Increased metabolite production by deletion of an HDA1-type histone deacetylase in the phytopathogenic fungi, Magnaporthe oryzae (Pyricularia oryzae) and Fusarium asiaticum.

Histone deacetylases (HDACs) play an important role in the regulation of chromatin structure and gene expression. We found that dark pigmentation of Magnaporthe oryzae (anamorph Pyricularia oryzae) ΔMohda1, a mutant strain in which an orthologue of the yeast HDA1 was disrupted by double cross-over homologous recombination, was significantly stimulated in liquid culture. Analysis of metabolites in a ΔMohda1 mutant culture revealed that the accumulation of shunt products of the 1,8-dihydroxynaphthalene melanin and ergosterol pathways were significantly enhanced compared to the wild-type strain. Northern blot analysis of the ΔMohda1 mutant revealed transcriptional activation of three melanin genes that are dispersed throughout the genome of M. oryzae. The effect of deletion of the yeast HDA1 orthologue was also observed in Fusarium asiaticum from the Fusarium graminearum species complex; the HDF2 deletion mutant produced increased levels of nivalenol-type trichothecenes. These results suggest that histone modification via HDA1-type HDAC regulates the production of natural products in filamentous fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural products of fungi have significant impacts on human welfare, in both detrimental and beneficial ways. Although HDA1-type histone deacetylase is not essential for vegetative growth, deletion of the gene affects the expression of clustered secondary metabolite genes in some fungi. Here, we report that such phenomena are also observed in physically unlinked genes required for melanin biosynthesis in the rice blast fungus. In addition, production of Fusarium trichothecenes, previously reported to be unaffected by HDA1 deletion, was significantly upregulated in another Fusarium species. Thus, the HDA1-inactivation strategy may be regarded as a general approach for overproduction and/or discovery of fungal metabolites.

Effects of acivicin on growth, mycotoxin production and virulence of phytopathogenic fungi.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 µmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.

An Arabidopsis sigma factor (SIG2)-dependent expression of plastid-encoded tRNAs in chloroplasts.

A eubacteria-type RNA polymerase (PEP) plays crucial roles for chloroplast development in higher plants. The core subunits are encoded on plastid DNA (rpo genes) while the regulatory sigma factors are encoded on the nuclear DNA (SIG genes). However, the definite gene specificity of each sigma factor is unknown. We recently identified an Arabidopsis recessive pale-green mutant abc1 in which T-DNA is inserted in SIG2 (sigB). In this mutant, almost normal etioplasts were developed under dark conditions while the small chloroplasts with poor thylakoid membranes and stacked lamellar were developed under light conditions. The sig2-1 mutant was deficient in accumulating enough photosynthetic and photosynthesis-related proteins as well as chlorophyll. However, mRNAs of their structural genes were not significantly reduced. Further analyses revealed that several plastid-encoded tRNAs including trnE-UUC that has dual function for protein and ALA biosyntheses were drastically reduced in the sig2-1 mutant. In contrast, nucleus-encoded T7 phage-type RNA polymerase (NEP)-dependent gene transcripts were steadily accumulated in the mutant. These results indicate that progress of chloroplast development requires SIG2-dependent expression of plastid genes, particularly some of the tRNA genes.

Splitting and penetration of the optic nerve by an aneurysm arising from the anterior wall of internal carotid artery: case report.

Aneurysms arising from the anterior wall of the internal carotid artery (ICA) are uncommon. There have not been any reports demonstrating the anatomical relation of such aneurysms to the optic nerve. An aneurysm arising from the anterior wall of the ICA splitting and penetrating the optic nerve is reported. A 73 year old woman presented with severe headache due to subarachnoid haemorrhage. She had never experienced a visual disturbance. At surgery, the aneurysm was shown to arise from the anterior wall of the left internal carotid artery and to split and penetrate the left optic nerve. The aneurysm was not related to arterial bifurcation of any branches and was safely neck clipped. Given the evidence of a split and penetration of the optic nerve, the pathogenesis of such an aneurysm may be due to the persistence of an embryonic vessel.

Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.

SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157:H7.

