Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene.

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus x euramericana ('Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic 'Ogy'. The enzyme activity in the transformed 'Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic 'Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic 'Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from 'Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.

Design of self-processing antimicrobial peptides for plant protection.

Small antimicrobial peptides are excellent candidates for inclusion in self-processing proteins that could be used to confer pathogen resistance in transgenic plants. Antimicrobial peptides as small as 22 amino acids in length have been designed to incorporate the residual amino acids left from protein processing by the tobacco etch virus'(TEVs') NIa protease. Also, by minimizing the length of these peptides and the number of highly hydrophobic residues, haemolytic activity was reduced without affecting the peptide's antimicrobial activity.

Linear mitochondrial plasmids of Fusarium oxysporum contain genes with sequence similarity to genes encoding a reverse transcriptase from Neurospora spp.

Two linear mitochondrial plasmids called pFOXC1 and pFOXC2 from the fungus Fusarium oxysporum were previously described. DNA sequence comparisons indicated that the derived amino acid sequences of both plasmids exhibit similarity to the reverse transcriptase of the Mauriceville and Varkud plasmids of Neurospora spp. The derived amino acid sequence of pFOXC2 has 51% similarity and 32% identity to the Neurospora reverse transcriptase; sequence similarity was greatest for seven blocks of amino acids that are conserved in reverse transcriptases from a wide range of biological sources. Northern analysis suggests that full-length RNAs corresponding to the plasmids are found in representative isolates.

Synthetic antimicrobial peptide design.

To guide the design of potential plant pathogen-resistance genes, synthetic variants of naturally occurring antimicrobial gene products were evaluated. Five 20-amino acid (ESF1, ESF4, ESF5, ESF6, ESF13), one 18-amino acid (ESF12), and one 17-amino acid (ESF17) amphipathic peptide sequences were designed, synthesized, and tested with in vitro bioassays. Positive charges on the hydrophilic side of the peptide were shown to be essential for antifungal activity, yet the number of positive charges could be varied with little or no change in activity. The size could be reduced to 18 amino acids, but at 17 amino acids a significant reduction in activity was observed. ESF1, 5, 6, and 12 peptides were inhibitory to the germination of conidia from Cryphonectria parasitica, Fusarium oxysporum f. sp. lycopersici, and Septoria musiva but did not inhibit the germination of pollen from Castanea mollissima and Salix lucida. ESF12 also had no effect on the germination of Malus sylvestris and Lycopersicon esculentum pollen, but inhibited the growth of the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Pseudomonas syringae. The minimal inhibitory concentrations of the active ESF peptides were similar to those of the naturally occurring control peptides, magainin II and cecropin B. The significant differential in sensitivity between the microbes and plant cells indicated that the active ESF peptides are potentially useful models for designing plant pathogen-resistance genes.

DNA fingerprinting and analysis of population structure in the chestnut blight fungus, Cryphonectria parasitica.

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).

Effect of follicular aspiration on hormonal parameters in patients undergoing ovarian stimulation.

The effect of follicular aspiration and oocyte retrieval on hormonal parameters was examined in women undergoing ovarian stimulation for in-vitro fertilization (IVF) compared to induced ovulation in women undergoing ovarian stimulation for intrauterine insemination (IUI). Blood samples were collected immediately before and 1 h after oocyte retrieval and 48 h later on the day of embryo transfer in 25 IVF patients and before the insemination and 48 h later in 20 IUI patients. A highly significant fall in serum levels of oestradiol (E2), progesterone (P) and human chorionic gonadotrophin (HCG), (P less than 0.001) was observed in the IVF group 1 h after follicular aspiration. The decline in serum E2 levels was maintained at 48 h. In contrast, there was no significant change in serum E2 levels in the IUI group during 48 h. The immediate decline in E2 levels after follicular aspiration might play a role in preventing ovarian hyperstimulation syndrome.

In vivo rearrangement of foreign DNA by Fusarium oxysporum produces linear self-replicating plasmids.

Particular combinations of fungal strains and transformation vectors allow for fungal rearrangement of normally integrative plasmids, resulting in the creation of linear self-replicating plasmids in Fusarium oxysporum. The rearrangement results in the addition of fungal DNA, including telomere consensus sequences, to plasmid termini. The mechanism by which this rearrangement occurs is unclear, but it has similarities to extrachromosomal gene amplification. A DNA fragment which allows for linear autonomous replication upon reintroduction to the fungus was subcloned and sequenced. This DNA sequence contains the repeated telomeric sequence TTAGGG flanked by a region of twofold symmetry consisting primarily of pUC12 DNA. Isolation and identification of this sequence is the first step toward development of vectors that function as artificial chromosomes in filamentous fungi. This sequence was shown to promote autonomous replication and enhance transformation in several strains of F. oxysporum, Nectria haematococca, and Cryphonectria parasitica.

Interleukin-1 stimulates human chorionic gonadotropin secretion by first trimester human trophoblast.

Interleukin-1 (IL-1) has been reported to stimulate LH, GH, ACTH, and TSH release from cultured pituitary cells. IL-1 also has been found to be secreted in significant amounts by placental macrophages. To determine the possible role of IL-1 within the placenta, we studied the effects of human recombinant IL-1 on hCG release by long term cultures of human first trimester trophoblast and the JAR (human choriocarcinoma) cell line. IL-1 in concentrations ranging from 10(-11)-10(-9) mol/L stimulated hCG release from trophoblast cultures. This stimulatory effect was not mediated by prostaglandin E2 (PGE2), as indicated by the inability of indomethacin to block this stimulation as well as a lack of IL-1 effect on PGE2 release by trophoblast cells. At these doses, IL-1 exerted no effect on hCG release by JAR cells. PGE2, when used in high concentrations (10(-6)-10(-5) mol/L), stimulated the release of hCG by the trophoblasts as well as by the JAR cells. Neither IL-1 nor PGE2 stimulated the proliferation, [3H]thymidine incorporation, or differentiation (syncytium formation) of trophoblast or JAR cells. These results suggest that IL-1 may be an important local regulator of hCG secretion by first trimester trophoblasts.

