Antenatal corticosteroid therapy in late preterm delivery: a nationwide population-based retrospective study in Taiwan.

OBJECTIVE: To investigate whether antenatal corticosteroid therapy improves neonatal and maternal outcomes in late preterm delivery. DESIGN: Population-based retrospective study. SETTING: The linkages of Taiwan's National Health Insurance Research Database, National Birth Reporting Database, and the Taiwan Maternal and Child Health Database. POPULATION: All births at risk for late preterm deliveries in Taiwan between 2004 and 2011. METHODS: For every birth at risk for late preterm delivery, five controls randomly matched by maternal and gestational ages and birthweight were included. A conditional logistic regression analysis was applied for risk estimation, with births without corticosteroids as the reference group. Odds ratios were adjusted for caesarean section, parity, sex, gestational hypertension and gestational diabetes mellitus. MAIN OUTCOME MEASURES: Neonatal outcomes, maternal outcomes and the utilisation of healthcare services. RESULTS: The outcomes of 5745 women treated with corticosteroids between 34+0  weeks and 36+6  weeks of gestation were compared with those of 28 135 untreated controls. Compared with the controls, births from women administered corticosteroids reduced the need for continuous positive airway pressure, the number of neonatal intensive care unit admission, and the need for glucose administration, as well as the risk of neonatal respiratory distress, but increased the risk of neonatal sepsis and the number of outpatient visits. CONCLUSIONS: Antenatal corticosteroid therapy in women at risk of late preterm delivery may significantly reduce the need for respiratory support and glucose supply, and respiratory complication risk in neonates. TWEETABLE ABSTRACT: Antenatal corticosteroids in late preterm delivery reduced the risk of neonatal respiratory complications in Taiwan.

TRAIL suppresses gut inflammation and inhibits colitogeic T-cell activation in experimental colitis via an apoptosis-independent pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cell apoptosis by transducing apoptosis signals. Recently, accumulating evidence demonstrated that TRAIL regulates autoimmune inflammation and immune cell homeostasis in several autoimmune animal models, suggesting a novel immunoregulatory role of TRAIL in autoimmune diseases. However, the impact of TRAIL in inflammatory bowel disease is yet undefined. This study is to address the therapeutic effects and immunoregulatory role of TRAIL in autoimmune gut inflammation. We demonstrated herein that TRAIL significantly suppressed gut inflammation and reduced the severity of colitis in a dextran sodium sulfate (DSS)-induced colitis model. Suppression of gut inflammation was not due to induction of apoptosis in colonic T cells, dendritic cells, or epithelium cells by TRAIL. In contrast, TRAIL directly inhibited activation of colitogenic T cells and development of gut inflammation in an adoptive transfer-induced colitis model. The anti-inflammatory effects of TRAIL on colitis were abolished when T cells from TRAIL receptor (TRAIL-R) knockout mice were adoptively transferred, suggesting that TRAIL regulates autoreactive colitogenic T-cell activation in the development of gut inflammation. Our results demonstrate that TRAIL effectively inhibited colonic T-cell activation and suppressed autoimmune colitis, suggesting a potential therapeutic application of TRAIL in human inflammatory bowel disease.

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP.

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.

Genetic polymorphism in milk fat globule-EGF factor 8 (MFG-E8) is associated with systemic lupus erythematosus in human.

Milk fat globule-EGF factor 8 (MFG-E8) is a molecule implicated in phagocytic clearance of apoptotic cells by bridging between macrophages and apoptotic cells. Defects in MFG-E8 cause lupus-like disease in murine models. The aim of our study is to determine whether genetic variation in MFG-E8 predisposes human to systemic lupus erythematosus (SLE). A case-control study of MFG-E8 genetic polymorphism was performed on 147 SLE patients and 146 non-lupus control subjects. Single nucleotide polymorphisms (SNPs) in the coding sequence of human MFG-E8 gene were investigated. SNPs on MFG-E8 residues 3 (3(Arg or Ser)) and 76 (76(Leu or Met)) did not show genetic linkage. Genetic polymorphism on MFG-E8 residue 76 correlated significantly to SLE. The MFG-E8-76(Met) allele predisposed subjects to SLE in a recessive mode (odds ratio: 2.1, P = 0.020), while carriage of MFG-E8-76(Leu) were negatively associated with SLE. The MFG-E8 genotypic combinations with 3(Ser) and 76(Leu) showed the most pronounced protective effect on SLE when compared to the most predisposing genotype 3(Arg/Arg)-76(Met/Met) (OR: 0.29, P = 0.007). According to our result, MFG-E8 is associated with SLE predisposition in Taiwanese. Our study implicates that the impairment of phagocytic clearance of apoptotic cells through phosphotidylserine-dependent MFG-E8 system may lead to the development of human SLE.

