Developing Transposable Element Marker System for Molecular Breeding.

Transposable element (TE) marker system was developed considering the useful properties of the transposable elements such as their large number in the animal and plant genomes, high rate of insertion polymorphism, and ease of detection. Various methods have been employed for developing a large number of TE markers in several crop plants for genomics studies. Here we describe some of these methods including the recent whole genome search. We also review the application of TE markers in molecular breeding.

High-dose zoledronic acid narrows the periodontal space in rats.

The aim of this experiment was to evaluate the histological effects of zoledronic acid on the periodontal space in rats. 40 male Wistar rats were divided into three zoledronic acid groups and a control group. Zoledronic acid was injected subcutaneously at doses of 10, 50, or 500 µg/kg once a week for 3 weeks. The rats were killed 1 or 9 weeks after the last injection. Histological examination of the periodontal space around the incisor tooth revealed that zoledronic acid did not inhibit tooth development. In the rats killed 1 week after treatment discontinuation, the periodontal space gradually narrowed in response to increasing zoledronic acid doses, and the changes were statistically significant according to ANOVA but not according to ANOVA with post hoc tests. The changes persisted in the high-dose zoledronic acid group despite zoledronic acid discontinuation, with significant differences identified by ANOVA and ANOVA with post hoc tests. Therefore, although zoledronic acid had an insignificant effect on tooth development, it had a significant effect on the periodontal space when high doses were administered. The results of this experiment may provide useful information for future investigations on the role of zoledronic acid in the osteonecrosis of the jaw.

Enhancement of laser interaction with vacuum for a large angular aperture.

We study the nonlinear interaction of laser light with vacuum for a large angular aperture at electromagnetic field strengths far below the Schwinger limit. The polarization and magnetization in vacuum irradiated by a focused laser beam clearly differ from those in matter. This is due to the dependence on the Lorentz invariant, which results in a ring-shaped radiation distribution in vacuum. The number of the radiated photons increases nonlinearly with increasing angular aperture.

Betulinic acid augments the inhibitory effects of vincristine on growth and lung metastasis of B16F10 melanoma cells in mice.

We examined the antitumour effect of a combination of betulinic acid (BA) and vincristine (VCR) on murine melanoma B16F10 cells in vitro and in vivo. Betulinic acid, a pentacyclic triterpene, showed a synergistic cytotoxic effect on melanoma cells by combinational use of VCR. Betulinic acid and VCR induced cell cycle arrest at different points (BA at G1 phase and VCR at G2/M phase) and caused apoptosis in B16F10 melanoma cells. In the in vivo study, VCR inhibited metastasis of tumour cells to the lung. The addition of BA to VCR augmented suppression of the experimental lung metastasis of melanoma cells in C57BL/6 mice. The number of lung nodules of more than 1 mm in diameter in mice treated with BA and VCR was less than that in mice treated with VCR alone. These results suggest that BA is an effective supplement for enhancing the chemotherapeutic effect on malignant melanoma.

Treatment of non-inflamed obstructive meibomian gland dysfunction by an infrared warm compression device.

AIM: To test the short term efficacy and safety of an infrared warm compression device (IWCD, Eye Hot, Cept Co, Tokyo, Japan) as treatment for non-inflamed meibomian gland dysfunction (MGD). METHODS: 37 subjects with non-inflamed obstructive MGD, with and without aqueous tear deficiency (ATD) dry eye, participated in a prospective non-comparative interventional case series. Symptom scores, face scores, tear evaporation rates, fluorescein and rose bengal vital staining, tear break up time (BUT), Schirmer test, meibomian gland obstruction, and meibography were compared before and after 2 weeks of therapy. RESULTS: In a total of 37 cases, total subjective symptom scores and subjective face scores improved significantly, from 12.3 (SD 5.9) to 8.4 (6.1), and from 7.0 (1.7) to 5.3 (2.0) (both p <0.0001). The results for tear evaporation rates during forced blinking (p = 0.002), fluorescein staining (p = 0.03), rose bengal staining (p = 0.03), BUT (p <0.0001), and meibomian gland orifice obstruction score (p <0.0001) had also improved significantly at the end of the 2 week period of infrared thermotherapy. No complaints and/or complications of the IWCD were reported. CONCLUSION: The IWCD was effective and safe for the treatment of MGD. Improved tear stability associated with release of meibum is a possible mechanism of this treatment.

