[Comparison of application effects of colonoscopy, fecal immunochemical test and a novel risk-adapted screening approach in colorectal cancer screening in Xuzhou population].

Objective: To compare the application effect of the colonoscopy, fecal immunochemical test (FIT) and novel risk-adapted screening approach in colorectal cancer screening in Xuzhou population. Methods: From May 2018 to April 2019, 4 280 subjects aged 50-74 were recruited from Gulou district, Yunlong district and Quanshan district of Xuzhou. They were randomly assigned to the colonoscopy group (n=863), FIT group (n=1 723) and novel risk-adapted screening approach group (n=1 694) according to the ratio of 1∶2∶2. For the novel risk-adapted screening approach group, after the risk assessment, high-risk subjects were invited to undergo colonoscopy and low-risk subjects were invited to undergo FIT examination. All FIT positive subjects were invited to undergo colonoscopy. Colonoscopy participation rate [(the number of colonoscopies completed/the number of colonoscopies invited to participate)×100%], detection rate of colorectal lesions [(the number of diagnosed patients/the number of colonoscopies completed)×100%], colonoscopy resource load (the number of colonoscopies completed/the number of diagnosed advanced tumors) and FIT resource load in each group were calculated and compared. Results: The age of all subjects was (61±6) years old, including 1 816 males (42.43%). There was no statistically significant difference in the socio-demographic characteristics of the subjects in different screening groups. The colonoscopy participation rate was 22.60% (195/863) in the colonoscopy group, 57.04% (77/135) in the FIT group, and 33.94% (149/439) in the novel risk-adapted screening approach group, respectively. The colonoscopy participation rate was higher in the FIT group than in the colonoscopy group and the novel risk-adapted screening approach group (P<0.001). The colonoscopy participation rate of novel risk-adapted screening group was significantly higher than the colonoscopy group (P<0.001). The detection rates of advanced tumors were 6.67% (13/195), 9.09% (7/77) and 8.72% (13/149), respectively, and the difference was not statistically significant (P>0.05). The colonoscopy resource load (95%CI) was 15 (13-17) in the colonoscopy group, 11 (9-14) in the FIT group and 11 (10-13) in the novel risk-adapted screening approach group, respectively. Among them, the colonoscopy resource load of high-risk individuals in the novel risk-adapted screening approach group was 12 (9-15). FIT resource loads (95%CI) were 207 (196-218) and 88 (83-94) in the FIT group and the novel risk-adapted screening approach group. Conclusion: The combined application of risk-adapted screening approach and FIT may have a good application effect in colorectal cancer screening.

Adamts18 Deficiency Causes Spontaneous SMG Fibrogenesis in Adult Mice.

Chronic sclerosing sialadenitis of the submandibular gland (also known as Küttner tumor) is characterized by concomitant swelling of the submandibular glands secondary to strong lymphocytic infiltration and fibrosis. The pathogenesis of this disease has been unclear, but it is associated with immune disorders. ADAMTS18 is a member of the ADAMTS superfamily of extracellular proteinases. In this study, we showed that Adamts18 is highly expressed in submandibular salivary gland (SMG) during embryonic development and decreases but is retained in adult SMG tissue in mice. Adamts18 deficiency led to reduced cleft formation and epithelial branching in embryonic SMG before embryonic day 15.5 in mice. No significant histologic changes in the later stages of branching or the morphology of SMG were detected in Adamts18-/- mice. However, Adamts18 deficiency causes spontaneous SMG fibrogenesis and fibrosis in adult mice. At 8 wk of age, Adamts18-/- mice began to manifest the first signs of pathologic changes of mild fibrosis and CD11b+ cell infiltration in SMG tissues. At ≥8 mo, all male and female Adamts18-/- mice developed unilateral or bilateral SMG scleroma that is similar to patients with chronic sclerosing sialadenitis of the submandibular gland. Adamts18-/- mice also showed secretory dysfunction and severe dental caries. Histologically, SMG scleroma is characterized by progressive periductal fibrosis, acinar atrophy, irregular duct ectasis, and dense infiltration of IgG-positive plasma cells. A significant infiltration of CD4+ T lymphocytes and CD11b+ monocytes and macrophages was also detected in the SMG scleroma of Adamts18-/- mice. The levels of TGF-ß1, IL-6, and IL-33 were significantly increased in Adamts18-/- SMGs, which induces chronic inflammation and myofibroblast activation, ultimately leading to fibrosis. This study indicates that Adamts18 regulates the early branching morphogenesis of embryonic SMG and plays a role in protecting from spontaneous SMG fibrogenesis via modulating local inflammation, autoimmune reaction, and myofibroblast activation in adult mice.

