Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass.

Lolitrems are potent tremorgenic mycotoxins that are synthesised by clavicipitaceous fungal endophytes of the Epichloë/Neotyphodium group in association with grasses. These indole-diterpenes confer major ecological benefits on the grass-endophyte symbiotum. A molecular signature for diterpene biosynthesis is the presence of two geranylgeranyl diphosphate (GGPP) synthases. Using degenerate primers for conserved domains of fungal GGPP synthases, we cloned two such genes, ltmG and ggsA, from Neotyphodium lolii. Adjacent to ltmG are two genes, ltmM and ltmK, that are predicted to encode an FAD-dependent monooxygenase and a cytochrome P450 monooxygenase, respectively. The cluster of ltm genes is flanked by AT-rich retrotransposon DNA that appears to have undergone extensive repeat induced point (RIP) mutation. Epichloë festucae, the sexual ancestor of N. lolii, contains an identical ltm gene cluster, but lacks the retrotransposon "platform'' on the right flank. Associations established between perennial ryegrass and an E. festucae mutant deleted for ltmM lack detectable levels of lolitrems. A wild-type copy of ltmM complemented this phenotype, as did paxM from Penicillium paxilli. Northern hybridization and RT-PCR analysis showed that all three genes are weakly expressed in culture but strongly induced in planta. The relative endophyte biomass in these associations was estimated by real-time PCR to be between 0.3 and 1.9%. Taking this difference into account, the steady-state levels of the ltm transcripts are about 100-fold greater than the levels of the endogenous ryegrass beta-tubulin (beta -Tub1) and actin (Act1) RNAs. Based on these results we propose that ltmG, ltmM and ltmK are members of a set of genes required for lolitrem biosynthesis in E. festucae and N. lolii.

Functionally associated molecular genetic marker map construction in perennial ryegrass (Lolium perenne L.).

A molecular marker-based map of perennial ryegrass (Lolium perenne L.) has been constructed through the use of polymorphisms associated with expressed sequence tags (ESTs). A pair-cross between genotypes from a North African ecotype and the cultivar Aurora was used to generate a two-way pseudo-testcross population. A selection of 157 cDNAs assigned to eight different functional categories associated with agronomically important biological processes was used to detect polymorphic EST-RFLP loci in the F(1)(NA(6) x AU(6)) population. A comprehensive set of EST-SSR markers was developed from the analysis of 14,767 unigenes, with 310 primer pairs showing efficient amplification and detecting 113 polymorphic loci. Two parental genetic maps were produced: the NA(6) genetic map contains 88 EST-RFLP and 71 EST-SSR loci with a total map length of 963 cM, while the AU(6) genetic map contains 67 EST-RFLP and 58 EST-SSR loci with a total map length of 757 cM. Bridging loci permitted the alignment of homologous chromosomes between the parental maps, and a sub-set of genomic DNA-derived SSRs was used to relate linkage groups to the perennial ryegrass reference map. Regions of segregation distortion were identified, in some instances in common with other perennial ryegrass maps. The EST-derived marker-based map provides the basis for in silico comparative genetic mapping, as well as the evaluation of co-location between QTLs and functionally associated genetic loci.

tA single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.

The rice blast resistance (R) gene Pi-ta mediates gene-for-gene resistance against strains of the fungus Magnaporthe grisea that express avirulent alleles of AVR-Pita. Using a map-based cloning strategy, we cloned Pi-ta, which is linked to the centromere of chromosome 12. Pi-ta encodes a predicted 928-amino acid cytoplasmic receptor with a centrally localized nucleotide binding site. A single-copy gene, Pi-ta shows low constitutive expression in both resistant and susceptible rice. Susceptible rice varieties contain pi-ta(-) alleles encoding predicted proteins that share a single amino acid difference relative to the Pi-ta resistance protein: serine instead of alanine at position 918. Transient expression in rice cells of a Pi-ta(+) R gene together with AVR-Pita(+) induces a resistance response. No resistance response is induced in transient assays that use a naturally occurring pi-ta(-) allele differing only by the serine at position 918. Rice varieties reported to have the linked Pi-ta(2) gene contain Pi-ta plus at least one other R gene, potentially explaining the broadened resistance spectrum of Pi-ta(2) relative to Pi-ta. Molecular cloning of the AVR-Pita and Pi-ta genes will aid in deployment of R genes for effective genetic control of rice blast disease.

Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.

Rice expressing the Pi-ta gene is resistant to strains of the rice blast fungus, Magnaporthe grisea, expressing AVR-Pita in a gene-for-gene relationship. Pi-ta encodes a putative cytoplasmic receptor with a centrally localized nucleotide-binding site and leucine-rich domain (LRD) at the C-terminus. AVR-Pita is predicted to encode a metalloprotease with an N-terminal secretory signal and pro-protein sequences. AVR-Pita(176) lacks the secretory and pro-protein sequences. We report here that transient expression of AVR-Pita(176) inside plant cells results in a Pi-ta-dependent resistance response. AVR-Pita(176) protein is shown to bind specifically to the LRD of the Pi-ta protein, both in the yeast two-hybrid system and in an in vitro binding assay. Single amino acid substitutions in the Pi-ta LRD or in the AVR-Pita(176) protease motif that result in loss of resistance in the plant also disrupt the physical interaction, both in yeast and in vitro. These data suggest that the AVR-Pita(176) protein binds directly to the Pi-ta LRD region inside the plant cell to initiate a Pi-ta-mediated defense response.

The renal hemodynamic basis of diabetic nephropathy.

Diabetic nephropathy occurs in approximately one third of individuals with insulin-dependent diabetes mellitus (IDDM), recent studies suggest that a similar proportion of non-insulin-dependent diabetes mellitus (NIDDM) patients develop this serious complication as well. Of the many risk factors identified in the pathogenesis of nephropathy, hemodynamic alterations have been particularly well studied. Increases in glomerular filtration rate (GFR), largely driven by increases in plasma flow and glomerular capillary pressure, are apparent in early IDDM and NIDDM. Furthermore, the elevation in capillary pressure may be damaging to glomerular endothelial, epithelial and mesangial cells, thereby initiating and contributing to the progression of diabetic nephropathy. Numerous mediators of diabetic hyperfiltration have been proposed, and this phenomenon likely reflects a mutilfactorial etiology. The purpose of this article is to examine the hemodynamic alterations characteristic of diabetic nephropathy, their etiology, and their role in the development and progression of diabetic nephropathy.

Comparison of fungi within the Gaeumannomyces-Phialophora complex by analysis of ribosomal DNA sequences.

Four ascomycete species of the genus Gaeumannomyces infect roots of monocotyledons. Gaeumannomyces graminis contains four varieties, var. tritici, var. avenae, var. graminis, and var. maydis. G. graminis varieties tritici, avenae, and graminis have Phialophora-like anamorphs and, together with the other Gaeumannomyces and Phialophora species found on cereal roots, constitute the Gaeumannomyces-Phialophora complex. Relatedness of a number of Gaeumannomyces and Phialophora isolates was assessed by comparison of DNA sequences of the 18S rRNA gene, the 5.8S rRNA gene, and the internal transcribed spacers (ITS). G. graminis var. tritici, G. graminis var. avenae, and G. graminis var. graminis isolates can be distinguished from each other by nucleotide sequence differences in the ITS regions. The G. graminis var. tritici isolates can be further subdivided into R and N isolates (correlating with ability [R] or inability [N] to infect rye). Phylogenetic analysis of the ITS regions of several oat-infecting G. graminis var. tritici isolates suggests that these isolates are actually more closely related to G. graminis var. avenae. The isolates of Magnaporthe grisea included in the analysis showed a surprising degree of relatedness to members of the Gaeumannomyces-Phialophora complex. G. graminis variety-specific oligonucleotide primers were used in PCRs to amplify DNA from cereal seedlings infected with G. graminis var. tritici or G. graminis var. avenae, and these should be valuable for sensitive detection of pathogenic isolates and for diagnosis of take-all.

Metabolic reduction of novel 3,4-dichloro-5-nitrofurans in Salmonella typhimurium.