[Thymic carcinoma].

Thymic epithelial tumors are mainly consisted of thymoma, thymic carcinoma, and thymic carcinoid. And thymic carcinoma is very rare neoplasm. The classification of thymic carcinoma has remained a subject of controversy for many years. The outline of thymic carcinoma has been clarified by "Atlas of Tumor Pathology: Tumors of the Mediastium (AFIP)" and "Histrogical Typing of Tumours of the Thymus (WHO)" published recently. Squamous cell carcinoma is by far the most common in Japan. Thymic carcinoma is a predilection for male. The peak of age was 40-60 years. Thymic carcinoma already had contiguous invasion around neighbor organs, dissemination, and lymph node metastases or distant metastases at diagnosis. Two third of patients with thymic carcinoma performed surgery, and most of them performed adjuvant radiotherapy or chemotherapy. 5-year survival of thymic carcinoma was 33-50%. Histologic tumor type, type of tumor margin, growth pattern, nuclear atypia, necrosis and mitotic activity were correlated with survival. In this paper thymic carcinoma is reviewed mainly based on recently literatures and results obtained from a questionnaire on thymic epithelial tumors in Japan.

Effects of bile acids on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced aberrant crypt foci and DNA adduct formation in the rat colon.

The effects of deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced aberrant crypt foci (ACF) in the rat colon were examined. The effect of these bile acids on DNA adduct formation by PhIP in the colon was then analyzed, since the main action of PhIP is the formation of DNA adducts and subsequent gene mutations. For the ACF study, male F344 rats were administered PhIP-HCl (75 mg/kg, 10 doses) by gavage, and a diet containing bile acid (0.4% DCA or UDCA) was provided from 3 days before the first dose of PhIP for 8 weeks. The mean number of ACF per colon of DCA, UDCA and controls were 9.9, 2.4 and 5.5, respectively. The ACF number was significantly increased by DCA and decreased by UDCA (P<0.001). To examine the effect of bile acids on DNA adduct formation, male F344 rats were fed a diet supplemented with bile acids (0.1 or 0.4% of DCA and UDCA) 7 days prior to the PhIP administration. All rats were administered a single dose of PhIP-HCl (50 mg/kg) by gavage and sacrificed 48 hours later. DNA adduct levels of the 0.1% UDCA, 0.1% DCA and controls were 2.93 (adducts/10(7) nucleotides), 2.65 and 1.10, respectively. Those of 0.4% UDCA, 0.4% DCA and controls were 1.64, 1.30 and 1.00, respectively. The PhIP-DNA adduct level was significantly increased by administration of 0.1% UDCA, 0.1% DCA (P<0.05) and 0.4% UDCA (P<0.01). The increasing effect of both DCA and UDCA on PhIP-induced DNA adduct formation was unexpected, and was not directly associated with ACF formation.

Suppression of mediastinal metastasis by uracil-tegafur or cis-diamminedichloroplatinum(II) using a lymphogenous metastatic model in a human lung cancer cell line.

PURPOSE AND EXPERIMENTAL DESIGN: The extent of lymphatic metastasis is the most important factor in the prognosis for non-small cell lung cancer (NSCLC). Therefore, suppression of lymphatic metastasis provides an improvement in survival time in lung cancer patients. We established a new patient-like model for lung cancer metastasis by orthotopic implantation in severe combined immunodeficiency (SCID) mice and demonstrated the lymphogenous spread histologically using human NSCLC cell lines. The cardinal features of this model are a simple procedure and a similarity to the metastatic form of human lung cancer. The purpose of this study is to assess the inhibitory action of uracil-tegafur (UFT) and cis-diamminedichloroplatinum(II) (CDDP) on lymphatic metastasis and life span prolongation in our lymphogenous metastatic model system using SCID mice. RESULTS: The inhibition ratios of mediastinal lymph node metastasis were 86.2, 94, and 92.1% for 12 mg/kg body UFT, 17 mg/kg body UFT, and 10 mg/kg body CDDP, respectively. The administration of anticancer drugs prolonged the life span by 4.6 days (17 mg/kg body UFT) and 8 days (10 mg/kg body CDDP) in MST. CONCLUSION: We demonstrated that UFT alone and CDDP alone suppressed mediastinal metastasis and prolonged the life span in our lymphogenous metastatic model. Regardless of the administration route and characteristics of anticancer drugs, cytostatic or cytotoxic, our model is capable of evaluating the inhibitory effect of drugs on lymphatic metastasis. This model should make an important contribution to our understanding of the mechanism and selection of drugs for antilymphatic metastasis in lung cancer.

