Use of Yarrowia lipolytica hexokinase for the quantitative determination of trehalose 6-phosphate

Abstract This paper describes a procedure for the quantitative determination of trehalose 6-phosphate (T6P) based on its ability to inhibit hexokinase from Yarrowia lipolytica . The assay is linear between 1 nmol and at least 8 nmol. The concentration of T6P in wild-type Saccharomyces cerevisiae (0.15 mM) and in ras2 mutants (0.25 mM) remained unchanged in the exponential or stationary phase of growth or after heat shock. A tps1 mutant affected in T6P synthase did not show detectable T6P. Heat shock increased the concentration of T6P in Schizosacharomyces pombe from 0.43 to 0.75 mM.     

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum （T22 ＋ P. ultimum） treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, aftel treatment with T22 ＋ P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 ＋ P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDITOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 ＋ P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS （pathogenesis-related maize seed）, GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host.     

The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia

Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor-recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.     

The antimetastatic function of concomitant antitumor immunity. II. Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells

Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen. This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter. It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice). Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor. By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice. However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90%. After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity. These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease. Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node. Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.     

Turgor, temperature and the growth of plant cells: using Chara corallina as a model system

Rapid changes in turgor pressure (P:) and temperature (T:) are giving new information about the mechanisms of plant growth. In the present work, single internode cells of the large-celled alga Chara corallina were used as a model for plant growth. P was changed without altering the chemical environment of the wall while observing growth without elastic changes. When P: was measured before any changes, the original growth rate bore no relationship to the original P. However, if P of growing cells was decreased, growth responded immediately without evidence for rapid changes in wall physical properties. Growth occurred only above a 0.3 MPa threshold, and increasing P caused small increases in growth that became progressively larger as P rose, resulting in a curvilinear response overall. The small changes in growth close to the threshold may explain early failures to detect these responses. When T was lowered, the elastic properties of the cell were unaffected, but growth was immediately inhibited. The lower T caused P to decrease, but returning P to its original value did not return growth to its original rate. The decreased P at low T occurred because of T effects on the osmotic potential of the cell. At above-normal P, growth partially resumed at low T Therefore, growth required a P-sensitive process that was also T-sensitive. Because elastic properties were little affected by T, but growth was markedly affected, the process is likely to involve metabolism. The rapidity of its response to P and T probably excludes the participation of changes in gene expression.     

Effects of triiodothyronine treatments on body and organ growth and the development of immune function in dwarf chickens

The effects of triiodothyronine (T3) treatments on general body growth, long bone growth, primary lymphoid organ development, antibody production, and serum growth hormone (GH) and thyroid hormone levels were examined in two dwarf strains (sex-linked dwarf--SLD, and autosomal dwarf--ADW) and in a normal-growing control strain (K) of White Leghorn chickens. One-day-old male chicks from each of these strains were assigned to either an untreated control group or to one of the groups receiving a T3 supplement ranging from 0.01 to 1.0 ppm. General body growth and long bone growth were significantly (P less than 0.05) stimulated only within the SLD strain by the intermediate T3 dosages. The 1.0-ppm T3 dosage level resulted in depressed body weights within both the K and ADW strains but produced no significant changes within the SLD strain. Thymic growth was significantly stimulated due to treatments of 0.1 ppm T3 in the SLD strain (P less than 0.05) and 1.0 ppm T3 in both the SLD and ADW strains (P less than 0.001 and P less than 0.05, respectively). Bursal growth was significantly depressed (P less than 0.05) at all T3 dosage levels within the SLD strain while 0.01 and 0.1-ppm T3 treatments resulted in significant bursal growth stimulation in the K and ADW strains, respectively. Concomitant with the depressed bursal growth, antibody production was significantly depressed (P less than 0.05) within the SLD strain at the 1.0-ppm T3 dosage level. Antibody production was not significantly affected by any of the T3 treatments within the control K or ADW strains. Serum T3 levels were significantly increased in all strains by the T3 supplementation but thyroxine (T4) serum levels were affected only within the SLD strain. The 0.01-ppm T3 treatment resulted in a significant increase (P less than 0.05) in serum T4 levels within this strain and treatment group. The only increase (P less than 0.05) in GH levels due to T3 treatments occurred within the same SLD treatment group. The higher T3 treatments resulted in serum GH levels being severely depressed (P less than 0.01) in all strains.     

