Biomedical subjects

M Kondo

Publications and source records attributed to M Kondo.

At least 55 records · Page 3Linked to original sources

PubMed2001

Validity and safety of sputum induction by inhaled uridine 5'-triphosphate.

UNLABELLED: Inhalation of hypertonic saline during sputum induction causes bronchoconstriction. We studied the validity and safety of sputum induction by uridine 5'-triphosphate (UTP). Sputum was induced by a 5-min inhalation of hypertonic saline (3%) on Day 1 and UTP (5 mg/ml in 0.9% saline) on Days 8 and 15 in 16 healthy subjects and 16 patients with mild-to-moderate asthma. Inhaled UTP produced twofold greater amounts of sputum than did hypertonic saline. There were significant differences in oxygen desaturation and bronchoconstriction during the procedure between the two methods: the maximal fall in Sa(O(2)), the AUC of the Sa(O(2))-time response, and the fall in PEF were less in the subjects who received UTP than in those who received hypertonic saline. Sputum total cell and differential cell counts, with a high proportion of eosinophils in asthmatics, were similar between specimens obtained by hypertonic saline and UTP. When we compared two consecutive measurements on the UTP-induced sputum samples, the reproducibility calculated by the intraclass correlation coefficient was high for the proportion of eosinophils, neutrophils, and macrophages. Therefore, inhalation of UTP aerosols may provide an effective, relatively noninvasive, valid, and reproducible method of sputum induction for the assessment of airway inflammation in asthma. KEYWORDS: uridine triphosphate; induced sputum; airway inflammation; bronchoconstriction; asthma

Administration, Inhalation↗

PubMed2001

Chloroplasts have a novel Cpn10 in addition to Cpn20 as co-chaperonins in Arabidopsis thaliana.

Previously, we characterized a mitochondrial co-chaperonin (Cpn10) and a chloroplast co-chaperonin (Cpn20) from Arabidopsis thaliana (Koumoto, Y., Tsugeki, R., Shimada, T., Mori, H., Kondo, M., Hara-Nishimura, I., and Nishimura, M. (1996) Plant J. 10, 1119-1125; Koumoto, Y., Shimada, T., Kondo, M., Takao, T., Shimonishi, Y., Hara-Nishimura, I., and Nishimura, M. (1999) Plant J. 17, 467-477). Here, we report a third co-chaperonin. The cDNA was 603 base pairs long, encoding a protein of 139 amino acids. From a sequence analysis, the protein was predicted to have one Cpn10 domain with an amino-terminal extension that might work as a chloroplast transit peptide. This novel Cpn10 was confirmed to be localized in chloroplasts, and we refer to it as chloroplast Cpn10 (chl-Cpn10). The phylogenic tree that was generated with amino acid sequences of other co-chaperonins indicates that chl-Cpn10 is highly divergent from the others. In the GroEL-assisted protein folding assay, about 30% of the substrates were refolded with chl-Cpn10, indicating that chl-Cpn10 works as a co-chaperonin. A Northern blot analysis revealed that mRNA for chl-Cpn10 is accumulated in the leaves and stems, but not in the roots. In germinating cotyledons, the accumulation of chl-Cpn10 was similar to that of chloroplastic proteins and accelerated by light. It was proposed that two kinds of co-chaperonins, Cpn20 and chl-Cpn10, work independently in the chloroplast.

Amino Acid Sequence↗

PubMed2001

FK506 inhibits Cl- secretion in airway epithelium via calcineurin-independent mechanism.

FK506 (tacrolimus)-binding protein (FKBP) is associated with intracellular Ca2+ release channel and modulates its function. To elucidate the effect of FK506 on Ca2+ dynamics and Ca2+-mediated Cl- secretion in airway epithelium, we studied intracellular Ca2+ ([Ca2+]i) concentration and Cl(-)-dependent short-circuit current (Isc), in cultured bovine tracheal epithelial cells. Addition of ATP induced an increase in [Ca2+]i, and this response was dose dependently inhibited by FK506. Rapamycin, which binds FKBP with high affinity, likewise inhibited the [Ca2+]i rise, but cyclosporin A, a specific calcineurin inhibitor, did not. In Cl- secretion studies using Ussing chamber, ATP increased Ca2+-mediated Isc in amiloride-treated cells, an effect that was inhibited by FK506 and rapamycin but not by cyclosporin A. Therefore, FK506 inhibits Ca2+ mobilization in airway epithelium via FKBP but not calcineurin-dependent mechanism, which may result in the suppression of Ca2+-activated Cl- secretion.