Sentinel lymph node biopsy in patients with papillary thyroid carcinoma.

BACKGROUND: It remains controversial whether modified radical neck dissection (MRND) for patients with papillary thyroid carcinoma improves prognosis. However, it is highly probable that the incidence of local recurrence is reduced by lymph node dissection. Sentinel lymph node (SLN) biopsy (SLNB) for patients with melanoma and breast carcinoma has been validated as an accurate method for assessing lymph node status. The objective of this study was to determine the feasibility of SLNB for the evaluation of cervical lymph node status in patients with papillary thyroid carcinoma. METHODS: After injection of methylene blue around the tumor in 22 patients with papillary thyroid carcinoma, blue-stained lymph nodes were dissected as SLNs. After the SLNB, all patients also underwent subtotal thyroidectomy and MRND. SLNs and other lymph nodes were investigated with regard to their number, distribution, size, lymph node status, and ratio of metastatic area. RESULTS: There was concordance between the SLN findings and the regional lymph node status in 19 of 21 patients (90.5%; 7 patients had both positive SLN and regional lymph node results, and 12 patients had both negative SLN and regional lymph node results). Two patients had negative SLN results but, in the end, had positive nonsentinel lymph nodes (NSLNs). The overall reliability rate of SLNB was 86.3% (19 of 22 patients). The authors experienced no complications with the use of methylene blue for the detection of SLNs. CONCLUSIONS: SLNB using methylene blue is feasible technically and is safe, and the findings correlate with cervical lymph node status. Therefore, SLNB is a good technique for estimating the status of cervical lymph nodes in patients with papillary thyroid carcinoma.

Malolactomycin D, a potent inhibitor of transcription controlled by the Ras responsive element, inhibits Ras-mediated transformation activity with suppression of MMP-1 and MMP-9 in NIH3T3 cells.

To search for anti-cancer agents, a screening system for Ras signal inhibitors was developed using a NIH3T3 cell line with an introduced reporter gene which is controlled by the Ras-responsive element (RRE). With this screening system, malolactomycin D was identified as a selective inhibitor of transcription from the RRE. This compound was found to preferentially inhibit the anchorage-independent growth rather than the anchorage-dependent growth of Ras-transformed NIH3T3 cells. The expression of matrix metalloproteinases MMP-1 and MMP-9, which have RRE in their promoters, were reduced by treatment with malolactomycin D at the translational and transcriptional levels. Analysis of the activity of mitogen-activated protein (MAP) kinases, which play important roles in transduction of the Ras signal, showed that malolactomycin D inhibits the activation of p38 MAP kinase and Jun N-terminal-kinase (JNK) but not extracellular signal-regulated kinase 1 or 2 (ERK1 or 2). These findings suggest that by inhibiting the pathway that leads to the activation of p38 MAP kinase and JNK, malolactomycin D suppresses the expression of MMPs. Since MMPs play important roles in metastasis and maintenance of the microenvironment of tumor cells, both of which facilitate tumor growth, the inhibition of MMPs by malolactomycin D is believed to contribute to its ability to inhibit Ras-mediated tumorigenesis.

Multilocular thymic cyst associated with Sjögren's syndrome.

A 61-year-old woman, who had been diagnosed as having Sjögren's syndrome, developed an anterior mediastinal mass. She was diagnosed with Sjögren's syndrome with thymoma, preoperatively. Extended thymectomy was performed. Macroscopically, the mediastinal mass showed thick-walled multiloculated cavities filled with turbid yellow fluid. Microscopically, the cyst lining was continuous with thymic lobules in the wall with inflammatory process, cholesterol granuloma formation, and prominent lymph follicular hyperplasia. She was diagnosed with multilocular thymic cysts associated with Sjögren's syndrome.