The quorum-sensing system in bacteria is a well-known regulatory system that controls gene expression in a cell density-dependent manner. A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram-negative bacteria. In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E. coli (EHEC) O157:H7. The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression. SdiA has a DNA-binding motif in its C-terminal part and can bind to the promoter regions of the esp and eae genes in vitro. Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N-terminal part of SdiA in vivo and in vitro. O157:H7 overproducing the N-terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N-terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors. These results indicate that a quorum-sensing system including the SdiA protein controls colonization by O157:H7.

Chloroplast development in Arabidopsis thaliana requires the nuclear-encoded transcription factor sigma B.

Development of plastids into chloroplasts, the organelles of photosynthesis, is triggered by light. However, little is known of the factors involved in the complex coordination of light-induced plastid gene expression, which must be directed by both nuclear and plastid genomes. We have isolated an Arabidopsis mutant, abc1, with impaired chloroplast development, which results in a pale green leaf phenotype. The mutated nuclear gene encodes a sigma factor, SigB, presumably for the eubacterial-like plastid RNA polymerase. Our results provide direct evidence that a nuclear-derived prokaryotic-like SigB protein, plays a critical role in the coordination of the two genomes for chloroplast development.

Chronic hydrocephalus presenting with bilateral ptosis after minor head injury: case report.

OBJECTIVE AND IMPORTANCE: Some patients with hydrocephalus may exhibit various signs of oculomotor dysfunction. However, ptosis has not previously been described in chronic hydrocephalus patients. CLINICAL PRESENTATION: We report a 50-year-old woman who was diagnosed with chronic hydrocephalus based on an evaluation for bilateral ptosis after a minor head injury. She exhibited bilateral ptosis and upward gaze paralysis, but other oculomotor functions were normal. Neuroimages revealed chronic hydrocephalus with no traumatic abnormalities. INTERVENTION: The eyelid dysfunction resolved after placement of a right ventriculoperitoneal shunt with a programmable pressure valve. CONCLUSION: The resolution of eyelid dysfunction by cerebrospinal fluid diversion suggests that chronic hydrocephalus was involved in the development of ptosis after the minor head injury. A mild but sudden cerebrospinal fluid pressure change at the time of minor head injury might induce functional impairment at the level of vulnerable periaqueductal structures, which barely withstood the longstanding ventriculomegaly, resulting in the clinical features observed in our patient.

Isolation and characterization of mini-Tn5Km2 insertion mutants of enterohemorrhagic Escherichia coli O157:H7 deficient in adherence to Caco-2 cells.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to intestinal epithelium is essential for initiation of the infection. To identify genes involved in adherence, an EHEC O157:H7 strain (O157Sakai) was mutagenized by mini-Tn5Km2, where Km refers to kanamycin resistance, and 4,677 insertion mutants were screened for their ability to form microcolonies (MC) on Caco-2 cells. The less adherent mutants were divided into three groups: those with no adherent ability (designated as class 1 mutants, n = 10), those less adherent than the wild type (class 2 mutants, n = 16), and those unable to form MC but which adhered in a diffuse manner (class 3 mutants, n = 1). The sites of insertion in class 1 mutants were all found within genes of the locus for enterocyte effacement (LEE) thought to be required for type III protein secretion. Indeed, the class 1 mutants failed to secrete type III secreted proteins such as EspA and Tir into the culture medium. The insertions in class 2 mutants were outside the LEE, and all the mutants except one were able to secrete type III proteins into the culture medium. The class 3 mutant had the insertion in the tir gene in the LEE and was deficient in Tir and intimin expression, suggesting that in the absence of intimin-Tir, O157Sakai can still adhere to Caco-2 cells but in a diffused manner. This was confirmed by construction of a nonpolar eae (encoding intimin) mutant. Examination of the eae mutant together with O157Sakai and one of the class 1 mutants for the ability to form MC revealed that EHEC initially adhered diffusely at 1.5 h after infection. Following washing out of the nonadherent bacteria, while wild-type EHEC bacteria developed MC for another 2 to 3 h on Caco-2 cells, the eae mutant diffusely adhered throughout the infection without forming MC. MC with O157Sakai but not the diffusely adherent eae mutant could evoke F-actin condensation beneath the bacterium. Our results suggest that EHEC encodes additional adherence-associated loci and that the type III secreted proteins are involved in the initial diffuse adherence, while the intimin-Tir interaction is required for the subsequent development of MC.