To gain insight on biochemical mechanisms of mutagenesis and carcinogenesis by the experimental carcinogens, 5-nitrofurans, a new series of 3,4-dichloro-5-nitrofurans, comprised of 3,4-dichloro-5-nitro-2-acetylfuran (I), 3,4-dichloro-5-nitro-2-bromoacetylfuran (II), methyl 3,4-dichloro-5-nitro-2-furoate (III), were synthesized and tested for their activation to mutagenic forms in the standard plate assay using Salmonella typhimurium TA98, TA100, and TA100NR, a derivative of TA100 deficient in nitroreductase activity. The mutagenic responses in TA98 were 2- to 6-fold lower compared to TA100. Furthermore, I and II were less active in TA100NR, while compound III was about four times more mutagenic in TA100NR compared to the parent strain TA100. Incubation of III with NADPH and bacterial lysates showed that the extent of reduction was greater in TA100 compared to TA100NR. High-pressure liquid chromatography analysis of the ethyl acetate extract obtained from incubation of III with lysates of TA100 revealed the formation of four metabolites with retention times of about 4.0, 5.7, 10.0, and 14.3 minutes. The spectroscopic and chromatographic properties of the components with retention times of 10.0 and 14.3 minutes were identical to two derivatives obtained by chemical reduction of III, and thus represent nitroreduction products. These derivatives have been identified as cis- and trans-oxime isomers of methyl 3,4-dichloro-2-furoate, based on spectroscopic analyses. These oximes were not mutagenic for TA100. Furthermore, III was more mutagenic under anaerobic conditions, suggesting that secondary superoxide or nitroanion free radicals generated from nitroreduction are not responsible for the mutagenicity of III. In addition, the higher mutagenic response in TA100NR, and the lack of mutagenic activities of the amino and the oxime analogs of III suggest that the mutagenic activation of III might be due to the nitroso intermediate or involve mechanisms other than nitroreduction.

Phase II study of echinomycin in the treatment of renal cell carcinoma ECOG study E2885.

Seventeen patients were treated with echinomycin for metastatic renal cell carcinoma. Echinomycin is a bifunctional DNA intercalating agent with broad preclinical antitumor activity. It was given at 1200 mg/m2 by intravenous infusion over 30-60 min weekly for 4 weeks. The treatment was repeated every 6 weeks. There were no responses observed in the study. No life threatening or lethal toxicity was documented in 13 eligible patients. The median survival of these patients was 13.7 months. We conclude that echinomycin is not active against metastatic renal cell carcinoma at the dose and schedule tested.

Pharmacodynamics of biological response in vivo after single and multiple doses of interferon-beta.

Interferons (IFNs) induce gene regulation in vivo that may be used to identify effective doses, schedules, and potential correlates of therapeutic response. To critically examine minimum effective dose, duration of response, and cumulative effects of repetitive doses, a range of subcutaneous doses of IFN beta ser was studied in 32 healthy human volunteers. IFN-induced products of gene regulation assessed were beta 2-microglobulin, neopterin, and tryptophan in serum and 2',5'-oligoadenylate (2-5A) synthetase activity in peripheral blood mononuclear cells. Eight subjects per group received 0.09, 0.9, 9, or 45 MU of IFN beta. Responses were measured at 24, 48, and 72 h after single and multiple doses. The lowest biologically effective dose was 0.9 MU; significant (p < 0.02) increases were observed at 24 h in beta 2-microglobulin and cellular 2-5A synthetase activity. At the two higher doses, 9 and 45 MU, changes were observed at 24 h in all products (p < 0.01). A dose response (p < 0.01) over the range of 0.09-45 MU was observed for all these serum and intracellular gene products. Changes in neopterin, beta 2-microglobulin, and cellular 2-5A synthetase correlated significantly with each other. The response to a single dose of IFN beta was as great in magnitude as the response to multiple doses, suggesting an alternate-day schedule would maintain biological response.

Inhibition of murine tumor growth by an interferon-inducing imidazoquinolinamine.