Long non­coding RNA XIST promotes cell proliferation of pancreatic cancer through miR­137 and Notch1 pathway.

OBJECTIVE: Long non-coding ribonucleic acids X-inactive specific transcript (lncRNA XIST) is one lncRNAs which involved in multiple human cancers. However, the functions and potential molecular regulatory mechanisms of XIST/microRNA-137 (miR­137) in pancreatic cancer (PC) still need to explore. PATIENTS AND METHODS: PC tissues and cell lines were analyzed for XIST, miR-137 and Notch1 expressions through quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Nude mouse xenograft tumor assay was used to detect XIST effects on pancreatic tumorigenesis in vivo. Cell Counting Kit (CCK-8) assay was performed to detect PC cell proliferation. Dual-Luciferase reporter assay, qRT-PCR, RNA immunoprecipitation (RIP) and Western blot assays were applied to validate the target relationship of XIST, miR­137 and Notch1. RESULTS: Results demonstrated that XIST expression was increased in PC tissues and cells. XIST knockdown inhibited PC cell proliferation in vitro and also repressed the tumor growth in vivo. XIST directly interacted with miR-137 and negatively regulated its expression. Notch1 was identified as a target gene of miR-137 and XIST acted as a competitive endogenous RNA (ceRNA) to positively regulate Notch1 expression by suppressing miR-137. In addition, we detected miR-137 was negatively correlated with XIST and Notch1 respectively, and a positive correlation between Notch1 and XIST expression in PC tissues. Furthermore, Notch1 overexpression could offset the suppressing effect of XIST knockdown or miR-137 overexpression on cell proliferation. Therefore, XIST may play an important role in promoting cell proliferation through miR­137 and Notch1 pathway in PC. CONCLUSIONS: To sum up, these results proposed that XIST functioned as an endogenous sponge in promoting PC cell proliferation through competing for miR-137 to regulate Notch1 expression, and may provide more therapeutic targets for the patients with PC.

MiRNA-92a promotes cell proliferation and invasion through binding to KLF4 in glioma.

OBJECTIVE: Glioma is one of the most frequent brain tumors in adults, and it has a low 5-year survival rate. MicroRNA-92a (miR-92a) has been reported to be upregulated and acted as an oncogene in many cancers. The purpose of this study was to explore the molecular mechanisms of miR-92a and kruppel-like factor 4 (KLF4) in glioma. PATIENTS AND METHODS: Western blotting assay and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) were applied to calculate the relative expression of interest proteins and mRNAs. Luciferase ability assay was conducted to evaluate whether miR-92a was targeting to KLF4. RESULTS: A higher expression of miR-92a was observed in glioma tissues compared with the corresponding adjacent non-tumor tissues. The upregulation of miR-92a predicted poor prognostic characteristics of glioma. The overexpression miR-92a significantly promoted cell proliferation an invasion, while the knockdown of miR-92a presented the opposite results. MiR-92a bound to KLF4 and mediated the expression of KLF4 in glioma cells. The knockdown of miR-92a inhibited cell invasion-mediated EMT. Furthermore, the knockdown of miR-92a suppressed cell proliferation through the KLF4/AKT/mTOR signal pathway. CONCLUSIONS: MiR-92a promoted the proliferation through the KLF4/AKT/mTOR signal pathway in glioma. The newly identified miR-92a/KLF4/AKT/mTOR axis provides novel insight into the pathogenesis of glioma.

[Effect and mechanism of silibinin on the inhibition of ALK positive NSCLC cells by sensitizing crizotinib].