Three new nuclear genes, sigD, sigE and sigF, encoding putative plastid RNA polymerase sigma factors in Aarabidopsis thaliana.

Three new nuclear genes (sigD, sigE and sigF) of Arabidopsis thaliana, encoding putative plastid RNA polymerase sigma factors, were identified and analyzed. Phylogenetic analysis revealed that higher plant sigma factors fell into at least four distinct subgroups within a diverse protein family. In addition, Arabidopsis sig genes contained conserved chromosomal intron sites, indicating that these genes arose by DNA duplication events during plant evolution. Transcript analyses revealed two alternatively spliced transcripts generated from the sigD region, one of which is predicted to encode a sigma protein lacking the carboxy-terminal regions 3 and 4. Finally, the amino-terminal sequence of the sigF gene product was shown to function as a plastid-targeting signal using green fluorescent protein fusions.

Prognostic significance of p53 and p21WAF1/CIP1 immunoreactivity and tumor micronecrosis for recurrence of meningiomas.

Recurrence is an important factor for prognosis of meningioma patients, this also occurring with some lesions diagnosed histopathologically as benign. To analyze their relationships with clinicopathological factors, p53 and p21WAF1/CIP1 immunoreactivity, 80 meningiomas were classified into four groups with regard to the World Health Organization (WHO) histological classification and recurrence: 40 cases of Group I (typical type)-NR (no recurrence); five cases of Group I-R (recurrence); 20 cases of Group II (atypical or anaplastic type)-NR and 15 cases of Group II-R. Micronecrosis was detected in 25% of Group II-NR and 73.3% of Group II-R (P = 0.007, odds ratio (OR) = 8.25, 95% confidence interval (CI) = 1.79-38.01). Patients receiving radiation therapy had a lower risk of recurrence (P = 0.041, OR = 0.20, 95% CI = 0.05-0.85). Immunoreactivity for p53 protein was positive in 22% of Group I and 54% or Group II (P = 0.005), and in 80% of Group I-R and 15% of Group I-NR (P = 0.006, OR = 22.7, 95% CI = 2.15-239.4). p21WAF1/CIP1 protein was detected in 22% of Group I and 48% of Group II (P = 0.017), but with no link to recurrence. Multivariate analysis also showed p53 immunoreactivity in Group I (benign lesions) and micronecrosis in Group II (atypical/anaplastic meningiomas) to be strong prognostic factors for recurrence (P < 0.05). These results indicate that p53 immunoreactivity and micronecrosis can help predicting recurrence of meningiomas.

Regulation of virulence factors of enterohemorrhagic Escherichia coli O157:H7 by self-produced extracellular factors.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes serious diarrhea and hemolytic uremic syndrome in humans. The expressions of EspD and intimin by O157:H7 have now been shown to be down-regulated by medium conditioned by O157:H7 grown at stationary phase. Preparation of conditioned medium showing the effect on the amount of EspD was not dependent on temperature or growth medium, but was dependent on growth phase. Inhibition of EspD and intimin expression was also induced by medium conditioned by E. coli K-12 strains and homoserine lactone, a signal molecule of the quorum-sensing system in gram-negative bacteria. These results suggest the possibility that the quorum-sensing system mediated by self-produced extracellular factors plays an important role in control of colonization of EHEC O157:H7.

Chloroplast targeting, distribution and transcriptional fluctuation of AtMinD1, a Eubacteria-type factor critical for chloroplast division.

In Arabidopsis thaliana, a mature mesophyll cell contains approximately 100 chloroplasts. Although 12 arc mutants (accumulation and replication of chloroplasts) and two chloroplast division genes homologous to eubacterial ftsZ have been isolated from A. thaliana, the molecular mechanism underlying the chloroplast division is still unclear. We characterized AtMinD1, a eubacterial minD homolog, for chloroplast division in A. thaliana. AtMinD1-green fluorescent protein targeted to the chloroplasts and possibly associated with the envelope membranes in vivo. During the seed germination, the AtMinD1 transcripts were accumulated twice, just after release from cold treatment and at the beginning of rapid greening, in similar fashion to AtFtsZs. Furthermore the transcript level in a severest chloroplast division mutant, arc6, was 3-5-fold higher than that in wild-type.