Two nonhomologus viruses of Cryphonectria (Endothia) parasitica reduce accumulation of specific virulence-associated polypeptides.

Double-stranded RNA responsible for transmissible hypovirulence of Cryphonectria (Endothia) parasitica affected the accumulation of specific polypeptides. Nonhomologous hypovirulence-causing double-stranded RNAs, originating in Europe or North America, affected accumulation of the same polypeptides. Fewer than 5% of detectable proteins were affected, indicating that hypovirulence is probably not the result of general debilitation of the fungus.

Differential accumulation of poly(A)+ RNA between virulent and double-stranded RNA-induced hypovirulent strains of Cryphonectria (Endothia) parasitica.

The double-stranded RNA responsible for transmissible hypovirulence in Cryphonectria (Endothia) parasitica was found to affect the accumulation of specific poly(A)+ RNA. Using differential hybridization techniques, two genes were isolated, Vir1 and Vir2, which were specifically expressed as poly(A)+ RNAs in the virulent cells. The highly expressed RNA sequences from these genes were not found in total RNA isolated from either American or European hypovirulent strains, although the genes were present in their genomes. Other virulence- and hypovirulence-specific RNA sequences were also detected. One isolated hypovirulence-specific RNA sequence was expressed in both virulent and hypovirulent cells, but in a two- to fourfold-higher concentration in the hypovirulent cells. The results show that hypovirulence is associated with concurrent changes in a few highly expressed poly(A)+ RNAs, which suggests a specific effect of the double-stranded RNA on fungal gene expression.

Effects of steroids on progesterone output by explants of human chorion.

Human chorion can synthesize and metabolize progesterone, and changes in progesterone synthesis by chorion at term might be important in the processes leading to parturition. We examined whether other steroids present within the maternal compartment and amniotic fluid during late pregnancy influence progesterone output by explants of chorion. We also sought differences in steroid effects on progesterone output in association with labor. Explants were prepared from chorion collected after the spontaneous onset of labor and vaginal delivery and chorion collected after cesarean section without active labor. To study the short-term effects of steroids on progesterone output by chorion, explants were incubated for 4 h with 3 microM pregnenolone and 3 microM of a potential interacting steroid. Other explants were preincubated for 24 h with steroid, then rinsed and incubated for 4 h with 3 microM pregnenolone and 3 microM of the same steroid as during preincubation. Under these conditions, dehydroepiandrosterone and androstenedione inhibited progesterone output by explants of chorion obtained at spontaneous labor and at cesarean section. Testosterone also inhibited progesterone output, but only in cesarean section chorion. If explants were preincubated for 24 h with steroid and then rinsed and incubated for 4 h with pregnenolone only, progesterone synthesis returned to control values. This finding indicates that the mechanism of action of these inhibitory steroids is likely through an effect on 3 beta-HSD activity and not due to a change in the rate of enzyme synthesis. We also noted apparent stimulatory effects of steroids.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of 20 alpha-dihydroprogesterone on progesterone output by human chorion explants.

The output of progesterone by explants of chorion incubated without exogenous precursor was increased in the presence of 20 alpha-dihydroprogesterone (20 alpha-diHP). We have now examined the nature of the 20 alpha-diHP effect. Explants of chorion were incubated with [3H]progesterone and 3 microM non-radiolabelled 20 alpha-diHP to examine the effect of 20 alpha-diHP on progesterone metabolism. Significantly more radioactivity remained as unmetabolized [3H]progesterone compared to chorion incubated in the absence of non-radiolabelled 20 alpha-diHP. There was also a decrease in the output of [3H]-20 alpha-diHP by the explants but no change in the output of a second metabolite of progesterone [3H]-5 alpha-pregnanedione. When explants were incubated with 20 alpha-diHP in the presence of the 3 beta-hydroxysteroid dehydrogenase inhibitor, trilostane, the output of progesterone was significantly greater than with explants incubated with trilostane but without addition of 20 alpha-diHP. Radiochemically pure [3H]progesterone was produced by explants of chorion incubated with [3H]-20 alpha-diHP. We conclude that 20 alpha-diHP may serve as substrate for chorionic progesterone production through 20 alpha-hydroxysteroid dehydrogenase. 20 alpha-diHP, formed from progesterone, may also exert product feedback inhibition on this enzyme.

Progesterone production by human fetal membranes: an in vitro incubation system for studying hormone production and metabolism.

We have established an in vitro tissue explant incubation system to study endocrine functions of human amnion, chorion, and decidua. By means of this technique, tissues remain histologically similar for at least 72 hours, actively use glucose for at least 48 hours, and demonstrate no evidence of release of lactate dehydrogenase into the medium by 24 hours. All three tissues produced progesterone, measured by specific radioimmunoassay, in a dose-dependent fashion from added pregnenolone. However, chorion was many times more active in this respect than were the other tissues. These results were corroborated by demonstrating conversion of 3H-pregnenolone to radiochemically pure 3H-progesterone. This activity was inhibited by a 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) enzyme inhibitor, trilostane. Histochemical staining identified the site of 3 beta HSD activity as being located predominantly in the trophoblast layer of the chorion. We conclude that: (1) this in vitro system is a simple and reliable method by means of which to study endocrine function of amnion, chorion, and decidua; and (2) human fetal membranes, particularly the trophoblast layer of the chorion, can produce progesterone, and hence may be an important regulator of local progesterone levels, which subsequently may affect myometrial contractility.