Adenosine Triphosphate↗

PubMed2001

Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression.

BACKGROUND: The recently identified RASSF1 locus is located within a 120-kilobase region of chromosome 3p21.3 that frequently undergoes allele loss in lung and breast cancers. We explored the hypothesis that RASSF1 encodes a tumor suppressor gene for lung and breast cancers. METHODS: We assessed expression of two RASSF1 gene products, RASSF1A and RASSF1C, and the methylation status of their respective promoters in 27 non-small-cell lung cancer (NSCLC) cell lines, in 107 resected NSCLCs, in 47 small-cell lung cancer (SCLC) cell lines, in 22 breast cancer cell lines, in 39 resected breast cancers, in 104 nonmalignant lung samples, and in three breast and lung epithelial cultures. We also transfected a lung cancer cell line that lacks RASSF1A expression with vectors containing RASSF1A complementary DNA to determine whether exogenous expression of RASSF1A would affect in vitro growth and in vivo tumorigenicity of this cell line. All statistical tests were two-sided. RESULTS: RASSF1A messenger RNA was expressed in nonmalignant epithelial cultures but not in 100% of the SCLC, in 65% of the NSCLC, or in 60% of the breast cancer lines. By contrast, RASSF1C was expressed in all nonmalignant cell cultures and in nearly all cancer cell lines. RASSF1A promoter hypermethylation was detected in 100% of SCLC, in 63% of NSCLC, in 64% of breast cancer lines, in 30% of primary NSCLCs, and in 49% of primary breast tumors but in none of the nonmalignant lung tissues. RASSF1A promoter hypermethylation in resected NSCLCs was associated with impaired patient survival (P =.046). Exogenous expression of RASSF1A in a cell line lacking expression decreased in vitro colony formation and in vivo tumorigenicity. CONCLUSION: RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

Adult↗

PubMed2001

Impact of HIV type 1 protease, reverse transcriptase, cleavage site, and p6 mutations on the virological response to quadruple therapy with saquinavir, ritonavir, and two nucleoside analogs.

Genotype alterations of HIV-1 protease, reverse transcriptase, cleavage sites p7/p1 and p1/p6, as well as p6(gag) and transframe protein p6* were studied in an observational cohort of 42 individuals who received antiretroviral therapy consisting of saquinavir, ritonavir, and two nucleoside analogs. In a multivariate logistic regression analysis, the prior protease inhibitor experience (odds ratio, 6.20; 95% CI, 1.22-31.38) and the presence of primary protease mutations (odds ratio, 9.99; 95% CI, 1.05-94.72) were independently associated with virological failure. Moreover, a trend was observed in that individuals with N-terminal amino acid insertions in the proline-rich motif of the p6(gag) protein were less likely to experience virological failure (OR, 0.17; 95% CI, 0.02-1.35; p = 0.09). In contrast, the presence of secondary protease, reverse transcriptase, or cleavage site mutations was not independently associated with treatment failure. However, mutations at cleavage site p7/p1 (p = 0.01) and C-terminal p6* mutations (p = 0.02) were both associated with primary protease mutations. In conclusion, the presence of primary protease mutations was the most important predictor of the subsequent virological response. Moreover, there is some evidence that insertions in the proline-rich area of the p6(gag) protein may affect the virological response. The relationship between mutations of cleavage sites or C-terminal p6* residues and protease mutations suggests that these alterations may serve a compensatory role, increasing viral fitness.

Adult↗

PubMed2001

Effects of peptide dimerization on pore formation: Antiparallel disulfide-dimerized magainin 2 analogue.

