Randomised clinical trial: oesophageal radiofrequency energy delivery versus sham for PPI-refractory heartburn.

BACKGROUND: Oesophageal radiofrequency reduces use of proton pump inhibitors (PPIs) in patients with gastro-oesophageal reflux disease responding to PPIs. AIM: To determine the efficacy of oesophageal radiofrequency in patients with PPI-refractory heartburn. METHODS: A randomised, double-blind, sham-controlled multicentre study was designed to assess the efficacy of oesophageal radiofrequency in PPI non-responding patients with heartburn. Patients had moderate-to-severe heartburn defined by at least 3 occurrences a week, and not improved by continuous PPI treatment. The primary endpoint was clinical success at week 24, defined by intake of less than 7 PPI doses over the 2 preceding weeks and adequate symptom control determined by the patient. RESULTS: Sixty two patients were randomised, 29 to the oesophageal radiofrequency group and 33 to the sham group. Intention-to-treat analysis showed that 1/29 (3.4%) and 5/33 (15.1%) achieved the primary endpoint in the oesophageal radiofrequency and sham groups, respectively (NS). There was no significant difference between oesophageal radiofrequency and sham regarding the number of days without heartburn, days with PPI consumption in the last 2 weeks, and patients not taking PPIs. No pH-impedance parameter was associated with clinical response. The occurrence of adverse events was similar in both groups. CONCLUSION: This sham-controlled, randomised study did not demonstrate any efficacy of oesophageal radiofrequency for the treatment of PPI-refractory heartburn regarding symptom relief or consumption of PPIs. ClinicalTrials.gov NCT01682265.

Attenuation of radiation toxicity by the phosphine resistance factor dihydrolipoamide dehydrogenase (DLD).

Phosphine gas is an excellent fumigant for disinfesting stored grain of insect pests, but heavy reliance on phosphine has led to resistance in grain pests that threatens its efficacy. Phosphine-resistance was previously reported to be mediated by the enzyme DLD. Here we explore the relationship between phosphine toxicity and genotoxic treatments with the goal of understanding how phosphine works. Specifically, we utilized mutant lines either sensitive or resistant to phosphine, gamma irradiation or UV exposure. The phosphine-resistance mutation in the enzyme of energy metabolism, dihydrolipoamide dehydrogenase exhibited cross-resistance to UV and ionizing radiation. Two radiation-sensitive mutants that are defective in DNA repair as well as a mutant that is defective in the activation of the DAF-16 stress response transcription factor all exhibit sensitivity to phosphine that exceeds the sensitivity of the wild type control. A radiation resistance mutation in cep-1, the p53 orthologue, that is deficient in double strand break repair of DNA and is also deficient in apoptosis causes radiation-resistance results but sensitivity toward phosphine.

Three Cases of Levodopa-Resistant Parkinsonism After Radiation Therapy.

BACKGROUND Unequivocal brain radiation-induced parkinsonism has so far been reported in only in two pediatric patients. However, with the rising incidence rates for brain tumors in industrialized countries and the consequential increased exposure to cranial radiotherapy, clinicians might become more exposed to this entity. CASE REPORT Three patients were treated for intraparenchymal brain tumor with resection, chemotherapy, and whole brain radiation. One patient developed leukoencephalopathy and parkinsonism within one year of treatment, one developed it seven years after treatment completion, and one developed dementia, parkinsonism and cerebral infracts 40 years after whole brain radiation. Brain MRIs and a DaTscan were obtained. All patients failed a trial of carbidopa/levodopa. We suggest that the brain radiation exposure was responsible for levodopa resistant parkinsonism, cognitive decline, and diffuse leukoencephalopathy. CONCLUSIONS Although rare, radiation therapy-induced parkinsonism might be responsible for levodopa-resistant parkinsonism.

Short Time Efficiency of Rhinophototherapy in Management of Patients with Allergic Rhinitis Resistant to Medical Therapy.