Objective: To explore the synergistic effect of silibinin combined with crizotinib on anaplastic lymphoma kinase positive (ALK+ ) non-small cell lung cancer (NSCLC) cells and its mechanism. Methods: H2228 and H3122 cells were treated with silibinin, crizotinib alone or in combination. Cell proliferation was measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and colony formation assay. Migration or invasion ability was tested by wound healing assay or transwell assay, respectively. Expressions of E-Cadherin and vimentin protein were examined by immunofluorescence staining. The protein expressions of ALK, p-ALK, E-Cadherin and Vimentin were detected by western blotting.The anti-cancer effect of silibinin combined with crizotinib in vivo was determined by subcutaneously injecting 2×10(6) H2228 cells into immunodeficient nude mice. Results: The result of MTT assay showed that the cell viability of H2228 or H3122 treated with 100 µmol/L silibinin was (88.38±4.10)% or (72.27±3.62)%, respectively, marginally decreased compared with that of the control. The 50% inhibitory concentration (IC(50)) of H2228 cells treated with crizotinib alone or combined with 100 µmol/L silibinin was (917.10±7.75) nmol/L or (238.73±7.67) nmol/L, respectively. The IC(50) of H3122 cells treated with crizotinib alone or combined with 100 µmol/L silibinin was (472.50±15.70) nmol/L or (206.10±12.01) nmol/L, respectively. The IC(50s) of H2228 and H3122 cells were significantly decreased by combined treatment of crizotinib and silibinin compared to crizotinib treatment alone (P<0.01). When compared with the control group, colony forming ratios of H2228 cells were (83.34±2.72)% in 100 µmol/L silibinin treatment group, (69.42±3.06)% in 400 nmol/L crizotinib treatment group and (27.32±1.42)% in combined treatment group. When compared with the control group, colony forming ratios of H3122 cells were (84.45±5.67)% in 100 µmol/L silibinin treatment group, (45.02±5.83)% in 400 nmol/L crizotinib treatment group and (17.43±3.83)% in combined treatment group. Silibinin combined with crizotinib treatment significantly inhibited the colony formation ability of H2228 and H3122 cells (P<0.01). Migration and invasion results showed that combined treatment of crizotinib and silibinin markedly inhibited the migration and invasion ability of H2228 cells (P<0.01). Western blot results indicated that treated with silibinin alone or in combination of crozitinib for 48 hours, the protein level of E-cadherin in H2228 cells was upregulated, while the expressions of p-ALK and vimentin were downregulated, without obvious alteration of ALK protein expression. In the xenograft model, the mean tumor weight was (9.40±2.58)g in crizotinib treatment group and (4.58±1.07)g in the combined treatment group. The inhibitory effect of tumor growth in vivo of combined treatment was significantly superior to that of crizotinib treatment alone (P<0.05). Conclusion: Silibinin enhances the inhibitory effect of crizotinib on ALK positive NSCLC cells, which may be associated with suppression of ALK activity and mesenchymal-epithelial transition.

[Value of intravoxel incoherent motion diffusion-weighted imaging in differential diagnosis of benign and malignant hepatic lesions and blood perfusion evaluation].

Objective: To investigate the value of intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) in the differential diagnosis and blood perfusion evaluation of benign and malignant hepatic lesions. Methods: A retrospective analysis was performed for 86 patients (96 lesions) with pathologically or clinically confirmed hepatic lesions or hepatic lesions diagnosed based on follow-up results, among whom 48 had malignant lesions (53 lesions) and 38 had benign lesions (43 lesions). The patients underwent conventional magnetic resonance (MR) plain scan, contrast-enhanced scan, and diffusion-weighted imaging (DWI) with different b values (b = 0, 50, 100, 150, 200, 400, 600, 800, 1 000, and 1 200 s/mm2) to determine the parameters of the double exponential model for intravoxel incoherent motion (IVIM): fast diffusion coefficient Dfast, slow diffusion coefficient Dslow, and percentage of fast-diffusion constituent F value. The patients were divided into groups according to the blood supply to lesions on conventional MR plain scan and contrast-enhanced scan, and there were 47 lesions in abundant blood supply group and 49 in poor blood supply group. The data for analysis were Dfast, Dslow, and F values of benign/malignant lesion groups and abundant/poor blood supply groups. The independent samples t-test was used for statistical analysis; the independent samples non-parametric test Mann-Whitney U test was used for the comparison of F value; the receiver operating characteristic (ROC) curve was used to evaluate the value of above parameters in the differentiation of benign and malignant lesions and blood supply evaluation. Results: Compared with the malignant lesion group, the benign lesion group had significantly higher Dslow, and F values (P< 0.001 orP= 0.001) and a higher Dfast value (P= 0.053). Compared with the poor blood supply group, the abundant blood supply group had significantly higher Dfast and F values (P< 0.001 orP= 0.001) and a higher Dslow value (P= 0.185). According to the ROC curve, the cut-off values of Dslow, Dfast, and F values in the diagnosis of benign/malignant hepatic lesions and evaluation of abundant/poor blood supply were 1.18×10-3mm2/s, 27.20×10-3mm2/s, 20.25%, 1.17×10-3mm2/s, 20.30×10-3mm2/s, and 17.80%, respectively, with sensitivities, specificities, accuracy, and areas under the ROC curve of 90.69%/92.45%/91.66%/0.938, 46.51%/73.58%/61.45%/0.589, 74.41%/50.94%/62.50%/0.653, 59.57%/57.14%/58.33%/0.559, 55.32%/63.26%/59.37%/0.618, and 93.61%/89.79%/90.62%/0.961, respectively. Conclusion: The parameter of the double exponential model for IVIM, Dslow value, has a certain value in the differential diagnosis of benign and malignant hepatic lesions, and F value can show blood perfusion in benign and malignant hepatic lesions without the need for contrast-enhanced scan, which provides a reference for the qualitative diagnosis of liver tumor.