To elucidate the effects of peptide dimerization on pore formation by magainin 2 (MG2), a covalently linked antiparallel dimer of the MG2 analogue [(F5Y, L6C, F16W, I20C-MG2)(2): II] was synthesized based on the dimer structure revealed by our NMR study. The interactions of the dimer with lipid bilayers were investigated by CD and fluorescence in comparison with a monomer analogue (F5Y, F16W-MG2: I). Similar to I, II was found to form a peptide-lipid supramolecular complex pore accompanied with lipid flip-flop and peptide translocation. The pore formed by II was characterized by a slightly larger pore diameter and a threefold longer lifetime than that of I, although the pore formation rate of the dimer was lower than that of the monomer. The coexistence of the dimer and the monomer exhibited slight but significant synergism in membrane permeabilization, which was maximal at a monomer/dimer ratio of 3. Therefore, we concluded that a pentameric pore composed of one pore-stabilizing dimer and three monomers maximized the overall leakage activity in keeping with our kinetic prediction.

Amino Acid Sequence↗

PubMed2001

Feedback inhibition of amidophosphoribosyltransferase regulates the rate of cell growth via purine nucleotide, DNA, and protein syntheses.

To clarify the contributions of amidophosphoribosyltransferase (ATase) and its feedback regulation to the rates of purine de novo synthesis, DNA synthesis, protein synthesis, and cell growth, mutated human ATase (mhATase) resistant to feedback inhibition by purine ribonucleotides was engineered by site-directed mutagenesis and expressed in CHO ade (-)A cells (an ATase-deficient cell line of Chinese hamster ovary fibroblasts) and in transgenic mice (mhATase-Tg mice). In Chinese hamster ovary transfectants with mhATase, the following parameters were examined: ATase activity and its subunit structure, the metabolic rates of de novo and salvage pathways, DNA and protein synthesis rates, and the rate of cell growth. In mhATase-Tg mice, ATase activity in the liver and spleen, the metabolic rate of the de novo pathway in the liver, serum uric acid concentration, urinary excretion of purine derivatives, and T lymphocyte proliferation by phytohemagglutinin were examined. We concluded the following. 1) ATase and its feedback inhibition regulate not only the rate of purine de novo synthesis but also DNA and protein synthesis rates and the rate of cell growth in cultured fibroblasts. 2) Suppression of the de novo pathway by the salvage pathway is mainly due to the feedback inhibition of ATase by purine ribonucleotides produced via the salvage pathway, whereas the suppression of the salvage pathway by the de novo pathway is due to consumption of 5-phosphoribosyl 1-pyrophosphate by the de novo pathway. 3) The feedback inhibition of ATase is more important for the regulation of the de novo pathway than that of 5-phosphoribosyl 1-pyrophosphate synthetase. 4) ATase superactivity leads to hyperuricemia and an increased bromodeoxyuridine incorporation in T lymphocytes stimulated by phytohemagglutinin.

Adenosine Monophosphate↗

PubMed2001

Functional imaging of the brain in sedated newborn infants using near infrared topography during passive knee movement.

Near infrared topography was used for functional imaging of the sensorimotor cortex of newborn infants during passive knee movement under sedated sleep. Contralateral knee movement caused a marked increase in oxyhemoglobin and total hemoglobin from the baseline values at almost all locations in the primary sensorimotor area of all neonates and a decrease in local deoxyhemoglobin in six of seven neonates. During ipsilateral knee movement, oxyhemoglobin and total hemoglobin showed slighter changes at a few locations, equal to 30% (mean) and 29% (mean) of the changes that occurred with contralateral stimulation, respectively. The mean times corresponding to maximal changes were 11.9 s for oxyhemoglobin and 19.1 s for deoxyhemoglobin, demonstrating that oxyhemoglobin has a much faster response than does deoxyhemoglobin.

Brain Mapping↗

PubMed2001

Searching for microsatellite mutations in coding regions in lung, breast, ovarian and colorectal cancers.