The low-molecular-weight imidazoquinolinamine derivative, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod, previously described as R-837), induced alpha-interferon (IFN-alpha) in mice. IFN induction was identified at oral doses as low as 3 mg/kg. The 10% lethal dose for daily treatment with imiquimod was 200 mg/kg. Oral treatment with 30 mg/kg imiquimod once every three days significantly inhibited MC-26 colon carcinoma. Delay of treatment from day 1 to day 5, when tumors were easily palpable, did not reduce benefits. Ten daily treatments were slightly more effective than five. However, delivery of the same total dose of imiquimod either once every day for 20 days, once every 4 days, once every 7 days, or once every 10 days inhibited tumor growth to the same level. The antitumor effects of imiquimod were significantly abrogated by an antiserum to murine IFN-alpha, suggesting that the antitumor effect was to a substantial extent mediated by IFN induction. Imiquimod also significantly reduced the number of lung colonies in mice inoculated i.v. with MC-26 tumor cells. Combination of treatment with imiquimod and cyclophosphamide was significantly (P less than 0.01) better than treatment with either drug alone. Combination treatment with cyclophosphamide led to cures in some of the mice inoculated either s.c. or i.v. with MC-26 cells. Treatment with imiquimod also inhibited the growth of RIF-1 sarcoma and Lewis lung carcinoma but was ineffective for P388 leukemia. Imiquimod is an oral IFN-alpha inducer with antitumor effectiveness for transplantable murine tumors.

Correlation between N-acetyltransferase activities in uroepithelia and in vivo acetylator phenotype.

The relationship between in vivo acetylator phenotype of individuals and N-acetyltransferase (NAT) activity in the cytosol of their cultured uroepithelia was examined in four urology patients. In vivo acetylator phenotypes were assigned by determining the ratio of N-acetyl vs. total [N-acetyl+free] sulfamethazine in urine and blood following a single oral dose (1 gm) of sulfamethazine. From the same patients, a surgical specimen of the ureter was obtained, uroepithelial cells were cultured in vitro, and the cytosols prepared. NAT activities were determined by measuring the amount of 4-acetylaminobiphenyl formed from incubation of uroepithelial cytosol with the substrate, 4-aminobiphenyl, and the cofactor [14C]acetyl coenzyme A. The two individuals phenotyped as "slow acetylators" by the in vivo method had NAT activities of 8.3 and 16.2 pmol 4-acetylaminobiphenyl/mg protein/min. In contrast, the two individuals phenotyped as "rapid acetylators" showed activities of 50.9 and 109.5 pmol 4-acetylaminobiphenyl/mg protein/min. The rapid acetylators exhibit about 6-fold greater uroepithelial NAT activities than slow acetylators, thus showing a direct correlation between the NAT activity in the uroepithelium, the target tissue of the human bladder carcinogen 4-aminobiphenyl, and the in vivo acetylator phenotype. These results imply that susceptibility of individuals to arylamine-induced bladder cancer might be associated with NAT activities in their target cells and that in vivo acetylator phenotyping could serve as a useful and relevant biochemical screening marker to assess the risk of developing bladder cancer.

Height, infant-feeding practices and cardiovascular functioning among 3 or 4 year old children in three ethnic groups.

Barker recently hypothesized that factors affecting prenatal and infant growth are related to adult blood pressure and CVD mortality. Predictions from Barker's hypothesis in regard to infant feeding were tested among a sample of 3 or 4 year old children. The relationship of infant-feeding characteristics (duration of breast-feeding, times of introduction of high fat, high carbohydrate, high potassium foods and table salt) to indicators of cardiovascular functioning (resting blood pressures and heart rates, and heart rate response to graded activity) while controlling for anthropometric (height, sum of seven skinfolds, BMI) and demographic (ethnicity, gender, social status) characteristics revealed that infant-feeding practices were not related to CV functioning in the predicted directions among this sample of 3 or 4 year old children. Furthermore, the positive relationship between height and systolic blood pressure was inconsistent with the Barker hypothesis.

Trimetrexate in advanced renal cell carcinoma. An ECOG phase II trial.