Success of dental implants in vascularised fibular osteoseptocutaneous flaps used as onlay grafts after marginal mandibulectomy.

We have evaluated the survival of dental implants placed in vascularised fibular flap onlay grafts placed over marginal mandibulectomies and the effects on marginal bone loss of different types of soft tissue around implants under functional loading. From 2001-2009 we studied a total of 11 patients (1 woman and10 men), three of whom had had ameloblastoma and eight who had had squamous cell carcinomas resected. A total of 38 dental implants were placed either at the time of transfer of the vascularised fibular ostoseptocutaneous flaps (nine patients with 30 implants) or secondarily (two patients with eight implants). Four patients were given palatal mucosal grafts to replace intraoral skin flaps around the dental implants (n=13), and the other seven had the skin flaps around the dental implants thinned (n=25) at the second stage of implantation of the osteointegrated teeth. All vascularised fibular osteoseptocutaneous flaps were successfully transferred, and all implants survived a mean (range) of 73 (33-113) months after occlusal functional loading. The mean (SD) marginal bone loss was 0.5 (0.3) mm on both mesial and distal sides in patients who had palatal mucosal grafts, but 1.8 (1.6) mm, and 1.7 (1.5) mm, respectively, on the mesial and distal sides in the patients who had had thinning of their skin flaps. This difference is significant (p=0.008) with less resorption of bone in the group who had palatal mucosal grafts. Palatal mucosa around the implants helps to reduce resorption of bone after functional loading of implants.

Multiple primary primitive neuroectodermal tumours within the spinal epidural space with non-concurrent onset.

A case of multiple primary primitive neuroectodermal tumours (PNETs), which occurred at different levels of the spinal epidural space successively over a period of 8 months, is reported. A 24-year-old male, presenting with rapidly progressive paralysis, hyperthesia and a posterior epidural mass extending from T8 to T10 revealed by magnetic resonance imaging (MRI), exhibited a good recovery after initial emergency surgery. Lower back pain, chest pain and paralysis were subsequently reported. Spinal MRI in month 7 revealed a mass extending from T12 to L1 and another mass extending from T4 to T5 was detected epidurally in month 8. Additional operations were performed and radiotherapy was given. Pathological findings were consistent with PNETs and symptoms improved with treatment, particularly following each surgical excision.

Metabolic and genotoxic interactions of 2-aminofluorene and 2,4-diaminotoluene.