RepX represents a new informatics approach to probe the UniGene database for potentially polymorphic repeat sequences in the open reading frame (ORF) of genes, 56% of which were found to be actually polymorphic. We now have performed mutational analysis of 17 such sites in genes not found to be polymorphic (<0.03 frequency) in a large panel of human cancer genomic DNAs derived from 31 lung, 21 breast, seven ovarian, 21 (13 microsatellite instability (MSI)+ and eight MSI-) colorectal cancer cell lines. In the lung, breast and ovarian tumor DNAs we found no mutations (<0.03-0.04 rate of tumor associated open reading frame mutations) in these sequences. By contrast, 18 MSI+ colorectal cancers (13 cancer cell lines and five primary tumors) with mismatch repair defects exhibited six mutations in three of the 17 genes (SREBP-2, TAN-1, GR6) (P<0.000003 compared to all other cancers tested). We conclude that coding region microsatellite alterations are rare in lung, breast, ovarian carcinomas and MSI (-) colorectal cancers, but are relatively frequent in MSI (+) colorectal cancers with mismatch repair deficits.

Base Pair Mismatch↗

PubMed2001

Inhibition of the visual cycle in vivo by 13-cis retinoic acid protects from light damage and provides a mechanism for night blindness in isotretinoin therapy.

Isotretinoin (13-cis retinoic acid) is frequently prescribed for severe acne [Peck, G. L., Olsen, T. G., Yoder, F. W., Strauss, J. S., Downing, D. T., Pandya, M., Butkus, D. & Arnaud-Battandier, J. (1979) N. Engl. J. Med. 300, 329-333] but can impair night vision [Fraunfelder, F. T., LaBraico, J. M. & Meyer, S. M. (1985) Am. J. Ophthalmol. 100, 534-537] shortly after the beginning of therapy [Shulman, S. R. (1989) Am. J. Public Health 79, 1565-1568]. As rod photoreceptors are responsible for night vision, we administered isotretinoin to rats to learn whether night blindness resulted from rod cell death or from rod functional impairment. High-dose isotretinoin was given daily for 2 months and produced systemic toxicity, but this caused no histological loss of rod photoreceptors, and rod-driven electroretinogram amplitudes were normal after prolonged dark adaptation. Additional studies showed, however, that even a single dose of isotretinoin slowed the recovery of rod signaling after exposure to an intense bleaching light, and that rhodopsin regeneration was markedly slowed. When only a single dose was given, rod function recovered to normal within several days. Rods and cones both showed slow recovery from bleach after isotretinoin in rats and in mice. HPLC analysis of ocular retinoids after isotretinoin and an intense bleach showed decreased levels of rhodopsin chromophore, 11-cis retinal, and the accumulation of the biosynthetic intermediates, 11-cis and all-trans retinyl esters. Isotretinoin was also found to protect rat photoreceptors from light-induced damage, suggesting that strategies of altering retinoid cycling may have therapeutic implications for some forms of retinal and macular degeneration.

Animals↗

PubMed2001

A second susceptibility gene for developing rheumatoid arthritis in the human MHC is localized within a 70-kb interval telomeric of the TNF genes in the HLA class III region.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with a multifactorial genetic basis. However, pathogenic genes for RA other than the human leukocyte antigen (HLA)-DRB1 gene have yet to be identified. Here, we investigated whether there is a second susceptibility locus for RA within the human major histocompatibility complex using 18 microsatellite markers distributed from the centromeric (HSET) to the telomeric end (P5-15) of the 3.6-Mb HLA region. Statistical studies of associated alleles on each microsatellite locus showed that one pathogenic gene for RA in the HLA region is localized in the DRB1 gene, as expected. Further, a second susceptibility gene of RA was suggested to be present in the HLA class III region, narrowed to 70 kb, that is just telomeric of the TNF gene cluster (TNFA and LTA) and that is located between the microsatellites TNFa and C1-2-A. In this critical segment, four expressed genes have been thus far identified, NFKBIL1 (IkappaBL), ATP6G, BAT1, and MICB, all of which are candidate genes for determining susceptibility to RA. These results exclude the possibility of involvement of the TNFA genes (TNF-alpha) in the development of RA, which was suggested previously to be a strong candidate for RA in the class III region.

Alleles↗

PubMed2001

Expression of TMEFF1 mRNA in the mouse central nervous system: precise examination and comparative studies of TMEFF1 and TMEFF2.

TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and two follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebral neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated differently and that they play region-specific roles in the central nervous system.

