Metabolism of verruculogen in rats.

Radiolabeled verruculogen was detected in a wide range of body tissues 6 min after intravenous administration, but after a further 20 min it was mainly being excreted via the biliary route. In isolated liver perfusion, [14C]verruculogen was rapidly taken up by the liver and metabolized completely, principally to the related tremorgen TR-2 but also to a desoxy derivative of verruculogen. In addition, a smaller amount of an isomer of TR-2 was detected. These metabolic products were excreted in the bile.     

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.     

Molecular characterization of a family of tandemly repeated DNA sequences,TR-1, in heterochromatic knobs of maize and its relatives

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B' (184 bp), 2/3B (115 bp), 2/3B' (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of approximately 180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B' may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B', probably via nonhomologous recombination; and (4) 69 bp at the 3' end of B or B', and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.     

Meiotic loss of the B chromosomes of maize is influenced by the B univalent co-orientation and the TR-1 knob constitution of the A chromosomes

The suppression of meiotic loss when the maize B chromosomes are unpaired is genetically determined. Two genotypes were selected in 1B x 0B crosses: the H line where the B transmission rate is Mendelian (50%) and the L line where the B is present in only about 40% of the progeny. Using the ZmBs probe located at the centromere and at the distal portion of the B chromosome in FISH, we found that the centromeric and telomeric ends of the B univalent co-orient at metaphase I. This feature seems to promote proper centromere orientation causing the lack of meiotic loss of the unpaired B. The co-orientation was observed in both lines, however in the L line the B univalents were not always properly oriented, showing amphitelic orientation in about 25% of the metaphase I cells. We also studied plants of the H and L lines with FISH to test the possible relation between the knob constitution and B loss. It has been found that the plants of both lines are similarly variable for the 180-bp knob repeat, but they differ in the TR-1 350-bp repeat, the L line having more TR-1 knobs. The use of a 45S rDNA probe which labels chromosome 6, allowed us to determine that this chromosome shows the main variability between the two lines: the L line has TR-1 in both arms, showing a large TR-1 knob on the long arm. The H line has only one, generally located on the short arm besides the NOR.     

Nonribosomal peptide synthetase genes pesL and pes1 are essential for Fumigaclavine C production in Aspergillus fumigatus

The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (ΔpesL) or pes1 (Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H(2)O(2), and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P < 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type and ΔpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.     

Protection of mice from wild-type Sendai virus infection by a trypsin-resistant mutant, TR-2.

A trypsin-resistant mutant of Sendai virus, TR-2, which could be activated by chymotrypsin but not by trypsin or the protease present in mouse lung, was inoculated intranasally into mice after being activated in vitro. TR-2 hardly brought about clinical illness or lung lesions in mice; the protease present in the lung could not activate the progeny virus, and the infection terminated after one-step replication. Nevertheless, the immunoglobulin A antibody against wild-type Sendai virus was produced in the respiratory tracts as well as the serum immunoglobulin G antibody, and the mice were protected from the challenge of the wild-type Sendai virus. On the basis of these results, TR-2 may provide a new model of live vaccine for paramyxoviruses; its availability as a live vaccine is also discussed.     