Thirty-four chemotherapy-naive, ambulatory patients with advanced renal cell cancer were treated with the non-classical antifol trimetrexate at the intravenous dose of 12 mg/m2 daily x 5 every three weeks (8 mg/m2 qd x 5 for greater than 30% bone marrow previously irradiated). One patient experienced a partial response lasting 24 weeks for a response rate of 3% (exact 95% CI, 0.1 to 15.3%). Toxicity was manageable and primarily myelosuppression, gastrointestinal, and mucosal. Trimetrexate has little activity in advanced renal cell carcinoma at this dose and schedule.

Nucleotide sequence of the coat protein gene of a strain of clover yellow vein virus from New Zealand: conservation of a stem-loop structure in the 3' region of potyviruses.

The sequence of the 3'-terminal 1492 nucleotides of the genome of a New Zealand isolate of clover yellow vein potyvirus (CYVV) has been determined. This sequence encodes a large open reading frame of 1314 nucleotides, the start of which was not identified, but which encodes a putative 272 amino acid coat protein. Downstream of the coat protein coding region is a 177 nucleotide untranslated sequence terminated by a polyadenylate tract. Comparison of the deduced CYVV-NZ coat protein amino acid sequence with two other strains of CYVV showed 86-93% similarity, suggesting CYVV-NZ should be regarded as a separate CYVV strain. CYVV-NZ shares with other CYVV strains a direct repeat of 14-16 nucleotides that is capable of forming a stem-loop structure. Examination of 35 strains of 15 other potyviruses showed a similar stem-loop structure conserved in all cases. A possible role in replication is hypothesized for the structure.

Mutagenicity of N- and O-acetyl derivatives of methyl 3,4-diphenyl-5-hydroxylamino-2-furoate and N-hydroxy-4-aminobiphenyl in Salmonella typhimurium.

Methyl 3,4-diphenyl-5-hydroxylamino-2-furoate (N-OH-MDPF) (I), methyl 3,4-diphenyl-5-acetoxyamino-2-furoate (N-OAc-MDPF) (II), methyl 3,4-diphenyl-N-hydroxy-5-acetylamino-2-furoate (N-OH-MDPAF) (III), and methyl 3,4-diphenyl-N-acetoxy-5-acetylamino-2-furoate (N-OAc-MDPAF) (IV) were synthesized and tested for mutagenic activity for Salmonella typhimurium TA98 and TA100. The hydroxylamine (I) and acetyl derivatives (II-IV) did not show mutagenic activity in TA98 or TA100. In contrast, the parent nitro compound, methyl 3,4-diphenyl-5-nitro-2-furoate (MDPNF) (V) was found to be equally active in TA98 and TA98-DNP, and more active in TA100 and TA104. The mutagenic activity in TA100 and TA104 decreased significantly under anaerobic conditions. Additionally, MDPNF was previously shown to be less mutagenic in the nitroreductase-deficient derivatives TA100NR and TA98NR, suggesting a requirement for nitro reduction. Incubation of V with NADPH and bacterial lysates of TA98 or TA98NR yielded a metabolite which was identified as I based on chromatographic and mass spectral characteristics. The rate of reduction by the lysate of TA98NR was about one-third that of TA98, showing a correlation between mutagenicity and nitroreductase activity. The lysates of TA98 did not reduce N-OH-MDPF further to the amine. In contrast to the lack of mutagenic activity of I-IV, N-hydroxy-4-aminobiphenyl (N-OH-ABP) and its acetyl derivatives were active in TA98, but less so in TA98-DNP. These data suggest that mechanisms involving O-acetylation of N-hydroxylamine to the acetoxyamine or acyl transfer reactions are not involved in the generation of mutagen from MDPNF. Furthermore, the differential mutagenic response of V in TA98 and TA98NR, its reduction to I, and the lack of activity of I suggest that the intermediates of reduction between the nitro and hydroxylamine, such as nitro or nitroso free radical anions, may be involved in mutagenesis. The decreased response of V under anaerobic conditions and increased response in TA104 suggest that secondary oxygen radicals generated from reduction intermediates may be responsible for the mutagenicity of MDPNF.

Ethnicity, infant-feeding practices, and childhood adiposity.