Identification and quantification of tamoxifen-DNA adducts in the liver of rats and mice.

A new HPLC gradient system was developed for (32)P-postlabeling analysis to identify and quantify hepatic tamoxifen-DNA adducts of rats and mice treated with tamoxifen. Four stereoisomers of alpha-(N(2)-deoxyguanosinyl)tamoxifen (dG(3')(P)-N(2)-TAM), alpha-(N(2)-deoxyguanosinyl)-N-desmethyltamoxifen (dG(3')(P)-N(2)-N-desmethyl-TAM), and alpha-(N(2)-deoxyguanosinyl)tamoxifen N-oxide (dG(3')(P)-N(2)-TAM N-oxide) were prepared by reacting either alpha-acetoxytamoxifen, alpha-acetoxy-N-desmethyltamoxifen or alpha-acetoxytamoxifen N-oxide with 2'-deoxyguanosine 3'-monophosphate, and used as standard markers for (32)P-postlabeling/HPLC analysis. Our HPLC gradient system can separate the above 12 nucleotide isomers as nine peaks; six peaks representing two each trans epimers (fr-1 and fr-2) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide, and three peaks representing a mixture of two cis epimers (fr-3 and fr-4) of nucleotides. Tamoxifen was given to female F344 rats and DBA/2 mice by gavage at doses of 45 mg/kg/day and 120 mg/kg/day, respectively, for 7 days. Totally 15 and 17 tamoxifen-DNA adducts were detected in rats and mice, respectively; among them 13 adducts were observed in both rats and mice. trans-dG-N(2)-TAM (fr-2) and trans-dG(3')(P)-N(2)-N-desmethyl-TAM (fr-2) were two major adducts in both animals. Except for these two adducts, trans-dG-N(2)-TAM N-oxide (fr-2) was the third abundant adduct that accounted for 6.4% of the total adducts in mice, while this accounted for only 0.3% in rats. A trans-isomer (fr-1) and cis-isomers (fr-3 and -4) of dG(3')(P)-N(2)-TAM, dG(3')(P)-N(2)-N-desmethyl-TAM and dG(3')(P)-N(2)-TAM N-oxide were also detected as minor adducts in both animals except for cis-form of dG-N(2)-TAM N-oxide in rats. Although the administered dose for rats was 2.7-fold less than that for mice, the total adduct level of rats (216 adducts/10(8) nucleotides) were 3.8-fold higher than mice (56.2 adducts/10(8) nucleotides). Thus, these three types of tamoxifen adducts accounted for 95.0 and 92.5% of the total DNA adducts of the rats and mice, respectively. The formation of tamoxifen adducts primarily resulted from alpha-hydroxylation of tamoxifen.

Prognostic value of CD4+ lymphocytes in pleural cavity of patients with non-small cell lung cancer.

BACKGROUND: For patients with non-small cell lung cancer the TNM staging system and other conventional prognostic factors fail to predict accurately the outcome of treatment and survival. This study attempts to determine the prognostic value for survival of the proportions of CD4+ lymphocytes in the pleural cavity (PLY) of patients with resectable non-small cell lung cancer. METHODS: Lymphocytes in the pleural cavity separated from 51 patients with non-small cell lung cancer were examined by flow cytometry to measure the proportions of CD4+ PLY. Univariate and multivariate analyses were performed to assess the association between the proportion of CD4+ PLY and survival. RESULTS: The 5 year survival rate of patients with percentage CD4+ PLY of < or = 30% was 84% whereas that of patients with %CD4+ PLY > 30% was 26.9%. The difference in survival between the %CD4+ PLY < or = 30% and %CD4+ PLY > 30% groups was significant (p < 0.0001). The %CD4+ PLY in those who survived for 5 years was significantly lower than that in the patients who died within 5 years (p < 0.0001). The difference in survival between patients with stage IA and IB lung cancer with %CD4+ PLY < or = 30% and those with %CD4+ PLY > 30% was also significant (p = 0.015). Multivariate analysis showed that the proportion of CD4+ PLY (hazard ratio = 6.9, 95% CI 0.045 to 0.47) and nodal status (hazard ratio = 22.7, 95% CI 0.006 to 1.806) are significant and independent prognostic factors for the survival of patients with lung cancer. CONCLUSIONS: The proportion of CD4+ PLY may help to select patients who are likely to have a poorer prognosis after surgery and therefore may be suitable for consideration of adjuvant treatments. These results need confirmation in a larger prospective study.