Animals↗

PubMed2001

Nicotinamide-N-methyltransferase is higher in the lumbar cerebrospinal fluid of patients with Parkinson's disease.

Parkinson's disease (PD) may be initiated or precipitated by endogenous toxins with a structure similar to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in genetically-predisposed individuals. Nicotinamide N-methyltransferase (NNMT) catalyzes N-methylation of nicotinamide and other pyridines to form pyridinium ions. The protein amount of NNMT was measured in the lumbar cerebrospinal fluid of PD patients by immunoblot analysis using anti-human NNMT antibody. In younger (65 years old or younger) PD patients, the relative level of NNMT protein was significantly higher than that in younger controls. The NNMT protein was significantly affected by aging: the amount decreased along with aging in PD patients. These findings suggested that excess NNMT in the central nervous system might be implicated in the PD pathogenesis.

Aging↗

PubMed2001

Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress.

In the present study, we find that cyclopentenone prostaglandins (PGs) of the J(2) series, naturally occurring derivatives of PGD(2), are potential inducers of intracellular oxidative stress that mediates cell degeneration. Based on an extensive screening of diverse chemical agents on induction of intracellular production of reactive oxygen species (ROS), we found that the cyclopentenone PGs, such as PGA(2), PGJ(2), Delta(12)-PGJ(2), and 15-deoxy-Delta(12,14)-PGJ(2), showed the most potent pro-oxidant effect on SH-SY5Y human neuroblastoma cells. As the intracellular events that mediate the PG cytotoxicity, we observed (i) the cellular redox alteration represented by depletion of antioxidant defenses, such as glutathione and glutathione peroxidase; (ii) a transient decrease in the mitochondrial membrane potential (Deltapsi); (iii) the production of protein-bound lipid peroxidation products, such as acrolein and 4-hydroxy-2-nonenal; and (iv) the accumulation of ubiquitinated proteins. These events correlated well with the reduction in cell viability. In addition, the thiol compound, N-acetylcysteine, could significantly inhibit the PG-induced ROS production, thereby preventing cytotoxicity, suggesting that the redox alteration is closely related to the pro-oxidant effect of cyclopentenone PGs. More strikingly, the lipid peroxidation end products, acrolein and 4-hydroxy-2-nonenal, detected in the PG-treated cells potently induced the ROS production, which was accompanied by the accumulation of ubiquitinated proteins and cell death, suggesting that the membrane lipid peroxidation products may represent one of the causative factors that potentiate the cytotoxic effect of cyclopentenone PGs by accelerating intracellular oxidative stress. These data suggest that the intracellular oxidative stress, represented by ROS production/lipid peroxidation and redox alteration, may underlie the well documented biological effects, such as antiproliferative and antitumor activities, of cyclopentenone PGs.

Aldehydes↗

PubMed2001

Outcome prediction by molecular detection of minimal residual disease in bone marrow for advanced neuroblastoma.

BACKGROUND: We have determined whether sequential molecular detection of minimal residual disease (MRD) in bone marrow (BM) could predict the outcome of patients with advanced neuroblastoma (NB). PROCEDURE: Bone marrow samples from 19 patients over 12 months of age with stage 4 neuroblastoma were sequentially examined for tumor cell contamination by detecting tyrosine hydroxylase (TH) mRNA using reverse transcription-polymerase chain reaction (RT-PCR). All patients received repetitive multi-drug chemotherapy including cisplatin, cyclophosphamide or ifosphamide, adriamycin, and etoposide or vincristine. Seventeen patients received myeloablative therapy with hematopoietic stem cell transplantation after achieving complete remission. RESULTS: All but one patient were histologically positive for tumor cells in BM samples at diagnosis, and they became negative for tumor cells within 3 months histologically. By the RT-PCR analysis, all patients were positive for TH mRNA in BM samples at diagnosis, and they became negative for TH mRNA 1 to 13 months after the start of chemotherapy. Six patients whose BM samples became negative for TH mRNA within 4 months after the start of chemotherapy remained alive without evidence of disease (median 61 months, range 20-76). In contrast, 12 of 13 patients whose BM samples remained positive at that time developed relapse and 10 of them died of disease (median 24 months, range 13-43). There was a statistically significant difference in survival between the two groups (P < 0.05). No significant difference of clinical characteristics by the MRD positivity at 4 months after the start of chemotherapy. CONCLUSIONS: Persistence of MRD in BM at 4 months after the start of chemotherapy could predict poor prognosis in advanced neuroblastoma.