Elevated serum decoy receptor 3 with enhanced T cell activation in systemic lupus erythematosus.

Decoy receptor 3 (DcR3/TR6) is a decoy receptor for the Fas ligand (FasL) and can inhibit FasL-induced apoptosis. It has been reported recently that DcR3 can induce T cell activation via co-stimulation of T cells, suggesting that DcR3 may be involved in the pathophysiology of autoimmune diseases. This study aims to analyse the serum DcR3 in patients with systemic lupus erythematosus (SLE) and to investigate the role of DcR3 in the pathogenesis of SLE. Significantly elevated serum DcR3 was observed in SLE patients, and the mean serum DcR3 level was significantly higher for those with active disease [SLE disease activity index (SLEDAI) >/= 10] compared with that in patients with inactive disease (SLEDAI < 10). In addition to reducing activation-induced cell death in activated T cells via neutralization of the FasL, soluble DcR3-Fc enhanced T cell proliferation and increased interleukin-2 and interferon-gamma production via co-stimulation of T cells. Moreover, enhanced T cell reactivity to DcR3-induced co-stimulation was demonstrated in lymphocytes from patients with SLE, suggesting the elevated serum DcR3 may associate with enhanced T cell activation in vivo. These findings are the first to demonstrate that serum DcR3 concentrations are increased in SLE patients, and this may imply a possible role of DcR3 in the pathogenesis of SLE via enhanced T cell hyperreactivity and reduced apoptosis in activated T cells.

Analysis of trinucleotide repeats in different SCA loci in spinocerebellar ataxia patients and in normal population of Taiwan.

OBJECTIVE: To identify various subtypes of spinocerebellar ataxias (SCAs) among autosomal dominant cerebellar ataxia (ADCA) patients referred to our research center, SCA1, SCA2, SCA3/MJD (Machado-Joseph disease), SCA6, SCA7, SCA8 and SCA12 loci were assessed for expansion of trinucleotide repeats. PATIENTS AND METHODS: A total of 211 ADCA patients, including 202 patients with dominantly inherited ataxia from 81 Taiwanese families and nine patients with sporadic ataxia, were included in this study and subjected to polymerase chain reaction (PCR) analysis. The amplified products of all loci were analyzed on both 3% agarose gels and 6% denaturing urea-polyacrylamide gels. PCR-based Southern blots were also applied for the detection of SCA7 locus. RESULTS: The SCA1 mutation was detected in six affected individuals from one family (1.2%) with expanded alleles of 50-53 CAG repeats. Fourteen individuals from nine families (11%) had a CAG trinucleotide repeat expansion at the SCA2 locus, while affected SCA2 alleles have 34-49 CAG repeats. The SCA3/MJD CAG trinucleotide repeat expansion in 60 affected individuals from 26 families (32%) was expanded to 71-85 CAG repeats. As for the SCA7 locus, there were two affected individuals from one family (1.2%) possessed 41 and 100 CAG repeats, respectively. However, we did not detect expansion in the SCA6, SCA8 and SCA12 loci in any patient. CONCLUSIONS: The SCA3/MJD CAG expansion was the most frequent mutation among the SCA patients. The relative prevalence of SCA3/MJD in Taiwan was higher than that of SCA2, SCA1 and SCA7.

Enhanced proliferation and increased IFN-gamma production in T cells by signal transduced through TNF-related apoptosis-inducing ligand.