Allergic rhinitis is one of the most common health problems with a major effect on the quality of life. We intended to treat Allergic Rhinitis (AR) in patients who are either unresponsive to antihistamines or their job requires optimal alertness that may be disturbed by antihistamine's side effects and those who do not comply with the regular use. We tried short term phototherapy and evaluated its effect on AR. As phototherapy is effective in the treatment of atopic dermatitis (AD) and the same allergens can produce both AD and AR, phototherapy is proposed as a new tool in the AR treatment. In AD, phototherapy causes induction of apoptosis in infiltrating T cells and other immunomodulatory effects. We performed a randomized single-blind study to investigate the effect of low-dose phototherapy in AR patients. Among AR patients who did not respond to local and systemic therapy, we chose 62 allergic patients all above 25 years of age with moderate to severe AR whose disease was verified by allergy skin test or specific IgE to allergens; then, they were randomly divided into 31 patients as treatment group and 31 patients as control group. In treatment groups, we used a mixture of UVA, UVB and visible light. In the control group, we used visible light alone as placebo. Then we evaluated the level of response to treatment in two groups and compared them according to Total Nasal Symptom scores (TNSS) and Global Severity Scores (GSS) and Rhinoconjunctivitis Quality of Life Questionnaires (RQLQ) symptom scores. We found out that phototherapy in the treatment group in comparison with placebo was effective in treatment of AR (p-value <0.001). However, we recommend that for substantiation of the claim, further investigations are still required.

Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80 % of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC- 9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC (AU)

No disponible

Co-Delivery of Cisplatin Prodrug and Chlorin e6 by Mesoporous Silica Nanoparticles for Chemo-Photodynamic Combination Therapy to Combat Drug Resistance.

Combination therapy shows great promise in circumventing cisplatin resistance. We report herein the development of a novel nanoscale drug delivery system (nDDS) based nanotherapeutic that combines chemotherapy and photodynamic therapy (PDT) into one single platform to achieve synergistic anticancer capacity to conquer cisplatin resistance. Mesoporous silica nanoparticle (MSNs) was used as the drug delivery vector to conjugate cisplatin prodrug and to load photosensitizer chlorin e6 (Ce6) to afford the dual drug loaded delivery system MSNs/Ce6/Pt. The hybrid nanoparticles have an average diameter of about 100 nm and slightly positive surface charge of about 18.2 mV. The MSNs/Ce6/Pt nanoparticles can be efficiently internalized by cells through endocytosis, thereby achieving much higher cellular Pt uptake than cisplatin in cisplatin-resistant A549R lung cancer cells. After 660 nm light irradiation (10 mW/cm(2)), the cellular reactive oxygen species (ROS) level in MSNs/Ce6/Pt treated cells was elevated dramatically. As a result of these properties, MSNs/Ce6/Pt exhibited very potent anticancer activity against A549R cells, giving a half-maximal inhibitory concentration (IC50) value for the combination therapy of 0.53 µM, much lower than that of cisplatin (25.1 µM). This study suggests the great potential of nDDS-based nanotherapeutic for combined chemo-photodynamic therapy to circumvent cisplatin resistance.

SPECT de perfusión, SISCOM y PET 18F-FDG en la valoración del paciente epiléptico fármaco-resistente candidato a cirugía de epilepsia / Perfusion SPECT, SISCOM and PET 18F-FDG in the assessment of drug- refractory epilepsy patients candidates for epilepsy surgery