Adrenal Gland Neoplasms↗

PubMed2001

Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1.

Genes related to the Drosophila melanogaster doublesex and Caenorhabditis elegans mab-3 genes are conserved in human. They are identified by a DNA-binding homology motif, the DM domain, and constitute a gene family (DMRTs). Unlike the invertebrate genes, whose role in the sex-determination process is essentially understood, the function of the different vertebrate DMRT genes is not as clear. Evidence has accumulated for the involvement of DMRT1 in male sex determination and differentiation. DMRT2 (known as terra in zebrafish) seems to be a critical factor for somitogenesis. To contribute to a better understanding of the function of this important gene family, we have analyzed DMRT1, DMRT2, and DMRT3 from the genome model organism Fugu rubripes and the medakafish, a complementary model organism for genetics and functional studies. We found conservation of synteny of human chromosome 9 in F. rubripes and an identical gene cluster organization of the DMRTs in both fish. Although expression analysis and gene linkage mapping in medaka exclude a function for any of the three genes in the primary step of male sex determination, comparison of F. rubripes and human sequences uncovered three putative regulatory regions that might have a role in more downstream events of sex determination and human XY sex reversal.

Amino Acid Sequence↗

PubMed2001

Estimation of regional cerebral blood flow distribution in infants by near-infrared topography using indocyanine green.

Near-infrared topography with indocyanine green was used to measure regional cerebral blood flow (rCBF) in the temporal lobes of infants. The mean rCBF in infants without neural abnormality was 14.5 +/- 3.1 ml/100 g/min, and the rCBFs in the fronto-temporal, temporal, and occipito-temporal regions were 15.1 +/- 3.9, 15.4 +/- 3.3, and 14.6 +/- 3.3 ml/100 g/min, respectively. Moreover, in one asphyxiated infant with infarction and one infant with subdural and intracerebellar hemorrhage, it was demonstrated that the area of defective blood flow could be detected as well as it can by SPECT. This technique makes it possible to estimate rCBF distribution in infants at the bedside. Thus, in the future, evaluation of various neonatal illnesses should be feasible.

Asphyxia Neonatorum↗

PubMed2001

Safety evaluation of phosphodiesterase produced from Penicillium citrinum: summary of toxicological data.

The toxicity of Enzyme RP-1, an enzyme preparation used to hydrolyze yeast RNA to produce flavor enhancers for use in the food industry, was evaluated in a series of studies. A 5-week dietary toxicity study in Wistar rats was conducted in which animals received Enzyme RP-1 in feed at concentrations of 0, 500, 2000, or 8000 mg/kg body wt/day. A 13-week dietary toxicity study in Sprague-Dawley rats was conducted in which animals received RP-1 concentrate at 0, 0.125, 0.5, or 2% in their diets. At the highest dose levels in both studies, submandibular glands in the oral cavity were enlarged, an effect attributed to protease activity of the enzyme preparation. The no-observed-effect level in rats in the 13-week study was 0.5%, equivalent to 317 mg/kg body wt/day for males and 346 mg/kg body wt/day for females. Based on estimated dietary exposure to the enzyme preparation, the margin of exposure is estimated to be greater than 38,000. Lack of genotoxic potential was demonstrated by an in vitro reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and in Escherichia coli strain WP2uvr and by an in vitro chromosome aberration test in CHL/IU cells derived from fibroblasts from the lungs of Chinese hamsters. Finally, the particular strain of Penicillium citrinum, the fungal strain used to prepare Enzyme RP-1, was shown to have low pathogenicity upon a single injection into the tail vein of rats of viable spores at doses up to 2.8x10(5) colony-forming units per animal. The results of these studies demonstrate that the enzyme preparation may be considered safe to workers and consumers when employed in the production of flavor enhancers from yeast.

Administration, Oral↗