There has been professional concern that the type of milk used for infant-feeding may lead to adiposity. Studies of the relationship between infant milk-feeding and adiposity, however, have led to inconsistent results. This study investigated the relationship of infant-feeding practices to three indicators of adiposity: body weight, body mass index (BMI) and sum of seven skinfolds. The sample includes children at 3 or 4 years of age, in three ethnic groups. Multivariate techniques assessed the relationship among practices of infant-feeding with three indicators of adiposity, while considering potential confounding variables. Although a weak bivariate relationship was detected between the duration of breastfeeding and body weight, none of the measures of infant-feeding were related to the three indicators of adiposity. Black-American girls had smaller skinfolds than Anglo- or Mexican-American girls, with no ethnic group differences among boys. Concerns about adiposity due to methods of infant-feeding can be allayed, at least among 3- or 4-year-old children.

Protection from carcinogen-induced murine bladder carcinoma by interferons and an oral interferon-inducing pyrimidinone, bropirimine.

Interferons (IFNs) have established activities as antivirals and inhibitors of viral and transplantable tumors. To establish whether IFNs or their inducers can affect induction of carcinogenesis in vivo, the bladder-specific carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was administered in the diet at 0.11 or 0.13% (w/w) to female C3H/He mice beginning at 7 weeks of age. Mice treated with the IFN-inducing bropirimine [2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone] i.p. twice a week for 14 weeks starting on day 30 of start of FANFT feeding developed fewer transitional cell carcinomas (TCC) than mice treated with the vehicle. Bropirimine (200 mg/kg twice a week) orally resulted in even greater effectiveness: 6 of 43 bladders with TCC for bropirimine-treated mice versus 24 of 39 for control glycine buffer-treated mice (P less than 0.01, x2 test). Mice treated i.p. daily on days 29 through 210 with 5,000 units of beta interferon (specific activity, 2.0 x 10(8) units/mg) had 0 of 15 TCC while control mice had 7 of 13 TCC (P less than 0.001). Bladders of untreated mice were also significantly heavier than those of beta interferon- or bropirimine-treated mice. This dose of IFN treatment was confirmed as effective in a second experiment, in which mice were treated daily on days 30-223 with 5,000 units alpha/beta interferon (specific activity, 1.2 x 10(7) units/mg). This resulted in 4 of 25 bladders with TCC versus 24 of 39 for control mice (P less than 0.001). A higher dose of IFN (50,000 units alpha/beta interferon daily) was toxic; 24 of 30 mice died within 2 months. IFN and an IFN inducer, bropirimine, inhibited development and progression of FANFT-induced bladder TCC in vivo and thus may have roles as chemopreventive modalities.

Hexachlorobenzene episode in Turkey.

During the period 1955-1959, approximately 4000 people in southeast Anatolia developed porphyria due to the ingestion of hexachlorobenzene (HCB), a fungicide added to wheat seedlings. These HCB exposures subsequently led to the development of bullae on sun-exposed areas, hyperpigmentation, hypertrichosis, and porphyrinuria. The condition was called kara yara or "black sore." Many of the breast-fed children under the age of 2 years whose mothers had ingested HCB-treated grain died from a disease known as pembe yara or "pink sore." In this follow-up study of 252 patients, 20-30 years postexposure, there were 162 males and 90 females, with an average current age of 35.7 years, an average of onset of 7.6 years, and a duration of 2.2 years. Many patients had dermatologic, neurologic, and orthopedic symptoms and signs. The observed clinical findings include scarring of the face and hands (83.7%), hyperpigmentation (65%), hypertrichosis (44.8%), pinched facies (40.1%), painless arthritis (70.2%), small hands (66.6%), sensory shading (60.6%), myotonia (37.9%), cogwheeling (41.9%), enlarged thyroid (34.9%), and enlarged liver (4.8%). Urine and stool porphyrin levels were determined in all patients, and 17 have at least one of the porphyrins elevated. A total of 56 specimens of human milk obtained from mothers with porphyria were analyzed for HCB. The average value was 0.51 ppm in HCB-exposed patients compared to 0.07 ppm in unexposed controls. Offspring of mothers with three decades of HCB-induced porphyria appear normal.