Occupational cancer genetics: infrequent ras oncogenes point mutations in lung cancer samples from chromate workers.

BACKGROUND: Chromium carcinogenicity and mutagenicity are no longer disputed. However, although chromium has various genetic effects that induce cancer, its mechanism of inducing lung cancer in humans is still not fully understood. p53, a tumor suppressor gene, was found to be infrequently mutated in samples of lung cancer in workers with long occupational exposure to chromium, suggesting other cancer-related genes to be targeted in such tumors. METHODS: To assess the contribution of the ras oncogenes in the pathogenesis of chromate-related lung cancer, we studied point mutations at the critical positions of codons 12, 13, and 61 of the Ha-ras and Ki-ras oncogenes in 38 lung cancer samples derived from Japanese patients who worked in the chromate industry for long periods. We used both radioactive isotope and non-radioisotope PCR-SSCP techniques. RESULTS: The results of this study demonstrated that activation of ras genes due to point mutations in chromate-related lung cancer is a rare event. CONCLUSIONS: Ras oncogenes activated by point mutations do not have a major role in the process of tumorigenesis of chromate-related lung cancer.

Effect of bile acids on formation of azoxymethane-induced aberrant crypt foci in colostomized F344 rat colon.

The effect of bile acids on the formation of azoxymethane induced aberrant crypt foci (ACF) was investigated using the fecal stream-excluded colons of colostomized F344 rats. The excluded colon was irrigated with saline or bile acids (1 mg/0.5 ml per day, 5 days/week) for 4 weeks. The mean numbers of ACF per colon in rats given cholic acid, deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), lithocholic acid, and ursodeoxycholic acid (UDCA) were 160.8, 118.2, 227.8, 150.7 and 87.3, respectively, while that of the control was 174.0. The number of ACF was significantly larger in CDCA, but smaller in UDCA and DCA-treated rats than the control (P<0.01). DCA did not induce apoptosis in the colon under the present conditions.

DNA adduct formation by 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) in rat colon.

A food-born carcinogen, 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) induces cancer in the rat colon. The mechanism for colonic DNA adduct formation leading to cancer by IQ was studied using a colostomized F344 rat model. In this model, the transverse colon of the rat was colostomized, which produced a fecal stream-positive proximal colon and a negative distal colon were produced. When IQ (50 mg/kg) was administered into the distal colon of the colostomized rats (n = 5), the ratio of the DNA adduct level of the distal colonic mucosa to the paired muscular layer 24 hr after dosage was 2.02, whereas that was 1.51 and 1.37 when IQ was administered into the stomach (n = 6) and the vein (n = 5), respectively. This suggested that luminal exposure of IQ induced DNA adduct formation. Since IQ (an amine form) has no reactivity toward DNA, these findings suggested that IQ was immediately activated in the absorbed mucosal cells and reacted with DNA. However, most of the IQ absorbed was metabolically activated in the liver, distributed by blood circulation, and formed DNA adducts in the colonic mucosa and muscular layer.

Activation of matrix metalloproteinase-2 is correlated with invasiveness in thymic epithelial tumors.

UNLABELLED: BACKGROUND AND OBJECTS: Matrix degradation, which is a critical event in the process of tumor invasion and metastasis, is considered to be caused by the action of proteolytic enzymes. METHODS: We examined the gelatinolytic activity of matrix metalloproteinase (MMP)-9, and the activity of active and inactive forms of MMP-2 in five thymi, five noninvasive thymomas, eight invasive thymomas, and five thymic carcinomas by quantitative gelatinolytic zymography. RESULTS: The gelatinolytic activity of active MMP-2 in five thymi was zero. The mean gelatinolytic activity of active MMP-2 was 0.020 +/- 0.015 in noninvasive thymoma, 0.084 +/- 0.098 in invasive thymoma and 0.246 +/- 0.194 in thymic carcinoma. The gelatinolytic activity of active MMP-2 correlated with the invasiveness of thymic epithelial tumors (Spearman rank correlation: r-value = 0.532). The gelatinolytic activity of active MMP-2 in three thymoma cases with microscopic capsular invasion was the same as that of noninvasive thymoma. When thymoma cases showing microscopic capsular invasion were classified into the "macroscopically noninvasive thymoma" group, the gelatinolytic activity of active MMP-2 correlated with the invasiveness of thymic epithelial tumors (Spearman rank correlation: r-value = 0.621). CONCLUSIONS: The gelatinolytic activity of active MMP-2 significantly correlated with the invasiveness in thymic epithelial tumors. J. Surg. Oncol. 2001;76:169-175.