Objetivos. La SPECT de perfusión ictal-interictal, subtraction ictal SPECT coregistered to MRI (SISCOM) y 18F-FDG-PET (interictal), desempeñan un papel fundamental en la valoración prequirúrgica del paciente epiléptico fármaco-resistente. Los objetivos de este trabajo fueron establecer la reproducibilidad del análisis visual de la SPECT y SISCOM y la capacidad de la SPECT, SISCOM y PET en la identificación del foco epileptógeno. Material y métodos. Se realizó una SPECT 99mTc-HMPAO (ictal-interictal) y SISCOM (Analyze 7.0) en 47 pacientes epilépticos fármaco-resistentes (24 M, 19-60 años). En 13 pacientes se repitió el SISCOM utilizando el programa FocusDET. El análisis de las imágenes fue realizado por 2 observadores. Se valoró la reproducibilidad utilizando el índice Kappa. Los resultados conjuntos de la SPECT, SISCOM y PET, en 16 pacientes, fueron comparados con la localización del área resecada y el seguimiento clínico poscirugía (escala de Engel) o con la estereo-EEG. Resultados. Grado de acuerdo interobservador de la SPECT 91% índice Kappa 0,86. Grado de acuerdo interobservador SISCOM Analyze 7.0 82%, índice Kappa 0,80. El Analyze 7.0 mostró un elevado número de resultados no concluyentes, superior al del análisis visual. El SISCOM FocusDET mostró un grado de acuerdo interobservador 92% con un índice Kappa 0,87 y menor número de resultados no concluyentes que el Analyze. La valoración conjunta SPECT, SISCOM y PET permitió identificar 87% focos epileptógenos: 79% temporales, 26% parieto-temporales y 7% frontales. Conclusión. La SPECT ictal-interictal y el SISCOM mostraron una elevada reproducibilidad. La valoración conjunta de la SPECT ictal-interictal, SISCOM y PET permitió mejorar la rentabilidad diagnóstica de la valoración individualizada (AU)

Aims. Brain perfusion SPECT (ictal-interictal), SPECT images and subtraction ictal SPECT coregistered to MRI (SISCOM) and 18F-FDG-PET (interictal), play an important role in the pre-surgical diagnosis of patients with medically refractory epilepsy. This study aimed to establish: the reproducibility of visual ictal-interictal SPECT and SISCOM analysis altogether with the capacity of SPECT, SISCOM and PET to determine the epileptogenic zone. Material and methods. 99mTc-HMPAO SPECT ictal-interictal and SISCOM (Analyze 7.0) were performed on 47 refractory epilepsy patients (24 F, 19-60 yrs). In 13 patients, SISCOM was also performed using a new program (Focus DET). Ictal-interictal SPECT and SISCOM images were analysed independently by two nuclear medicine physicians (observer 1 and 2). Kappa concordance coefficient was used to evaluate the reproducibility. In sixteen patients, SPECT, SISCOM and PET findings were compared with the resected area during the surgery, and surgical outcome using Engel scale or with the stereo EEG-(SEEG). Results. The ictal-interictal SPECT interobserver agreement was 91%, Kappa index 0.86, SISCOM (Analyze 7.0) interobserver agreement percentage was 82%, Kappa index 0.80, Analyze 7.0 showed a higher inconclusive results than visual SPECT analysis. SISCOM FocusDET interobserver agreement was 92%, Kappa index 0.87, with lower inconclusive results than Analyze 7.0. SPECT, SISCOM and PET combined findings identified 87% seizure onset zone: 79% temporal, 26% parieto-temporal and 7% frontal. Conclusions. Ictal-interictal SPECT and SISCOM showed a high reproducibility in this sample of patients with drug-refractory epilepsy. SPECT,SISCOM and PET combined findings improved detection of epileptogenic zone in comparison with the individual assessment (AU)

TLI in refractory chronic GVHD.

Refractory chronic GVHD (cGVHD) remains a major cause of morbidity after transplantation. Many drugs are used but there is no consensus on the standard of care. We investigated the efficacy of TLI in corticosteroid-refractory cGVHD. We analyzed retrospectively 31 patients receiving one or more TLI session for refractory cGVHD from 2000 to 2007. The main objective was to evaluate the response rate after TLI. Decreased corticosteroid doses and/or discontinued immunosuppressive agents were considered to be surrogate markers of response. All but one patient presented with severe cGVHD at the time of TLI. The median number of previous immunosuppressive treatment lines was 3 (range: 2-4). Fourteen patients (45%) achieved an objective response after TLI and 8 (25%) were cGVHD free at long-term follow-up. In all, 5 (29%) of the 17 nonresponsive patients did not show the features of progressive cGVHD and could decrease the amount of immunosuppressive drugs taken. Response after TLI significantly improved 5-year GVHD-related mortality (14% vs 42%, P=0.038) but not OS (58%vs 64% P=0.27). Regarding the promising response rate in this heavily pretreated population, we reasoned that TLI could be an alternative treatment for corticosteroid-refractory cGVHD.