美丽箬竹对模拟大气O3浓度倍增胁迫的生理响应

运用开顶式气室(OTCs)模拟当前环境大气O3浓度(对照,40～45 nL·L-1)、O3浓度倍增4倍(TR-1,92～106 nL·L-1)和O3浓度倍增2倍(TR-2,142～160 nL·L-1)胁迫,以叶片光合色素、MDA、可溶性蛋白质和可溶性糖含量及SOD和POD活性和相对电导率为指标,分析了美丽箬竹(Indocalamus decorus Q.H.Dai)对O3胁迫的生理响应规律.结果显示:随O3浓度的提高,叶片叶绿素a(Chla)、叶绿素b(Chlb)、总叶绿素和类胡萝卜素含量,SOD和POD活性,可溶性蛋白质和可溶性糖含量及二者的比值(SPC/SSC)均呈下降趋势;叶绿素a口与b的比值(Chla/Chlb)、MDA含量和相对电导率均呈增加趋势.TR-1条件下叶片POD活性和可溶性糖含量分别比对照下降38.86％和10.37％,差异显著;Chla/Chlb比值显著高于对照,其余指标均与对照无显著差异.而在TR-2条件下叶片Chla、Chlb、总叶绿素和类胡萝卜素含量显著低于对照,降幅分别为26.89％、40.91％、30.47％和20.37％;SOD和POD活性、可溶性蛋白质和可溶性糖含量以及PC/SSC比值也均显著低于对照,降幅分别为42.03％、46.62％、45.09％、11.52％和40.15％;Chla/Chlb比值、MDA含量和相对电导率均显著高于对照.另外,TR-1与TR-2处理组间光合色素含量和POD活性差异不显著,TR-2处理组MDA含量和相对电导率显著高于TR-1处理组,SOD活性显著低于后者.研究结果表明:美丽箬竹对O3胁迫具有强耐受性,可种植于O3浓度较高的环境中.     

Culture-Independent Assessment of Rhizobiales-Related Alphaproteobacteria and the Diversity of <Emphasis Type="Italic">Methylobacterium</Emphasis> in the Rhizosphere and Rhizoplane of Transgenic Eucalyptus

The rhizosphere is an ecosystem exploited by a variety of organisms involved in plant health and environmental sustainability. Abiotic factors influence microorganism–plant interactions, but the microbial community is also affected by expression of heterologous genes from host plants. In the present work, we assessed the community shifts of Alphaproteobacteria phylogenetically related to the Rhizobiales order (Rhizobiales-like community) in rhizoplane and rhizosphere soils of wild-type and transgenic eucalyptus. A greenhouse experiment was performed and the bacterial communities associated with two wild-type (WT17 and WT18) and four transgenic (TR-9, TR-15, TR-22, and TR-23) eucalyptus plant lines were evaluated. The culture-independent approach consisted of the quantification, by real-time polymerase chain reaction (PCR), of a targeted subset of Alphaproteobacteria and the assessment of its diversity using PCR–denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Real-time quantification revealed a lesser density of the targeted community in TR-9 and TR-15 plants and diversity analysis by principal components analysis, based on PCR–DGGE, revealed differences between bacterial communities, not only between transgenic and nontransgenic plants, but also among wild-type plants. The comparison between clone libraries obtained from the transgenic plant TR-15 and wild-type WT17 revealed distinct bacterial communities associated with these plants. In addition, a culturable approach was used to quantify the Methylobacterium spp. in the samples where the identification of isolates, based on 16S rRNA gene sequences, showed similarities to the species Methylobacterium nodulans, Methylobacterium isbiliense, Methylobacterium variable, Methylobacterium fujisawaense, and Methylobacterium radiotolerans. Colonies classified into this genus were not isolated from the rhizosphere but brought in culture from rhizoplane samples, except for one line of the transgenic plants (TR-15). In general, the data suggested that, in most cases, shifts in bacterial communities due to cultivation of transgenic plants are similar to those observed when different wild-type cultivars are compared, although shifts directly correlated to transgenic plant cultivation may be found.     

Cell-mediated immunity induced in mice after vaccination with a protease activation mutant, TR-2, of Sendai virus.

Our previous study has shown that, although a trypsin-resistant mutant of Sendai virus, TR-2, replicates only in a single cycle in mouse lung with a negligible lesion, the animal acquires a strong immunity against lethal infection with wild-type Sendai virus, suggesting that TR-2 could be used as a new type of live vaccine (M. Tashiro and M. Homma, J. Virol. 53:228-234, 1985). In the present study, we investigated the immunological response elicited in TR-2-infected mice, particularly with respect to cell-mediated immunity. Analyses of cytotoxic activities of spleen cells with 51Cr release assays revealed that Sendai virus-specific T lymphocytes (CTL), in addition to natural killer activity and antiviral antibodies, were induced in DBA/2 and C3H/He mice infected intranasally with TR-2. Proteolytic activation of the fusion glycoprotein F was required for the primary induction of CTL, though not necessarily for stimulation of natural killer and antibody responses. Memory of the CTL induced by TR-2 was long-lasting and was recalled in vivo immediately after challenge with wild-type Sendai virus. In contrast to TR-2, immunization with inactive split vaccine failed to induce the CTL response, but it elicited a high titer of serum antibody and a low level of natural killer activity.     