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.     

Antifungal effects of herbal essential oils alone and in combination with ketoconazole against Trichophyton spp

AIMS: To determine the effects of herbal essential oils on Trichophyton spp. growth and to evaluate the effects of Pelargonium graveolens oil and its main components citronellol and geraniol combined with ketoconazole against Trichophyton spp. METHODS AND RESULTS: Growth inhibition of six Trichophyton spp. by herbal essential oils was accessed and the combined effects of P. graveolens oil and its main components citronellol and geraniol were evaluated using a checkerboard microtitre assay against T. schoenleinii, T. erinacei and T. soudanense. The essential oil fraction of P. graveolens and its main components, geraniol and citronellol, exhibited strong synergism with ketoconazole against T. schoenleinii and T. soudanense, with fractional inhibitory concentration (FIC) indices in the range of 0.18-0.38. CONCLUSIONS: The antifungal effects of ketoconazole against Trichophyton spp. are enhanced significantly by administering it in combination with the essential oil fraction of P. graveolens or its main components, because of strong synergism, especially against T. soudanense and T. schoenleinii. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of ketoconazole and the essential oil fraction from P. graveolens or its main components for treatment of infections caused by Trichophyton species may reduce the minimum effective dose of ketoconazole, and thus minimize the side-effects of ketoconazole.     

PDGF-like growth factor induces EGF-potentiated phenotypic transformation of normal rat kidney cells in the absence of TGF beta

Using a growth factor defined assay for anchorage-independent growth (van Zoelen, E.J.J., van Oostwaard, Th.M.J., van der Saag, P.T. and de Laat, S.W. (1985) J. Cell. Physiol. 123, 151- 160, we have studied the ability of polypeptide growth factors produced by Neuro-2A neuroblastoma cells to induce anchorage-independent growth of normal rat kidney cells. Neuro-2A cells produce and secrete a PDGF-like growth factor in addition to TGF beta, which can be fully separated from each other by means of reverse-phase HPLC. Using a new, very sensitive technique for detection of TGF beta in growth factor samples based on its additional ability to act as a growth inhibitory factor, it is shown that the PDGF-like growth factor does not contain any detectable TGF beta. Still this neuroblastoma derived PDGF-like growth factor is able to induce anchorage-independent growth of NRK cells, particularly in the additional presence of EGF. It is concluded that under growth factor defined assay conditions TGF beta is not essential for phenotypic transformation of NRK cells.     

铜绿假单胞菌环介导等温扩增技术检测方法在实验小鼠微生物控制中的应用

目的建立铜绿假单胞菌Pseudomonas aeruginosa环介导等温扩增技术(LAMP)检测方法并初步应用于实验小鼠微生物控制。方法根据铜绿假单胞菌oprL基因设计LAMP特异性引物,优化反应条件,确立LAMP的检测体系;再通过对小鼠血清样本的检测,与《GB/T 14926. 17-2001实验动物绿脓杆菌检测方法》对比,阳性结果再用PCR方法验证。结果新建立的LAMP方法特异性强,灵敏度比普通PCR高10~3倍;当反应温度为66℃,内引物和环引物的浓度分别为70μmol·L^-1和30μmol·L^-1时,LAMP反应体系最佳;利用建立的LAMP方法检测87份小鼠血清样本,铜绿假单胞菌检出率为11. 5%(10/87),比《GB/T 14926. 17-2001实验动物绿脓杆菌检测方法》的高(0/87),阳性结果与PCR方法一致。结论本研究建立的LAMP方法特异性强、灵敏度高、可重复率高、稳定性好,为检测铜绿假单胞菌提供了新的研究手段。     

Molecular characterization of the essential response regulator protein YycF in Bacillus subtilis