Resistencia al tratamiento de primera línea de quimioterapia en un paciente diagnosticado de linfoma de Hodgkin de predominio linfocítico nodular identificada mediante 18F-FDG PET/TAC: implicaciones diagnósticas y terapéuticas / Resistance to first line chemotherapy treatment in a patient diagnosed of nodular lymphocyte-predominant Hodgkin's lymphoma identified by 18F-FDG PET/CT: diagnostic and therapeutic implications

El linfoma de Hodgkin de predominio linfocítico nodular (LHPLN) es una rara entidad que representa menos del 5% de los casos de linfoma de Hodgkin (LH) con rasgos morfológicos, inmunofenotípicos, genéticos y de comportamiento clínico distintos del LH clásico. En una minoría de pacientes, la evolución del LHPLN se complica por transformación a un linfoma no Hodgkin difuso B de células grandes (LNHDBCG) con implicaciones pronósticas y terapéuticas. Los cambios metabólicos precoces observados mediante 18F-FDG PET en pacientes con LH y LNH, después de 1-3 ciclos de quimioterapia predicen la respuesta final al tratamiento y la supervivencia libre de progresión. En el caso que presentamos, tanto si se trata de LHPLN transformado a LNHDBCG o la coexistencia de los dos tipos de linfoma en el mismo paciente, la exploración 18F-FDG PET/TAC fue determinante para identificar la resistencia del tumor a la primera línea de quimioterapia, orientar la toma de una segunda biopsia y modificar el régimen de quimioterapia(AU)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare entity that accounts for less than 5% of the cases of Hodgkin lymphoma (HL) with morphological, immunophenotypical, genetic and clinical behavior traits different from the classic HL. In a minority of patients, the NLPHL course is complicated by a transformation to a non-Hodgkin diffuse large B-cell lymphoma (NHDLBCL) with prognostic and therapeutic implications. Early metabolic changes observed by 18F-FDG PET in patients with HL and NHL, after 1-3 cycles of chemotherapy, predict the final response to treatment and progression-free survival. In the case we are presenting herein, whether NLPHL is transformed to NHDLBCL or the two types of lymphoma co-exist in the same patient, the 18F-FDG PET/CT scan was crucial for the identification of tumor resistance to first line chemotherapy and to guide a second biopsy decision and therefore modify the chemotherapy regimen(AU)

RAD53 is limiting in double-strand break repair and in protection against toxicity associated with ribonucleotide reductase inhibition.

The yeast Chk2/Chk1 homolog Rad53 is a central component of the DNA damage checkpoint system. While it controls genotoxic stress responses such as cell cycle arrest, replication fork stabilization and increase in dNTP pools, little is known about the consequences of reduced Rad53 levels on the various cellular endpoints or about its roles in dealing with chronic vs. acute genotoxic challenges. Using a tetraploid gene dosage model in which only one copy of the yeast RAD53 is functional (simplex), we found that the simplex strain was not sensitive to acute UV radiation or chronic MMS exposure. However, the simplex strain was sensitized to chronic exposure of the ribonucleotide reductase inhibitor hydroxyurea (HU). Surprisingly, reduced RAD53 gene dosage did not affect sensitivity to HU acute exposure, indicating that immediate checkpoint responses and recovery from HU-induced stress were not compromised. Interestingly, cells of most of the colonies that arise after chronic HU exposure acquired heritable resistance to HU. We also found that short HU exposure before and after treatment of G2 cells with ionizing radiation (IR) reduced the capability of RAD53 simplex cells to repair DSBs, in agreement with sensitivity of RAD53 simplex strain to high doses of IR. We propose that a modest reduction in Rad53 activity can impact the activation of the ribonucleotide reductase catalytic subunit Rnr1 following stress, reducing the ability to generate nucleotide pools sufficient for DNA repair and replication. At the same time, reduced Rad53 activity may lead to genome instability and to the acquisition of drug resistance before and/or during the chronic exposure to HU. These results have implications for developing drug enhancers as well as for understanding mechanisms of drug resistance in cells compromised for DNA damage checkpoint.

