FDA-approved drugs showed selective killing of BRCA1-mutated breast cancer cells (HCC1937), BRCA2-mutated ovarian cancer cells (PE01), and BRCA1-mutated ovarian cancer cells (UWB1.289). It was noteworthy that selective killing was seen in cells known to be resistant to PARP inhibitors (HCC1937 and UWB1 SYr13). A cell-based double-strand break (DSB) repair assay indicated that mitoxantrone significantly suppressed RAD52-dependent single-strand annealing (SSA) and mitoxantrone treatment disrupted the RPA:RAD52 PPI in cells. Furthermore, mitoxantrone reduced radiation-induced foci-formation of RAD52 with no significant activity against RAD51 foci formation. The results indicate that the RPA:RAD52 PPI could be a therapeutic target for HR-deficient cancers. These data also suggest that RAD52 is one of the targets of mitoxantrone and related compounds.

Human replication protein A-Rad52-single-stranded DNA complex: stoichiometry and evidence for strand transfer regulation by phosphorylation.

The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential in DNA metabolism and is phosphorylated in response to DNA-damaging agents. Rad52 and RPA participate in the repair of double-stranded DNA breaks (DSBs). It is known that human RPA and Rad52 form a complex, but the molecular mass, stoichiometry, and exact role of this complex in DSB repair are unclear. In this study, absolute molecular masses of individual proteins and complexes were measured in solution using analytical size-exclusion chromatography coupled with multiangle light scattering, the protein species present in each purified fraction were verified via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western analyses, and the presence of biotinylated ssDNA in the complexes was verified by chemiluminescence detection. Then, employing UV cross-linking, the protein partner holding the ssDNA was identified. These data show that phosphorylated RPA promoted formation of a complex with monomeric Rad52 and caused the transfer of ssDNA from RPA to Rad52. This suggests that RPA phosphorylation may regulate the first steps of DSB repair and is necessary for the mediator function of Rad52.

Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase.

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix alpha1, the loop before alpha6 and helix alpha7 to cap off the active site when substrate is bound.

Biophysical characterization and modeling of human Ecdysoneless (ECD) protein supports a scaffolding function.

The human homolog of Drosophila ecdysoneless protein (ECD) is a p53 binding protein that stabilizes and enhances p53 functions. Homozygous deletion of mouse Ecd is early embryonic lethal and Ecd deletion delays G1-S cell cycle progression. Importantly, ECD directly interacts with the Rb tumor suppressor and competes with the E2F transcription factor for binding to Rb. Further studies demonstrated ECD is overexpressed in breast and pancreatic cancers and its overexpression correlates with poor patient survival. ECD overexpression together with Ras induces cellular transformation through upregulation of autophagy. Recently we demonstrated that CK2 mediated phosphorylation of ECD and interaction with R2TP complex are important for its cell cycle regulatory function. Considering that ECD is a component of multiprotein complexes and its crystal structure is unknown, we characterized ECD structure by circular dichroism measurements and sequence analysis software. These analyses suggest that the majority of ECD is composed of α-helices. Furthermore, small angle X-ray scattering (SAXS) analysis showed that deletion fragments, ECD(1-432) and ECD(1-534), are both well-folded and reveals that the first 400 residues are globular and the next 100 residues are in an extended cylindrical structure. Taking all these results together, we speculate that ECD acts like a structural hub or scaffolding protein in its association with its protein partners. In the future, the hypothetical model presented here for ECD will need to be tested experimentally.

Culture and immortalization of pancreatic ductal epithelial cells.

Some populations of the epithelial cells from the duct and ductular network of the mammalian pancreas have been isolated and maintained in vitro for up to 3 mo. These cells express many of the surface factors that are unique to them in vivo. They also retain significant drug- and carcinogen-metabolizing capacity in vitro. In this chapter we review the progression of the methods for the isolation, culture and maintenance in vitro for these cells from the earliest when only duct/ductular fragments were obtainable to the current ones which provide epithelial cells. The critical steps in the isolation process are identified and strategies are provided to facilitate these steps. These include the selection of tissue digestive enzymes, the importance of extensive mincing before culture and the importance of roles of some co-factors used in the culture medium.

The OB-fold domain 1 of human POT1 recognizes both telomeric and non-telomeric DNA motifs.