Biliary excretion of flavopiridol and its glucuronides in the isolated perfused rat liver: role of multidrug resistance protein 2 (Mrp2)

Flavopiridol (FLAP) is a novel anticancer agent that is extensively glucuronidated in patients. Biliary excretion is the main elimination pathway of FLAP conjugates responsible for enterohepatic recirculation and for the main side effect diarrhea. To investigate the hepatic transport system for FLAP glucuronides, livers of Wistar and Mrp2-deficient TR- rats were perfused with FLAP (30 microM) in a single pass system. Biliary excretion and efflux into perfusate during a 60 min period greatly differ in TR- rats. While cumulative biliary excretion of M1 and M2 was significantly reduced to 4.3% and 5.4% efflux into perfusate was increased by 1.5 and 4.2-fold. This indicates that in control rats, M1 and M2 are almost exclusively eliminated into bile by Mrp2. Cumulative FLAP secretion into bile and perfusate, however, was non-significantly reduced by 36.7% and 43.2% in the mutant rat strain, suggesting that besides Mrp2, other transporters might also be involved in FLAP elimination. FLAP stimulates bile flow up to 24% in control rats, but secretion is nearly absent in TR- rats further supporting an efficient transport of FLAP glucuronides by Mrp2. FLAP (30 microM) also reversibly inhibited the Mrp2-mediated biliary elimination of bilirubin and bromsulphthalein in Wistar rats by 54% and 51%, respectively, indicating a competition with the elimination of Mrp2-specific substrates. In summary, we found that FLAP glucuronides are substrates of Mrp2 effectively inhibiting the biliary excretion of bilirubin. This may explain the increased serum bilirubin levels observed in cancer patients during FLAP therapy.     

Definition of a differentiation antigen on the surface of phagocytic cells of thymic reticulum which is down-regulated by interferon gamma

Monoclonal antibodies (MoAb) were raised against phagocytic cells of thymic reticulum (P-TR) grown in vitro. Each of the two MoAb (TR-1N, TR-3N) defined two polypeptides of 46-57 kDa on P-TR membrane. TR-1N and TR-3N recognize respectively 48 and 81% of P-TR, but do not recognize any cells in spleen, lymph node, thymic lymphocytes, or bone marrow. They bind to part of peritoneal macrophages and to macrophage cell lines J 774 and P 388 D1. Cell binding of TR-1N and TR-3N was compared by immunofluorescence to that of anti-CR3 antibody (Mac-1) which recognizes P-TR, a small number of cells in bone marrow and spleen, and a much higher percentage of peritoneal macrophages. The polypeptides recognized by TR-1N/TR-3N may be defined as differentiation antigens on accessory cells as they appear on bone marrow cells during maturation in vitro in the presence of L-cell supernatant which contains colony stimulating factor (CSF-1). Interferon gamma is able to down-regulate the expression of TR-1N/TR-3N antigen on P-TR membrane while that of Mac-1 is unchanged and that of Ia is up-regulated.     

Biosynthesis of radiolabeled verruculogen by Penicillium simplicissimum.

In surface culture of Penicillium simplicissimum, verruculogen was shown to be biosynthesized from the intact carbon skeletons of tryptophan and proline, isoprenoid derivatives of mevalonic acid, and a methyl group donated by methionine. Selected radiolabeled precursors (1 mCi) pulse-fed at the optimum stage of fermentation yielded verruculogen (specific activity, 5.89 X 10(2) microCi mmol-1) labeled in the prolyl and isoprenyl regions of the molecule and suitable for metabolic studies.     