The response regulator YycF is essential for cell growth in gram-positive bacteria including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. To study the function of YycF in the essential process, we characterized a YycF (H215P) mutation that caused temperature-sensitive growth in B. subtilis. The response regulators YycF and YycF (H215P) were analyzed using circular dichroism spectroscopy, whose T(m) values were 56.0 and 45.9 degrees C, respectively, suggesting that YycF (H215P) significantly affects the protein structure with an increase in temperature. Furthermore, using the gel mobility shift assay and DNase I footprinting, we investigated the effect of YycF (H215P) on binding to the YycF box of ftsAZ operon of B. subtilis. The replacement of the histidine 215 with proline resulted in a decrease of the DNA-binding ability of YycF in vitro. In vivo, using Escherichia coli two-hybrid and homodimerization assays, we clarified that His 215 of YycF plays a crucial role in the homodimerization of the protein. Thus the essential genes involved in growth of B. subtilis appear to be regulated by the homodimer of YycF. These results suggest that the YycF dimerization is an excellent target for the discovery of novel antibiotics.     

Hyper-susceptibility of a fusidic acid-resistant mutant of Salmonella to different classes of antibiotics

Fusidic acid resistance (Fus(R)) in Salmonella enterica serovar Typhimurium is caused by mutations in fusA, encoding elongation factor G (EF-G). Pleiotropic phenotypes are observed in Fus(R) mutants. Thus, the fusA1 allele (EF-G P413L) is associated with slow growth rate, reduced ppGpp and RpoS levels, reduced heme levels, and increased sensitivity to oxidative stress. The fusA1-15 allele, (EF-G P413L and T423I) derived from fusA1 in a selection for growth rate compensation, is partially compensated in each of these phenotypic defects but maintains its resistance to fusidic acid. We show here that the fusA1 allele is associated with sensitivity to ultraviolet light and increased susceptibility to the inhibitory action of several unrelated antibiotic classes (beta-lactam, fluoroquinolone, aminoglycoside, rifampicin, and chloramphenicol). The fusA1-15 allele, in contrast, is less susceptible to UV and to other antibiotics than fusA1. The hyper-susceptibility to multiple antibiotics associated with fusA1 and fusA1-15 is revealed in a novel growth competition assay at sub-MIC concentrations, but not in a standard MIC assay.     

Dietary thyrotropin-releasing hormone stimulates growth rate and increases the insulin: glucagon molar ratio of broiler chickens

The effects of thyroid manipulation on growth, feed efficiency, and plasma hormone levels were determined in rapidly growing chickens. Beginning at 3 weeks of age, eight broiler cockerels were provided with control feed (CF) or feed containing either 1 ppm of triiodothyronine (T3), 1 ppm of thyroxine (T4), 0.3% propylthiouracil (PTU), or 5 ppm of thyrotropin-releasing hormone (TRH) for 3 weeks. Blood samples were taken at 4, 5, and 6 weeks for determination of plasma levels of growth hormone, insulin-like growth factor, T3, T4, insulin, glucagon, glucose, and nonesterified fatty acids. Dietary TRH increased (P less than 0.05) the growth rate of chickens by 14% when compared with the CF group. Plasma growth hormone levels were reduced (P less than 0.05) 65% by dietary T3 and 33% by treatment with either T4 or TRH when compared with the CF group. Plasma insulin-like growth factor levels were 16% lower (P less than 0.05) in PTU-fed birds than the other treatment groups. Plasma T3 levels were elevated (P less than 0.05) 3-fold by dietary T3 and 38% by TRH whereas plasma T3 in the PTU group was 38% below the average of CF birds. Plasma T4 levels were increased (P less than 0.05) by 12-fold in T4-fed birds, decreased 48% in TRH-fed birds, and nondetectable in birds treated with either T3 or PTU. Compared with the other treatments, dietary PTU increased (P less than 0.01) plasma insulin levels 4.3-fold whereas TRH provided a 2.7-fold increase in plasma insulin. Plasma glucagon levels were 26% higher (P less than 0.05) in T3-fed birds than those fed either T4 or PTU. These observations indicate that thyroid activity plays an important role in regulating secretion of GH and the pancreatic hormones. Furthermore, our study demonstrates the potential use of TRH as an orally active growth promoter for poultry.     