Granulocyte/macrophage-colony-stimulating factor augments lymphokine-activated killer activity from lymphocytes via macrophages in lung cancer patients.

INTRODUCTION: Therapies with granulocyte/macrophage-colony-stimulating factor (GM-CSF) and interleukin(IL)-2 are designed to activate macrophages and lymphocytes. We investigated whether combined treatment with GM-CSF and IL-2 induced macrophage-mediated antitumor activity and/or T-cell-mediated antitumor activity in lung cancer patients. PATIENTS AND METHODS: Macrophages in the pleural cavity (PCM), lymphocytes in the pleural cavity (PLY), and peripheral blood lymphocytes (PBL) were separated from 48 patients with resectable lung cancer. Lymphokine-activated killer (LAK) activity was assayed by measuring 51Cr release. The proportion of PCM positive for the GM-CSF receptor (GM-CFSR) alpha chain was examined by flow-cytometric analysis and the expression of GM-CSFR alpha chain mRNA in PCM was examined by reverse transcription/polymerase chain reaction analysis. RESULTS: Treatment with GM-CSF developed no significant antitumor activity in PCM, PLY, or PBL. LAK activity was developed by PLY and PBL after incubation with IL-2. Stimulation with GM-CSF augmented LAK activities in PLY and PBL significantly, when these cells were cultured with autologous PCM (P < 0.05, P < 0.01). Moreover, GM-CSFR-alpha-chain-positive PCM had a higher potential to augmented LAK activities in PLY and PBL than had GM-CSFR-alpha-chain-negative PCM. CONCLUSION: These findings suggest that GM-CSF may stimulate GM-CSFR-alpha-chain-positive PCM and that these PCM may augment LAK activities developed by PLY and PBL. Combined treatment with GM-CSF and IL-2, therefore, may be a reasonable approach to treating lung cancer patients.

A mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine.

Alloproteins, proteins that contain unnatural amino acids, have immense potential in biotechnology and medicine. Although various approaches for alloprotein production exist, there is no satisfactory method to produce large quantities of alloproteins containing unnatural amino acids in specific positions. The tyrosine analogue azatyrosine, l-beta-(5-hydroxy-2-pyridyl)-alanine, can convert the ras-transformed phenotype to normal phenotype, presumably by its incorporation into cellular proteins. This provided the stimulus for isolation of a mutant tyrosyl-tRNA synthetase (TyrRS) capable of charging azatyrosine to tRNA. A plasmid library of randomly mutated Escherichia coli tyrS (encoding TyrRS) was made by polymerase chain reaction techniques. The desired TyrRS mutants were selected by screening for in vivo azatyrosine incorporation of E. coli cells transformed with the mutant tyrS plasmids. One of the clones thus isolated, R-6-A-7, showed a 17-fold higher in vivo activity for azatyrosine incorporation than wild-type TyrRS. The mutant tyrS gene contained a single point mutation resulting in replacement of phenylalanine by serine at position 130 in the protein. Structural modeling revealed that position 130 is located close to Asp(182), which directly interacts with tyrosyladenylate. Kinetic analysis of aminoacyl-tRNA formation by the wild-type and mutated F130S TyrRS enzymes showed that the specificity for azatyrosine, measured by the ratios of k(cat)/K(m) for tyrosine and the analogue, increased from 17 to 36 as a result of the F130S mutation. Thus, the high discrimination against azatyrosine is significantly reduced in the mutant enzyme. These results suggest that utilization of F130S TyrRS for in vivo protein biosynthesis may lead to efficient production of azatyrosine-containing alloproteins.