The POT1 protein plays a critical role in telomere protection and telomerase regulation. POT1 binds single-stranded 5'-TTAGGGTTAG-3' and forms a dimer with the TPP1 protein. The dimer is recruited to telomeres, either directly or as part of the Shelterin complex. Human POT1 contains two Oligonucleotide/Oligosaccharide Binding (OB) fold domains, OB1 and OB2, which make physical contact with the DNA. OB1 recognizes 5'-TTAGGG whereas OB2 binds to the downstream TTAG-3'. Studies of POT1 proteins from other species have shown that some of these proteins are able to recognize a broader variety of DNA ligands than expected. To explore this possibility in humans, we have used SELEX to reexamine the sequence-specificity of the protein. Using human POT1 as a selection matrix, high-affinity DNA ligands were selected from a pool of randomized single-stranded oligonucleotides. After six successive rounds of selection, two classes of high-affinity targets were obtained. The first class was composed of oligonucleotides containing a cognate POT1 binding sites (5'-TTAGGGTTAG-3'). The second and more abundant class was made of molecules that carried a novel non-telomeric consensus: 5'-TNCANNAGKKKTTAGG-3' (where K = G/T and N = any base). Binding studies showed that these non-telomeric sites were made of an OB1-binding motif (TTAGG) and a non-telomeric motif (NT motif), with the two motifs recognized by distinct regions of the OB1 domain. POT1 interacted with these non-telomeric binding sites with high affinity and specificity, even when bound to its dimerization partner TPP1. This intrinsic ability of POT1 to recognize NT motifs raises the possibility that the protein may fulfill additional functions at certain non-telomeric locations of the genome, in perhaps gene transcription, replication, or repair.

Human prostate epithelial cells metabolize chemicals of dietary origin to mutagens.

Human prostate epithelial cells from a 17- and 42-year-old donor and designated as HuPrEC(17) and HuPrEC(42), were used to metabolize 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3,8-dimethylimidazo[4.5-f]quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP). The ability of the HuPrEC to metabolize these chemicals was measured as the mutagenicity of the test chemicals in V79 cells. Arylamine N-acetyltransferase (NAT1 and NAT2) genotype and activity, cytochrome P4501A2 (CYP1A2) activity and genotype, and glutathione S-transferase (GSTM1, GSTP1 and GSTT1) genotype were measured. HUPrEC(17) expressed a slow form of NAT1 (*4/*3) and an intermediate form of NAT2 (*4/*6) while HuPrEC(42) expressed the rapid form of NAT1 (*10/*10) and an intermediate form of NAT2 (*4/*5). Both had comparable NAT1 activity (2.9 and 3.6 nmol substrate acetylated/mg protein/min) but neither had detectable NAT2 activity. Cells from both donors metabolized the pro-mutagens, although there were some significant differences in the extent of mutagenicity produced. HuPrEC(42) more efficiently converted the three heterocyclic amines to mutagens than the HuPrEC(17), the ratios being Glu-P-2 (2.3:1), MeIQx (1.6:1), and PhIP (7.3:1). These data show that human prostate epithelial cells can metabolize important dietary chemicals to mutagenic species.

Interplay of DNA damage and cell cycle signaling at the level of human replication protein A.

Replication protein A (RPA) is the main human single-stranded DNA (ssDNA)-binding protein. It is essential for cellular DNA metabolism and has important functions in human cell cycle and DNA damage signaling. RPA is indispensable for accurate homologous recombination (HR)-based DNA double-strand break (DSB) repair and its activity is regulated by phosphorylation and other post-translational modifications. HR occurs only during S and G2 phases of the cell cycle. All three subunits of RPA contain phosphorylation sites but the exact set of HR-relevant phosphorylation sites on RPA is unknown. In this study, a high resolution capillary isoelectric focusing immunoassay, used under native conditions, revealed the isoforms of the RPA heterotrimer in control and damaged cell lysates in G2. Moreover, the phosphorylation sites of chromatin-bound and cytosolic RPA in S and G2 phases were identified by western and IEF analysis with all available phosphospecific antibodies for RPA2. Strikingly, most of the RPA heterotrimers in control G2 cells are phosphorylated with 5 isoforms containing up to 7 phosphates. These isoforms include RPA2 pSer23 and pSer33. DNA damaged cells in G2 had 9 isoforms with up to 14 phosphates. DNA damage isoforms contained pSer4/8, pSer12, pThr21, pSer23, and pSer33 on RPA2 and up to 8 unidentified phosphorylation sites.

The paradox of conformational constraint in the design of Cbl(TKB)-binding peptides.

Solving the crystal structure of Cbl(TKB) in complex with a pentapeptide, pYTPEP, revealed that the PEP region adopted a poly-L-proline type II (PPII) helix. An unnatural amino acid termed a proline-templated glutamic acid (ptE) that constrained both the backbone and sidechain to the bound conformation was synthesized and incorporated into the pYTPXP peptide. We estimated imposing structural constraints onto the backbone and sidechain of the peptide and preorganize it to the bound conformation in solution will yield nearly an order of magnitude improvement in activity. NMR studies confirmed that the ptE-containing peptide adopts the PPII conformation, however, competitive binding studies showed an order of magnitude loss of activity. Given the emphasis that is placed on imposing structural constraints, we provide an example to support the contrary. These results point to conformational flexibility at the interface, which have implications in the design of potent Cbl(TKB)-binding peptides.