Inhibition of tumor growth mediated by lymphocytes sensitized in vitro to a syngeneic murine teratocarcinoma 402AX

TerC, a cell line derived from a strain 129 teratocarcinoma 402AX, was used to sensitize syngeneic 129 (H-2bc) splenic lymphocytes in vitro. The effector cells generated inhibited in vitro growth of TerC as measured by an 125I-IUDR ost-labeling technique. It was also shown, with a modified Winn assay, that the sensitized cells were effective in preventing TerC growth in vivo. The effector lymphocyte was nonadherent to nylon wool was sensitive to anti-Thy-1.2 + C, and was phenotypically Ly 1-2+. The anti-TerC effector T lymphocytes were not functional in a 51Cr-release assay. However, this failure to lyse appears not to be due to some intrinsic membrane resistance since both BCG and ConA-activated killers were able to lyse TerC. The TerC-sensitized lymphocytes displayed no H-2 restriction and were able to growth inhibit in vitro a wide range of tumorigenic cell lines, e.g., P815 (H-2d), EL-4 (H-2b),Sal (H-2a), and BALB/c (H-2d) 3T12. Mouse blastocyst cell lines were also inhibited. BALB/c 3T3 and mouse fibroblast cell strains were not growth inhibited. Thus, it appears that oncofetal antigens expressed on TerC are capable of initiating a cell-mediated response and that these antigenic specificities are shared by many transformed cell lines.     

Amplification of a specific repetitive DNA sequence for Trypanosoma rangeli identification and its potential application in epidemiological investigations

Trypanosoma rangeli can infect humans as well as the same domestic and wild animals and triatomine vectors infected by Trypanosoma cruzi in Central and South America. This overlapping distribution complicates the epidemiology of American trypanosomiasis due to the cross-reactivity between T. rangeli and T. cruzi antigens and the presence of conserved DNA sequences in these parasites. We have isolated a T. rangeli-specific DNA repetitive element which is represented in approximately 103 copies per parasite genome and is distributed in several chromosomal bands. The 542-bp nucleotide sequence of this element, named P542, was determined and a PCR assay was standardized for its amplification. The sensitivity of the assay is high, allowing the detection of one tenth of the DNA content of a single parasite. The presence of the P542 element was confirmed in 11 T. rangeli isolates from mammalian hosts and insect vectors originating from several countries in Latin America. Negative amplification was observed with different T. cruzi strains and other trypanosomatids. The potential field application of the P542 PCR assay was investigated in simulated samples containing T. rangeli and/or T. cruzi and intestinal tract and feces of Rhodnius prolixus. Epidemiological studies were conducted in DNA preparations obtained from the digestive tracts of 12 Rhodnius colombiensis insects collected in a sylvatic area in Colombia. Positive amplification of the P542 element was obtained in 9/12 insects. We have also compared in the same samples the diagnostic performance of two PCR assays for the amplification of the variable domain of minicircle kinetoplast DNA (kDNA) and of the large subunit (LSU) of the ribosomal RNA gene of T. cruzi and T. rangeli. Data indicate that the kDNA PCR assay does not allow diagnosis of mixed infections in most insects. On the other hand, the PCR assay of the LSU RNA gene showed lower sensitivity in the detection of T. rangeli than the PCR assay of the P542 element. It is predicted that the use of sensitive detection techniques will indicate that the actual distribution of T. rangeli in America is wider than presumed.     

High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE

Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F(2) population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.     

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.     

A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: determination of toluene-4-monooxygenase activity

A spectrophotometric method for the quantitative determination of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the aromatic ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (epsilon) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was determined experimentally to be 12,827 M(-1) cm(-1) with a linear range of quantification between 2.5 and 75 microM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as determined spectrophotometrically, were 61.8+/-4.4 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9+/-4.6 nmol/min/mg P. mendocina KR1 protein (using cell extracts Pseudomonas putida F6), and, as determined by HPLC analysis, 62.6+/-1.4 nmol/min/mg P. mendocina KR1 protein.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.