Monoclonal antibody to the thrombin receptor stimulates DNA synthesis in combination with gamma-thrombin or phorbol myristate acetate

Studies with various thrombin derivatives have shown that initiation of cell proliferation by thrombin requires two separate types of signals: one, generated by high affinity interaction of thrombin or DIP-thrombin (alpha-thrombin inactivated at ser 205 of the B chain by diisopropylphosphofluoridate) with receptors and the other, by thrombin's enzymic activity. To further study the role of high affinity thrombin receptors in initiation, we immunized mice with whole human fibroblasts and selected antibodies that blocked the binding of 125I- thrombin to high affinity receptors on hamster fibroblasts. One of these antibodies, TR-9, inhibits from 80 to 100% of 125I-thrombin binding, exhibits an immunofluorescent pattern indistinguishable from that of thrombin bound to receptors on these cells, and selectively binds solubilized thrombin receptors. By itself, TR-9 did not initiate DNA synthesis nor did it block thrombin initiation, but TR-9 addition to cells in the presence of alpha-thrombin, gamma-thrombin (0.5 microgram/ml), or PMA stimulated thymidine incorporation up to threefold over controls. In all cases, maximal stimulation was observed at concentrations of TR-9, ranging from 1 to 4 nM corresponding to concentrations required to inhibit from 30 to 100% of 125I-thrombin binding. These results demonstrate that the binding of the monoclonal antibody to the alpha-thrombin receptor can mimic the effects of thrombin's high affinity interaction with this receptor in stimulating cell proliferation.     

Photoreaction of indole-containing mycotoxins to fluorescent products

Photochemical reaction of the non-fluorescent mycotoxin cyclopiazonic acid (CPA) to fluorescent products was recently reported. Because CPA contains an indole moiety, believed to contribute to the fluorescence, it was of interest to determine whether the effect might be more generally applicable to indole-containing mycotoxins. Three indole-containing tremorgens (penitrem A, paxilline, verruculogen) that have not previously been reported to be fluorescent were rendered fluorescent by exposure to ultraviolet light in a photoreactor. Naturally fluorescent ergot alkaloids, which also contain an indole-moiety, exhibited a diminished response after exposure. This suggests that the phenomenon may be most useful for detection of indole-containing tremorgens that are non-fluorescent, rather than for the enhancement of materials that are already fluorescent, such as the ergot alkaloids. The extent to which fluorescence enhancement was seen was strongly influenced by the reaction environment, in particular the solvent used and whether cyclodextrins were present. In an HPLC format, placement of the photoreactor post-column allowed for the fluorescence detection of penitrem A, paxilline, and verruculogen. The ability to photoreact indole-containing tremorgens and detect them by fluorescence may open up new avenues for detection of these mycotoxins alone or in combination.     

Structure of Porriolide,a New Metabolite from Alternaria porri

Compounds from a soil isolate of Penicillium sp. MG-11 showed remarkable convulsive activity against silkworms. Activity-based fractionation of an acetone extract of the fermentation products resulted in the isolation of four compounds. One was identified as verruculogen, which was an active principle. Another compound, acetoxydehydroaustin, was new and was structurally characterized by spectral data and a single-crystal X-ray analysis. The two other compounds were identified as dehydroaustin and austin on the basis of the spectral data. Although the latter three compounds were not active themselves, they enhanced the convulsive activity of verruculogen in a silkworm bioassay. Three additional compounds with no identifiable activity were also isolated. Two of them were identified as austinol and dehydroaustinol, and the third was determined to be a new related compound named neoaustin by an X-ray analysis of crystals.     

Using three overlapped RILs to dissect genetically clustered QTL for fiber strength on Chro.D8 in Upland cotton

Fiber strength is an important trait among cotton fiber qualities due to ongoing changes in spinning technology. Major quantitative trait loci (QTL) for fiber quality enable molecular marker-assisted selection (MAS) to effectively improve fiber quality of cotton cultivars. We previously identified a major QTL for fiber strength derived from 7235 in Upland cotton. In the present study, in order to fine-map fiber strength QTL, we chose three recombinant inbred lines (RIL), 7TR-133, 7TR-132, and 7TR-214, developed from a cross between 7235 and TM-1 for backcrossing to TM-1 to develop three large mapping populations. Phenotypic data for fiber strength traits were collected in Nanjing (JES/NAU) and Xinjiang (BES/XJ) in 2006 and 2007. Three simple sequence repeat (SSR) genetic linkage maps on Chro.24(D8) were constructed using these three backcrossed populations. The SSR genetic maps were constructed using 907 individuals in (7TR-133 × TM-1)F₂ (Pop A), 670 in (7TR-132 × TM-1)F₂ (Pop B), and 940 in (7TR-214 × TM-1)F₂ (Pop C). The average distance between SSR loci was 0.62, 1.7, and 0.56 cM for the three maps. MapQTL 5 software detected five-clustered QTL (2.5 < LOD < 29.8) on Chro.D8 for fiber strength following analysis of three RIL backcrossed F₂/F_2:3 progenies at JES/NAU and BES/XJ over 2 years. Five QTL for fiber strength exhibited a total phenotypic variance (PV) of 28.8–59.6%.     