Peptide truncation leads to a twist and an unusual increase in affinity for casitas B-lineage lymphoma tyrosine kinase binding domain.

We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.

A real-time and hands-on research course in protein purification and characterization: Purification and crystal growth of human inosine triphosphate pyrophosphatase.

A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately undergraduate institution, and to complete an entire research project in a realistic timeframe via a time-intensive curriculum as a special summer session. The course content gives a strong background in protein structure/chemistry, purification principles, protocol development, optimization strategies, use and programming of an automated chromatography instrument, and characterization strategies with an emphasis on X-ray crystallography. The laboratory portion offers students the chance to purify a protein (human inosine triphosphate pyrophosphatase) from start to finish, program and use an ÄKTA fast protein liquid chromatography instrument, and to grow and analyze their own protein crystals using their purified protein. This innovative laboratory experience gives the participating students the opportunity to complete a miniresearch project in real time and enhances their overall understanding of important biochemical research techniques and the instrumentation involved, fostering a better understanding of the research process all within a classroom setting. Evaluations and feedback concerning this course indicated a positive learning environment, a retention of knowledge and skills, a belief that the skill set learned continues to be useful in current endeavors, and a sense of accomplishment in the completion of an actual research project within the confines of a class setting.

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18.

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [(3)H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.

The role of histone H4 biotinylation in the structure of nucleosomes.

BACKGROUND: Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p<0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. CONCLUSIONS/SIGNIFICANCE: The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation.

Functional study of the P32T ITPA variant associated with drug sensitivity in humans.

Sanitization of the cellular nucleotide pools from mutagenic base analogues is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic, and toxic incorporation of purine base analogues [i.e., ITP, dITP, XTP, dXTP, or 6-hydroxylaminopurine (HAP) deoxynucleoside triphosphate] into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele, 94C-->A, encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analogue drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by Western blot is severely diminished in a human fibroblast cell line with the 94C-->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability, and availability.

Association of NAT and GST polymorphisms with non-Hodgkin's lymphoma: a population-based case-control study.

Several chemicals have been associated with risk of non-Hodgkin's lymphoma (NHL), many of which are substrates for N-acetyltransferase (NAT) and glutathione S-transferase (GST) enzymes. We investigated the association between polymorphisms in genes coding for these enzymes and NHL risk in a population-based study (389 cases and 535 controls). NAT1 slow genotype was associated with a slightly increased risk in women [odds ratios (OR) = 1.4; 95% confidence interval (CI) = 0.9-2.3], but not in men. NAT2 slow genotype was not associated with risk in either sex. The two slow genotypes of NAT1 and NAT2 combined were associated with a minor increase of risk in women (OR = 1.4; 0.8-2.4). There was no association with the GSTM1 or GSTT1 null genotype in either sex, irrespective of histological subtypes. Individuals with GSTP1 Val homozygotes had non-significant excessive risk of marginal zone lymphoma (OR = 1.8; 0.6-5.1) and 'other' B-cell NHLs (OR = 1.6; 0.7-3.6), but lower risk of diffuse large B-cell lymphoma (OR = 0.2; 0.1-0.96). Risk did not elevate with an increasing number of high-risk GST alleles in either sex. In summary, although NAT1, NAT2, GSTM1, GSTT1, or GSTP1 polymorphisms do not appear to be associated with NHL risk overall, there might be gender-specific and subtype-specific associations that require confirmation.

Nitrosation of glycine ethyl ester and ethyl diazoacetate to give the alkylating agent and mutagen ethyl chloro(hydroximino)acetate.

Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test. We report here that treatment of glycine ethyl ester at 37 degrees C with excess nitrite acidified with HCl, followed by ether extraction, gave 30-40% yields of a product identified as ethyl chloro(hydroximino)acetate [ClC(=NOH)COOEt, ECHA] and a 9% yield of ethyl chloroacetate. The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA. Nitrosation of ethyl diazoacetate also yielded ECHA. Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA. ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate [PhN=C(NHOH)CO2Et]. This product was different from ethyl [(phenylamino)carbonyl]carbamate [PhNHC(=O)NHCO2Et], which was synthesized by reacting ethyl isocyanatoformate (OCN.CO2Et) with aniline. ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(=NOH)CO2Et derivative. ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 x control value for 12-18 microg/plats) and for V79 mammalian cells (1.5-1.7 x control value for 60-100 microM). In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.