TNF-related apoptosis-inducing ligand (TRAIL, also called Apo2L), a novel member of TNF superfamily, induces apoptosis in transformed cell lines of diverse origin. TRAIL is expressed in most of the cells, and the expression is up-regulated in activated T cells. Four receptors for TRAIL have been identified, and there is complex interplay between TRAIL and TRAIL receptors in vivo. The actual biological function of TRAIL/TRAIL receptor is still not clear. Growing evidence has demonstrated that members of TNF superfamily transduce signals after engagement with their receptors. Cross-linking of TRAIL by plate-bound rTRAIL receptor, death receptor 4-Fc fusion protein enhanced T cell proliferation and increased IFN-gamma production in conjunction with immobilized suboptimal anti-CD3 stimulation in mouse splenocytes. The increase of T cell proliferation by death receptor 4-Fc was dose dependent, and this effect could be blocked by soluble rTRAIL proteins, indicating the occurrence of reverse signaling through TRAIL on T cell. The enhanced secretion of IFN-gamma mediated via TRAIL could be blocked by SB203580, a p38 mitogen-activated protein kinase-specific inhibitor. Thus, in addition to its role in inducing apoptosis by binding to the death receptors, TRAIL itself can enhance T cell proliferation after TCR engagement and signal the augmentation of IFN-gamma secretion via a p38-dependent pathway. This provides another example of reverse signaling by a member of TNF superfamily. In conclusion, our data suggest that TRAIL can itself transduce a reverse signal, and this may shed light on the biological function of TRAIL.

Expression of human Fas ligand on mouse beta islet cells does not induce insulitis but is insufficient to confer immune privilege for islet grafts.

Fas (CD95) and Fas ligand (FasL/CD95L) are involved in programmed cell death and the regulation of host immune responses. FasL has been shown to provide immune privilege, thus prolonging the survival of unmatched grafts in a variety of tissues, such as eyes and testis. In murine FasL (mFasL) transgenic mice, FasL provoked granulocyte infiltration and insulitis in the pancreas. We intended to study whether the expression of human FasL, instead of mFasL, on mouse beta islet cells could avoid granulocyte infiltration, and whether islet cells transgenic for FasL could be used in islet transplantation. We produced transgenic mice in which the human FasL transgene was driven by rat insulin promoter and was expressed exclusively in the pancreas islet cells in ICR mice. In contrast to mFasL transgenic mice, histochemical staining showed that the pancreas was intact in human FasL transgenic ICR mice. However, when human FasL transgenic islet cells were transplanted into allogeneic mice with streptozotocin-induced diabetes, human FasL appeared not to prolong graft survival. Intensive granulocyte infiltration into the islet grafts was observed in recipients (Balb/c mice) which received islet grafts from human FasL transgenic mice, but not from nontransgenic, allogeneic ICR mice on day 31. Our observations suggest that FasL alone is insufficient to confer immune protection, and that other environmental factors might contribute to the formation of immune privilege sites in vivo

Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor.

Chain lengths and cyclization patterns of microbial polyketides are generally determined by polyketide synthases alone. Fungal polyketide melanins are often derived from a pentaketide 1,8-dihydroxynaphthalene, and pentaketide synthases are used for synthesis of the upstream pentaketide precursor, 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN). However, Aspergillus fumigatus, a human fungal pathogen, uses a heptaketide synthase (Alb1p) to synthesize its conidial pigment through a pentaketide pathway similar to that which produces 1,8-dihydroxynaphthalene-melanin. In this study we demonstrate that a novel protein, Ayg1p, is involved in the formation of 1,3,6,8-THN by chain-length shortening of a heptaketide precursor in A. fumigatus. Deletion of the ayg1 gene prevented the accumulation of 1,3,6,8-THN suggesting the involvement of ayg1 in 1,3,6,8-THN production. Genetic analyses of double-gene deletants suggested that Ayg1p catalyzes a novel biosynthetic step downstream of Alb1p and upstream of Arp2p (1,3,6,8-THN reductase). Further genetic and biochemical analyses of the reconstituted strains carrying alb1, ayg1, or alb1 + ayg1 indicated that Ayg1p is essential for synthesis of 1,3,6,8-THN in addition to Alb1p. Cell-free enzyme assays, using the crude Ayg1p protein extract, revealed that Ayg1p enzymatically shortened the heptaketide product of Alb1p to 1,3,6,8-THN. Thus, the protein Ayg1p facilitates the participation of a heptaketide synthase in a pentaketide pathway via a novel polyketide-shortening mechanism in A. fumigatus.

A developmentally regulated gene cluster involved in conidial pigment biosynthesis in Aspergillus fumigatus.