FOXO transcription factors enforce cell cycle checkpoints and promote survival of hematopoietic cells after DNA damage.

The PI3K/AKT signaling pathway contributes to cell cycle progression of cytokine-dependent hematopoietic cells under normal conditions, and it is absolutely required to override DNA damage-induced cell cycle arrest checkpoints in these cells. Phosphatidylinositol-3-kinase (PI3K)/AKT activity also correlates with Cdk2 activity in hematopoietic cells, suggesting that Cdk2 activation may be a relevant end point for this signaling pathway. However, mediators downstream of AKT in this pathway have not been defined. The forkhead transcription factor O (FOXO) family are negatively regulated by AKT-dependent phosphorylation and are known regulators of genes affecting cell cycle progression. We show that enhanced FOXO activity replicates the effect of PI3K inhibitors in enforcing G(1) and G(2) phase arrest after DNA damage. Conversely, knockdown of endogenous FOXO proteins increased Cdk2 activity and overrode DNA damage checkpoints in cells lacking PI3K activity. Moreover, loss of FOXO activity caused an increase in sensitivity to cisplatin-induced cell death, which was associated with failure to arrest cell cycle progression in the face of DNA damage caused by this chemotherapeutic agent. These cell cycle arrests were dependent on p27 expression when mediated by FOXO3a alone, but also involve p27-independent mechanisms when promoted by endogenous FOXO proteins. Together, these observations show that FOXO proteins enforce DNA damage-induced cell cycle arrest in hematopoietic cells. Inhibition of FOXO activity by cytokine-induced PI3K/AKT signaling is sufficient to override these DNA damage-induced cell cycle checkpoints, but may negatively impact hematopoietic cell viability.

Impact of temperature on susceptible and resistant strains of Culex quinquefasciatus to synthetic pyrethroids.

Effect of temperature on the physiological activity and resistance status of Culex quinquefasciatus was studied. The LT(50) (lethal time) of deltamethrin, lambdacyhalothrin resistant and susceptible 4th-instar larvae decreased with increasing temperature. The LT(50) of deltamethrin resistant strains was significantly lower than the susceptible strains but not so in lambdacyhalothrin resistant strains. The developmental period of the thermal exposed and non-exposed larvae of resistant strains were nearly equal, however a significant difference was observed in case of susceptible strain. The thermal exposed susceptible larvae developed 3.3 days faster than the thermal un-exposed population. The percentage of survival of thermal exposed and un-exposed resistant strains were nearly equal. However, the thermal exposed population survived 12.9% less than the thermal un-exposed susceptible population. Longevity of thermal exposed deltamethrin and lambdacyhalothrin resistant females was significantly higher than the un-exposed population. The longevity of thermal exposed susceptible population was lower than the un-exposed population. The resistance of both the deltamethrin and lambdacyhalothrin resistant strains increased when exposed to high temperature for 3h. The difference in longevity between thermal un-exposed and exposed resistant male population was not significantly different. Among the un-exposed population, highest pupation was recorded in the susceptible strains, and lowest in the deltamethrin resistant strain. The fact that the thermal exposed resistant strains were more successful than the un-exposed population could be related to the presence of resistant gene.

[Leptinotarsa decemlineata Say sensitivity to ionizing radiation and pesticides].

The effects of acute gamma-irradiation on the Colorado potato beetle (Leptinotarsa decemlineata Say) and sensitivity of insects, long time exposed on radioactively contaminated territories, to pesticides were studied. LD50 values of insects to ionizing radiation during the ontogenesis process was shown to increase (from 8 to 48 Gy). Differences were noted in the development rates of the natural population of beetles collected on agricultural lands with 137Cs contamination density of 0.05-1.2 MBq/m2, with the sensitivity to various pesticide groups being the same.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination.