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats

The canalicular multispecific organic anion transporter/multidrug resistance protein 2 (cMOAT/Mrp2) plays a major role in the transport of anionic xenobiotics across the bile canalicular membrane. Transport deficient rats (TR-) and Eisai-hyperbilirubinemic rats (EHBR), defective in Mrp2, are mutants of Wistar and Sprague Dawley (SD) rats, respectively. In this study, Phase I metabolic enzyme activities in liver microsomes prepared from these mutant male and female rats were compared to their corresponding non-mutant rats. The total cytochrome P450 contents and NADPH-cytochrome P450 reductase activity in male and female TR- rats were significantly higher than in Wistar rats. In male TR- rats, ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-deethylation (PROD), testosterone 2alpha, 7alpha and 16 alpha-hydroxylase activities were higher, but testosterone 6beta-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR- rats had higher 7alpha-hydroxylase activity, but EROD activity was lower in female Wistar rats. Similar studies conducted in EHBR versus SD rats demonstrated increased total cytochrome P450 content in male and female EHBR rats; NADPH-cytochrome P450 reductase activity was not significantly affected. Decreased PROD activity and the rate of androstenedione formation were observed in male and female EHBR rats. Furthermore, testosterone 6beta-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7alpha-hydroxylase activity was significantly higher in male and female EHBR rats. Thus, in addition to Mrp2 deficiency, differential expression of CYP isoforms and their potential impact on the metabolism and pharmacokinetics of compounds should be considered when interpreting data from these rat strains.     

组培条件下不同番茄品种及砧木自交系幼苗期硝酸盐耐性的比较

采用组培的方法,对15个番茄普通栽培品种的幼苗进行系列浓度Ca(NO₃)₂胁迫处理,10 d后调查不同品种单株幼苗的生长情况和盐害程度。结果表明,15个供试品种幼苗期硝酸盐耐性存在显著差异。上海903、苏红2003、阳光906、大禹中蔬4号、三星L402、番茄大红、中蔬4号、宝大903、霞粉、毛粉 802为硝酸盐敏感品种;早丰番茄、江蔬14号、宝粉和三星906为中等耐硝酸盐品种;日本大粉皇后为耐硝酸盐品种。同时对7个番茄砧木自交系（TR-1、TR-2、TR-3、TR-4、TR-5、TR-7、TR-8）进行了幼苗期高浓度Ca(NO₃)₂的耐盐分析。结果表明,砧木自交系的平均侧根数均显著高于同浓度下供试的普通番茄栽培品种,盐胁迫指数均显著低于普通栽培品种,具有较强的耐盐性;其中TR-8耐盐性最强。     

Biliary excretion of a stretched bilirubin in UGT1A1-deficient (Gunn) and Mrp2-deficient (TR-) rats

The metabolism and biliary excretion of a stretched bilirubin analog with a p-xylyl group replacing the central CH2 hinge were investigated in normal rats, Gunn rats deficient in bilirubin conjugation, and TR- rats deficient in bilirubin glucuronide hepatobiliary transport. Unlike bilirubin, the analog was excreted rapidly in bile unchanged in all three rat strains after intravenous administration. In TR- rats biliary excretion of the analog was diminished, but still substantial, demonstrating that the ATP-binding cassette transporter Mrp2 is not required for its hepatic efflux. These effects are attributable to differences in the preferred conformations of bilirubin and the analog.