We have reported previously that the rodent carcinogen 2,4-diaminotoluene (2,4-DAT) is not activated as a mutagen to the standard Ames S. typhimurium tester strains when oxidized by prostaglandin H synthase (PHS). 2,4-DAT does, however, enhance the bacterial mutagenicity of the potent mutagen 2-aminofluorene (2-AF) when both compounds are incubated with the PHS activating system. Enhancement of activation of 2-AF would provide a plausible mechanism for the observed co-mutagenicity of 2,4-DAT. Co-incubation with 100 microM 2,4-DAT, however, inhibited the total metabolism of 25 microM 2-AF by 60% in both the PHS/H2O2 system and PHS/arachidonic acid system. The inhibition included a 75% decrease in the formation of water-soluble and protein-bound metabolites and about a 35% decrease in production of the peroxidative metabolites 2-nitrofluorene (NF) and 2-aminodifluorenylamine (ADFA). Azofluorene (AzF) production was the most sensitive to the effects of 2,4-DAT, exhibiting an 80% decrease in both PHS-catalyzed systems. No new 2-AF derived products were observed in the presence of 2,4-DAT. This pronounced inhibition of 2-AF metabolism by 2,4-DAT also was observed in incubations of the aromatic amines with PHS in the presence of S. typhimurium strain TA98. Bacterial N-acetylation of 2-AF did not appear to be an important reaction in any of these incubations. 2,4-DAT not only inhibited 2-AF metabolism by PHS, but also decreased the level of 2-AF covalent binding to the bacterial DNA by as much as 81%. This stands in sharp contrast to the enhancement of the mutagenicity of 2-AF elicited by 2,4-DAT in these same incubations. This clear dissociation between the extent of peroxidative activation, and resultant covalent modification of bacterial DNA, by 2-AF and the subsequent mutagenic response indicates that a metabolic interaction is not involved in the co-mutagenicity of 2,4-DAT.

Prostaglandin H synthase-dependent genotoxicity of 2,4-diaminotoluene.

2,4-Diaminotoluene (2,4-DAT), a high volume synthetic compound, is moderately carcinogenic to rodents. We report here that 2,4-DAT is a substrate for the peroxidase activity of prostaglandin H synthase (PHS). In contrast to many aromatic amines which are activated as mutagens by PHS, we find that 2,4-DAT is not mutagenic to six S. typhimurium strains with this activation system. The strains tested include YG1006, YG1024, and YG1029, which are far more sensitive to the mutagenicity of aromatic amines and nitroarenes than are the standard tester strains. Although not mutagenic itself, 2,4-DAT does enhance the mutagenicity of 2-aminofluorene (2-AF) in the PHS-catalyzed system in strains TA98, YG1006, and YG1024, with maximal enhancement of 140%, 1831%, and 1216%, respectively. Half-maximal enhancement of 2-AF mutagenicity is observed at 15-20 microM 2,4-DAT for strains YG1006 and YG1024, and about 80 microM for TA98. Studies with compounds structurally related to 2,4-DAT revealed enhancement of 2-AF mutagenicity with 2,5-DAT and o-phenylenediamine (o-PD) but not for other DAT isomers, toluidines, and phenylenediamines. Maximal enhancement of 2-AF mutagenicity observed in TA98 with PHS-catalyzed activation was 110% for o-PD and 60% for 2,5-DAT. This comutagenic effect of 2,4-DAT appears quite specific for 2-AF, as it fails to enhance either the PHS-dependent mutagenicity of the aromatic amines benzidine and 2-naphtylamine, or the direct mutagenicity of N-acetoxy-acetylaminofluorene,2-nitrofluorene,4- nitroquinoline-N-oxide and 1,1,1-trichloropropene-2,3-oxide. Enhancement of 2-AF mutagenicity by 2,4-DAT is also observed with cytochrome P-450-dependent activation, however the half-maximal 2,4-DAT concentration was 400 microM, and the maximal enhancement was only 50%. The ability of 2,4-DAT, under conditions where it is not itself mutagenic, to enhance the genotoxicity of the potent carcinogen 2-AF comprises an intriguing toxicological interaction, and underscores the inherent difficulties in assessing the genotoxic risks posed by mixtures of compounds.

Mutagenicity of benzo[a]pyrene bay-region sulfonates.