Aspergillus fumigatus, a filamentous fungus producing bluish-green conidia, is an important opportunistic pathogen that primarily affects immunocompromised patients. Conidial pigmentation of A. fumigatus significantly influences its virulence in a murine model. In the present study, six genes, forming a gene cluster spanning 19 kb, were identified as involved in conidial pigment biosynthesis in A. fumigatus. Northern blot analyses showed the six genes to be developmentally regulated and expressed during conidiation. The gene products of alb1 (for "albino 1"), arp1 (for "aspergillus reddish-pink 1"), and arp2 have high similarity to polyketide synthases, scytalone dehydratases, and hydroxynaphthalene reductases, respectively, found in the dihydroxynaphthalene (DHN)-melanin pathway of brown and black fungi. The abr1 gene (for "aspergillus brown 1") encodes a putative protein possessing two signatures of multicopper oxidases. The abr2 gene product has homology to the laccase encoded by the yA gene of Aspergillus nidulans. The function of ayg1 (for "aspergillus yellowish-green 1") remains unknown. Involvement of the six genes in conidial pigmentation was confirmed by the altered conidial color phenotypes that resulted from disruption of each gene in A. fumigatus. The presence of a DHN-melanin pathway in A. fumigatus was supported by the accumulation of scytalone and flaviolin in the arp1 deletant, whereas only flaviolin was accumulated in the arp2 deletants. Scytalone and flaviolin are well-known signature metabolites of the DHN-melanin pathway. Based on DNA sequence similarity, gene disruption results, and biochemical analyses, we conclude that the 19-kb DNA fragment contains a six-gene cluster which is required for conidial pigment biosynthesis in A. fumigatus. However, the presence of abr1, abr2, and ayg1 in addition to alb1, arp1, and arp2 suggests that conidial pigment biosynthesis in A. fumigatus is more complex than the known DHN-melanin pathway.

The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence.

Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color. We identified a gene, alb1, which is required for conidial pigmentation. The alb1 gene encodes a putative polyketide synthase, and disruption of alb1 resulted in an albino conidial phenotype. Expression of alb1 is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage. The alb1 mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating that alb1 is involved in dihydroxynaphthalene-melanin biosynthesis. Scanning electron microscopy studies showed that the alb1 disruptant exhibited a smooth conidial surface, whereas complementation of the alb1 deletion restored the echinulate wild-type surface. Disruption of alb1 resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of the alb1 disruptant were ingested by human neutrophils at a higher rate than were those of the wild type. The alb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type. Importantly, the alb1 disruptant had a statistically significant loss of virulence compared to the wild-type and alb1-complemented strains in a murine model. These results suggest that disruption of alb1 causes pleiotropic effects on conidial morphology and fungal virulence.

Studies of the CAG repeat in the Machado-Joseph disease gene in Taiwan.

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by cerebellar ataxia and pyramidal signs associated in varying degrees with a dystonic-rigid extrapyramidal syndrome or peripheral amyotrophy. Unstable CAG trinucleotide repeat expansion in the MJD gene on the long arm of chromosome 14 has been identified as the pathological mutation for MJD. While investigating the distribution of CAG repeat lengths of the MJD gene in Taiwan's population, we have identified 18 MJD-affected patients and 12 at-risk individuals in seven families. In addition, we have analyzed the range of CAG repeat lengths in 96 control individuals. The CAG repeat number ranged from 13 to 44 in the controls and 72-85 in the affected and at-risk individuals. Our results indicated that the CAG repeat number was inversely correlated with the age of onset. The differences in CAG repeat length between parent and child and between siblings are greater with paternal transmission than maternal transmission. Our data show a tendency towards the phenomenon of anticipation in the MJD families but do not support unidirectional expansion of CAG repeats during transmission. We also demonstrated that PCR amplification of the CAG repeats in the MJD gene from villous DNA was possible and might prove useful as a diagnostic tool for affected families in the future.

The CAG repeats number of spinocerebellar ataxia type 1 gene in normal Taiwanese and in patients with dominant inherited ataxia.

Spinocerebellar ataxia type 1 (SCA 1) is an autosomal dominant disorder characterized by neurodegeneration of the cerebellum, spinal cord and brainstem. This neurodegeneration disease is associated with expansion of unstable CAG repeats within the coding region of the gene. We are conducting a local survey of the normal population and candidate patients to analyze the CAG repeats in SCA 1 gene. So far, we have collected peripheral blood from 78 normal individuals and 10 patients with dominant inherited ataxia disorders, and assayed the SCA1 CAG trinucleotide repeat using genomic polymerase chain reaction (PCR). Even though no local SCA 1 patients have been identified, we have established the distributions of the CAG repeat units of SCA 1 gene in the normal population in Taiwan. The normal range of CAG repeats is from 22 to 33 repeats, with the most common being 30 repeats. The range is relatively narrow compared to that reported for other ethnic groups. In addition, direct genomic PCR analysis of the SCA 1 gene from villous DNA has been successful in our laboratory. Screening of SCA 1 patients from patients with dominant inherited ataxia is currently underway in our laboratory. Here, we demonstrate that our molecular analysis technique makes possible the quick and accurate diagnosis of SCA1 patients and prenatal screening for SCA 1 families.