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.

McCune-Albright syndrome and acromegaly: effects of hypothalamopituitary radiotherapy and/or pegvisomant in somatostatin analog-resistant patients.

BACKGROUND: Acromegaly, which may be present in patients with McCune-Albright syndrome (MCAS), in association with café-au-lait spots, precocious puberty, and fibrous dysplasia, is often difficult to treat surgically because skull base bone dysplasia prevents the removal of the pituitary adenoma. Somatostatin analogs (SAs) generally give only partial responses. The use of radiotherapy (RT) is controversial because of a possible risk of bone sarcomatous transformation. AIM: This study was a retrospective analysis of the efficacy and adverse effects of different treatment modalities in six patients with both MCAS and acromegaly. PATIENTS AND METHODS: Because surgery was impossible and SA failed to normalize GH/IGF-I hypersecretion, five of the six patients received fractionated RT (45-55 Grays). Three patients (two with previous RT) were also prescribed pegvisomant. We analyzed the clinical features of acromegaly, GH, and IGF-I concentrations and bone radiological features. RESULTS: GH and IGF-I concentrations fell after RT (median follow-up, 5 yr; range, 0.5-9 yr). Symptoms of acromegaly improved in parallel. Bone sarcomatous transformation was only noted in one patient in a region (the mandible) outside the radiation field. RT alone and/or combined with SA failed to normalize GH/IGF-I levels in the five patients concerned. In contrast, IGF-I levels normalized very rapidly (5-9 months) in the three patients receiving pegvisomant (10-20 mg/d). CONCLUSION: RT may be an option for the treatment of acromegaly in patients with MCAS when surgery is impossible and SA therapy is ineffective. However, although no bone sarcomatous transformation was observed within the radiation field in this series, this risk cannot be ruled out. As shown in this small series of severely affected patients, pegvisomant therapy may thus be useful to normalize IGF-I levels rapidly.

A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells.

We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.

Characterization of the altered anthranilate synthase in 5-methyltryptophan-resistant rice mutants.

In an earlier investigation, homologous mutant lines resistant to growth inhibition by 5-methyltryptophan (5MT) were selected from a callus that had been irradiated with a 50-Gy gamma ray during embryo culture. In order to identify the 5MT-resistant mechanism, we have continued our investigations of these mutant lines and studied the anthranilate synthase activity of the M5) advanced lines by direct fluorometric detection of the anthranilate formed in both control plants and mutant lines grown on 500 microM 5MT. The anthranilate synthase activity of the mutant plants was 2.2- to 3-fold higher than that of the control. In a kinetic analysis with tryptophan, an anthranilate synthase of the mutant lines was insensitive to feedback inhibition. These lines showed an enhanced accumulation of storage proteins and amino acids. The increased rates of protein synthesis in the mutant lines, relative to that of the control seeds, were 17-28.5%. The amino acid contents were 2.4-fold (MRI-40-2) to 2.6-fold (MRI-110-6) higher in the MRI lines than in the control seeds, and 2.4-fold (MRII-12-5) to 3.5-fold (MRII-8-1) higher in the MRII lines than in the control seeds. Significant increases among the amino acids of the MR lines were observed for tryptophan, phenylalanine, and tyrosine, which had been biosynthesized through the shikimate pathway. The transcript levels of putative OASA2, which is one of the key-regulating enzyme subunits in the tryptophan biosynthesis pathway, were studied in the control and 5MT-resistant mutant lines subjected to inhibition by two tryptophan analogs (5MT and alphaMT) and to other abiotic stresses (ABA, NaCl, and cold). The putative OASA2 gene in the 5MT-resistant mutant lines was highly expressed in at a low 5MT concentration and at an early stage of the 5MT and alphaMT treatments. However, mRNA accumulation of the putative OASA2 gene in the mutant plants gradually decreased when the plants were subjected to abiotic stresses such as NaCl and cold. These results indicated that the 5MT resistance in the mutant lines is due to altered anthranilate synthase forms.