The interaction between the sulfite anion and specific benzo[a]pyrene (B[a]P) derivatives produces a novel class of benzo[a]pyrene sulfonates. (+/-)-7,8,9-Trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-sulfonate (B[a]PT-10-sulfonate) is formed in high yields in incubations containing (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (anti-BPDE) and sulfite, and sulfite strongly enhances the mutagenicity of the diolepoxide toward Salmonella typhimurium under those conditions. Although B[a]PT-10-sulfonate itself shows little direct mutagenicity over a 1-20 microM concentration range, this reactive bay-region intermediate does enhance the mutagenicity of anti-BPDE in strains TA98 and TA100 by up to 280%. No significant enhancement was seen when up to 20 microM B[a]PT-10-sulfonate was used in concert with another direct-acting mutagen, N-acetoxy-acetylaminofluorene (N-AcO-AAF). The isomeric product derived from sulfite and (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) is (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate (B[a]PT-9-sulfonate). Like B[a]PT-10-sulfonate, B[a]PT-9-sulfonate is not mutagenic to strains TA97, TA98 and TA100. This sulfonate exhibited little enhancing activity with anti-BPDE over a 1-20 microM concentration range, but did enhance the mutagenic response of strain TA98 to 0.2 microM N-Aco-AAF by up to 128%. Sulfite, anti-BPDE and B[a]PT-sulfonates were also examined for the ability to induce a forward mutation at the hgprt locus (8-azaguanine resistance) in strains of S.typhimurium. Sulfite caused a marked enhancement of forward mutation due to anti-BPDE in both TA98 and TA100. Surprisingly, concurrent administration of B[a]PT-10-sulfonate with anti-BPDE did not increase the number of mutant colonies. The extensive conversion of anti-BPDE to B[a]PT-10-sulfonate under conditions where sulfite enhances diolepoxide mutagenicity, when coupled with this enhancement of diolepoxide mutagenicity by B[a]PT-10-sulfonate in the reverse mutation assay, supports this novel B[a]P derivative as a mediator of the sulfite-dependent enhancement of B[a]P genotoxicity. Determining why this enhancing effect was not seen when selecting for mutation at the hgprt locus of S.typhimurium will require further study.

Ki-ras activation and expression in transformed mouse lung cell lines.

The Ki-ras proto-oncogene is activated by specific point mutations and is the transforming gene often identified in rodent and human lung tumors. An in vitro model to aid in the study of the consequences of Ki-ras activation and expression in mouse lung is needed. Accordingly, we have examined cell lines derived from chemically induced mouse lung tumors as well as spontaneous transformants of untreated mouse lung epithelial cells. The specific Ki-ras-activating gene mutations and the level of mRNA expression were examined for each cell line. Polymerase chain reaction and oligonucleotide hybridization were used to demonstrate that five of seven transformed lung cell lines contain codon 61 Ki-ras-activating mutations, resulting in an arginine substitution for wild-type glutamine. One transformed line contained this activating mutation and had also lost, or contained an altered, wild-type codon 61 Ki-ras allele. No codon 12 Ki-ras mutations were observed. Two transformed and two nontransformed epithelial lung cell lines contained only the wild-type codon 12 and 61 Ki-ras alleles. Northern blot analysis demonstrated that the Ki-ras mRNA was present in all the cell lines and was overexpressed in some, but not all, of the transformed lung cell lines. Those transformed lines with the highest levels of Ki-ras mRNA also expressed more H4-histone mRNA, suggesting that these cells have a greater proliferation rate. The level of Ki-ras mRNA increased during the proliferation of the nontransformed lung cells but then decreased upon reaching confluency. In contrast, the level of Ki-ras mRNA in the transformed lung cells was high during both growth and confluency, suggesting a potential defect in the regulation of Ki-ras in these cells. These lung cell lines will help provide a better understanding of the regulation of both the Ki-ras proto-oncogene and oncogene in the lung.

The status of tyrosyl residues in a Formosan cobra cardiotoxin.

Spectrophotometric titration of Formosan cobra cardiotoxin showed that two of the three tyrosyl residues were titrated freely with a normal apparent pKa of 9.6 whereas the remaining one ionized at pH above 11.0. Nitration of cardiotoxin in Tris . HCl buffer with tetranitromethane resulted in the selective nitration of tyrosine 11 and tyrosine 22. It also revealed that tyrosine 51 was the abnormal one in the spectrophotometric titration. Complete nitration occurred in the presence of 6.0 M guanidine hydrochloride. Compared with the conformation of native cardiotoxin, the peptide conformation of the partially nitrated cardiotoxin did not change significantly but the conformation of the completely nitrated cardiotoxin changed remarkably. The biological activity of cardiotoxin was indeed affected by nitration, but the immunological activity was nearly intact even when all the tyrosine residues were nitrated.