Aspergillus fumigatus arp1 modulates conidial pigmentation and complement deposition.

Aspergillus fumigatus is an important pathogen causing invasive pulmonary aspergillosis in immunocompromised patients. The fungus propagates by conidia, which are the infectious structures inhaled by the human host. Opsonophagocytosis is thought to contribute to clearance of the inhaled conidia, a process that is facilitated by complement deposition on conidial surfaces. We now show that conidial colour mutants exhibit significant increases in C3 binding capacity compared with wild type. A reddish-pink mutation that led to enhanced C3 binding was complemented by a cosmid clone. A 3.3 kb DNA fragment from the subsequently rescued cosmid was sufficient to restore the bluish-green conidial pigment. The bluish-green transformant exhibited a level of C3 binding similar to that of the parental strain. A gene, designated arp1, was responsible for the complementation. Comparison of the genomic and cDNA sequences of arp1 revealed that it has two introns and encodes a putative protein of 168 amino acids. Arp1 is very similar to scytalone dehydratase, an enzyme involved in 1,8-dihydroxynaphthalene-melanin synthesis in Colletotrichum lagenarium and Magnaporthe grisea. Northern hybridization analysis revealed that arp1 is developmentally regulated, being expressed during conidiation. Disruption of arp1 resulted in reddish-pink conidia and increased C3 binding. Our studies suggest that arp1 modulates the bluish-green pigmentation of conidia as well as complement deposition.

Further Characterization of Nociception-Related and Arterial Pressure-Related Neuronal Responses in the Nucleus Reticularis Gigantocellularis of the Rat.

The present study was undertaken to further characterize the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata in the central processing of nociceptive and cardiovascular signals, and its modulation by met-enkephalin. In Sprague-Dawley rats anesthetized with pentobarbital sodium, we found that all 125 spontaneously active NRGC neurons that responded to noxious stimuli (tail clamp) also exhibited arterial pressure-relatedness. Forty neurons additionally manifested cardiac periodicity that persisted even during nociceptive responses. While maintaining their cardiovascular responsive characteristics, the nociception-related NRGC neuronal activity was blocked, naloxone-reversibly (0.5 mg/kg, i.v.), by morphine (5 mg/kg, i.v.). Microiontophoretically applied met-enkephalin suppressed the responsiveness of NRGC neurons to individually delivered tail clamp or transient hypertension induced by phenylephrine (5 &mgr;g/kg, i.v.). Interestingly, in NRGC neurons that manifested both nociception and arterial pressure relatedness, the preferential reduction in the response to noxious stimuli upon simultaneous elevation in systemic arterial pressure was reversed to one that favored nociception in the presence of met-enkephalin. All actions of met-enkephalin were discernibly blocked by the opioid receptor antagonist, naloxone. Our results suggest that individual NRGC neurons may participate in the processing of both nociceptive and cardiovascular information, or in the coordination of the necessary circulatory supports during nociception. In addition, neuropeptides such as met-enkephalin may exert differential modulation on neuronal responsiveness according to the prevailing physiologic status of the animal. They also showed that NRGC may be a central integrator for pain and cardiovascular-related functions. Copyright 1996 S. Karger AG, Basel

The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis.

The first pathway-specific step of ergot alkaloid biosynthesis in the fungus, Claviceps purpurea, is catalyzed by the prenyltransferase, 4-(gamma,gamma-dimethylallyl)tryptophan synthase. Partial sequence information was obtained for the purified enzyme and a degenerate oligonucleotide mixture was used to identify and amplify segments of the gene, dmaW. The complete gene and near-full-length cDNA were cloned and sequenced. The cDNA was cloned in a yeast expression vector in sense and antisense orientations relative to the inducible GAL1 promoter. Extracts of yeast transformants with the sense constructs, but not antisense constructs or cloning vector, catalyzed production of 4-(gamma,gamma-dimethylallyl)tryptophan. The sequence of dmaW and its cDNA indicated that it encoded a 455 amino acid polypeptide with a predicted molecular mass of 51,824 Da and a putative prenyl diphosphate binding motif.