Plastidic RNA polymerase sigma factors in Arabidopsis.

In plant cells, plastid DNA is transcribed by at least two types of RNA polymerase, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is homologous to eubacterial transcription machinery, but its regulatory subunit, sigma (sigma) factor, is not encoded on the plastid DNA. We previously cloned the three nuclear-encoded sigma factor genes from Arabidopsis thaliana and designated them as sigA, sigB, and sigC. By means of RFLP mapping, sigA and sigB were mapped on chromosome I and sigC on the chromosome III. Based on comparison of the genomic structure of the three sig genes, intron sites in the 3' half of the genes were shown to be identical between sigB and sigC but divergent in sigA, consistent with the phylogenetic relevance of the three gene products. A transient expression assay of GFP fusions in Arabidopsis protoplasts showed that the N-termini of all three sig gene products functioned as chloroplast-targeting signals. We also constructed transgenic Arabidopsis lines harboring the sigA-promoter or the sigB-promoter uidA fusion. Both the sigA- and sigB-promoters were similarly activated at cotyledons, hypocotyls, rosette leaves, cauline leaves, sepals, and siliques but not at roots, seeds, or other flower organs. In addition, the two promoters were repeatedly activated in young seedlings under continuous light, possibly in an oscillated fashion.

Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats.

Delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) causes cerebral ischemia and infarction. To date, the pathogenesis and gene expression associated with vasospasm remain poorly understood. The present study used fluorescent differential display to identify differentially expressed genes in a rat model of SAH. By using quantitative RT-PCR, we found that heme oxygenase-1 (HO-1) mRNA was prominently induced in the basilar artery and modestly in brain tissue in a rat vasospasm model. A significant correlation was observed between the degree of vasospasm and HO-1 mRNA levels in the basilar arteries exhibiting vasospasm. Intracisternal injection of antisense HO-1 oligodeoxynucleotide (ODN) significantly delayed the clearance of oxyhemoglobin and deoxyhemoglobin from the subarachnoid space and aggravated angiographic vasospasm. Antisense HO-1 ODN inhibited HO-1 induction in the basilar arteries but not in the whole brain tissue. This phenomenon was not observed in the nontreated, sense HO-1 ODN-treated, or scrambled ODN-treated arteries. We report the protective effects of HO-1 gene induction in cerebral vasospasm after SAH, a finding that should provide a novel therapeutic approach for cerebral vasospasm.

Assessment of long-distance running performance in elite male runners using onset of blood lactate accumulation.

The purpose of this study was to evaluate the relationship between onset of blood lactate accumulation (OBLA) and long-distance running performance in order to examine whether OBLA can be a good predictor of long-distance running performance even in elite male runners with similar performance levels. Eleven highly-trained male long-distance runners participated in this study. The average running velocities of the individuals' running performance were 5.918 +/- 0.084 m.s-1 and 5.672 +/- 0.095 m.s-1 for 5000 m (V5000) and 10,000 m (V10000), respectively. The blood lactate concentrations and heart rate responses were measured immediately after field running, and the average value of running velocity corresponding to OBLA (VOBLA) was 5.447 +/- 0.132 m.s-1. Variations of these three velocities expressed as a coefficient of variance (CV) ranged from 1.4 to 2.4%. A strong inverse relationship between heart rate corresponding to OBLA (HROBLA) and performance was observed (r = -0.709, p < 0.02 for V5000 and r = -0.830, p < 0.01 for V10000), while there was a lack of significant relationship between VOBLA and performance (r = 0.293, NS for V5000 and r = 0.130, NS for V10000). Furthermore, the average value of HROBLA obtained in this study (174.5 +/- 8.2 b.min-1) was quite similar to that of the heart rate threshold reported by some previous researchers. In conclusion, VOBLA alone could not explain the small variation of long-distance running performance, and HROBLA should be used in place of VOBLA for evaluating long-distance running performance in elite runners with quite similar performance levels.