Origin of a fungal symbiont of perennial ryegrass by interspecific hybridization of a mutualist with the ryegrass choke pathogen, Epichloë typhina.

Seed-borne fungal symbionts (endophytes) provide many cool-season grass species with biological protection from biotic and abiotic stresses. The endophytes are asexual, whereas closely related sexual species of genus Epichloë (Clavicipitales) cause grass choke disease. Perennial ryegrass (Lolium perenne) is a host of two endophyte taxa, LpTG-1 (L. perenne endophyte taxonomic grouping one = Acremonium lolii) and LpTG-2, as well as the choke pathogen, Epichloë typhina (represented by isolate E8). Relationships among these fungi and other Epichloë species were investigated by analysis of gene sequences, DNA polymorphisms and allozymes. The results indicate that LpTG-2 is a heteroploid derived from an interspecific hybrid. The LpTG-2 isolates had two copies each of nine out of ten genes analyzed (the exception being the rRNA gene locus), and the profiles for seven of these were composites of those from E. typhina E8 and A. lolii isolate Lp5. Molecular phylogenetic analysis grouped the two beta-tubulin genes of LpTG-2 into separate clades. One (tub2-1) was related to that of E. typhina E8, and the other (tub2-2) to that of A. lolii. The mitochondrial DNA profile of LpTG-2 was similar to that of A. lolii, but its rRNA gene sequence grouped it with E. typhina E8. A proposed model for the evolution of LpTG-2 involves infection of a L. perenne-A. lolii symbiotum by E. typhina, followed by hybridization of the two fungi. Such interspecific hybridization may be a common and important mechanism for genetic variation in Epichloë endophytes.

Modulation by angiotensin III of nociception-related and arterial pressure-related neuronal responsiveness in the nucleus reticularis gigantocellularis of the rat.

We evaluated possible modulation by angiotensin III (AIII) of the interactive effect of noxious stimuli and elevation in systemic arterial pressure on the responsiveness of neurons in the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata. Combined extracellular single-neuron recording and microiontophoresis were carried out on male, adult Sprague-Dawley rats anesthetized with pentobarbital sodium. The responsiveness of NRGC neurons to nociception (tail clamp) and/or transient hypertension elicited by phenylephrine (5 micrograms/kg, i.v.), in the absence or presence of AIII, was used as the experimental index. Microiontophoretic application of the heptapeptide suppressed the responses of spontaneously active NRGC neurons to individually delivered nociception or hypertension. Interestingly, the preferential reduction in responsiveness to tail clamp upon simultaneous elevation in arterial pressure was reversed to one that favored nociception in the presence of AIII. These actions of the heptapeptide appeared to be receptor-specific, since they were discernibly blocked by its selective antagonist, Ile7-angiotensin III. Our results reveal that neuropeptides such as AIII may differentially modulate neuronal responsiveness according to the prevailing physiologic input(s) to the central nervous system of the animal.

Evolutionary diversification of fungal endophytes of tall fescue grass by hybridization with Epichloë species.

The mutualistic associations of tall fescue (Festuca arundinacea) with seed-borne fungal symbionts (endophytes) are important for fitness of the grass host and its survival under biotic and abiotic stress. The tall fescue endophytes are asexual relatives of biological species (mating populations) of genus Epichloë (Clavicipitaceae), sexual fungi that cause grass choke disease. Isozyme studies have suggested considerable genetic diversity among endophytes of tall fescue. Phylogenetic relationships among seven isolates from tall fescue, three from meadow fescue (a probable ancestor of tall fescue), and nine Epichloë isolates from other host species were investigated by comparing sequences of noncoding segments of the beta-tubulin (tub2) and rRNA (rrn) genes. Whereas each Epichloë isolate and meadow fescue endophyte had only a single tub2 gene, most tall fescue endophytes had two or three distinct tub2 copies. Phylogenetic analysis of tub2 sequences indicated that the presence of multiple copies in the tall fescue endophytes was a consequence of hybridization with Epichloë species. At least three hybridization events account for the distribution and relationships of tub2 genes. These results suggest that interspecific hybridization is the major cause of genetic diversification of the tall fescue endophytes.