Effects of tirilazad mesylate on vasospasm and phospholipid hydroperoxides in a primate model of subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Tirilazad mesylate has been used in the attempt to prevent cerebral vasospasm after subarachnoid hemorrhage (SAH), although the actual targets of this agent in vivo have thus far been controversial. Chemiluminescence/high-performance liquid chromatography provided a new method for direct measurements of phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) in vivo and showed that phosphatidylcholine is the lipid class most susceptible to lipid peroxidation. In the present study we measured those levels in a primate model of SAH for determination of the effects of tirilazad on vasospasm. METHODS: Fourteen Macaca monkeys of both sexes were randomly assigned into 2 groups: a tirilazad group receiving a dosage of 0.3 mg/kg and a placebo group receiving only the vehicle in which tirilazad was delivered. After the induction of experimental SAH around the right middle cerebral artery on day 0, tirilazad or vehicle was administered intravenously every 8 hours for 6 days. On day 7, the animals were killed after angiography and regional cerebral blood flow measurements were performed. The levels of PCOOH and PEOOH were measured in the clots, bilateral parietal cortices, right frontal cortex contact with clots, cerebellar hemispheres, bilateral middle cerebral arteries, and basilar arteries. RESULTS: In the placebo group, a significant vasospasm occurred in the cerebral arteries on both sides, but most prominently on the right side. The degree of vasospasm in the cerebral arteries was significantly attenuated in the tirilazad group (P<0.005). There were no significant differences in regional cerebral blood flow, PCOOH, and PEOOH levels in the clots, cerebral cortices, and cerebellar hemispheres between the 2 groups. In contrast, the levels of PCOOH in the cerebral arteries were significantly higher in the placebo group than in the tirilazad group (P<0.025). It was remarkable that the tirilazad treatments eliminated PCOOH in any vascular territory after SAH. CONCLUSIONS: PCOOH in the artery wall may be an important indicator for vasospasm, and the inhibition of PCOOH may explain the efficacy of tirilazad on vasospasm.

Ser-534 in the hinge 1 region of Arabidopsis nitrate reductase is conditionally required for binding of 14-3-3 proteins and in vitro inhibition.

14-3-3 proteins bind to the hinge 1 region of nitrate reductase (NR) and inhibit its activity. To determine which residues of NR are required for 14-3-3-inhibitory interactions, wild-type and mutant forms of Arabidopsis NR were examined in the yeast two-hybrid system and in vitro inhibition assays. NR fragments with or without hinge 1 were introduced into yeast with one of seven Arabidopsis 14-3-3 isoforms (called GF14s). NR fragments (residues 1-562 or 487-562) containing hinge 1 interacted with all GF-14s tested; an NR fragment (residues 1-487) lacking hinge 1 did not. GF14 binding to NR fragments was dependent on Ser-534, since Asp or Ala substitutions at this site blocked the interaction. Revertants with second site substitutions restoring interaction between GF14omega and the Ala- or Asp-substituted NR fragments were identified. One isolate had a Lys to Glu substitution at position 531, which is in hinge 1, and six isolates had Ile to Leu or Phe substitutions at 561 in the heme binding region. Double mutant forms of holo-NR (S534D plus K531E, I561F, or I561L) were constructed and found to be partially inhibited by protein extracts from Arabidopsis containing 14-3-3 proteins. Wild-type NR is phosphorylated and inhibited by these extracts, but S534D single mutant forms are not. These results show that inhibitory NR/14-3-3 interactions are dependent on Ser-534 but only in the context of the wild-type sequence, since substitutions at second sites render 14-3-3 binding and in vitro NR inhibition independent of Ser-534.

[Heme oxgenase-1 gene induction and the mechanism in the rat vasospasm model].

Using fluorescent differential display and quantitative reverse-PCR, we found in the rat vasospasm model that heme oxygenase-1 messenger RNA was induced in basilar artery. Intracistemal injection of antisense OH-1 oligodeoxynucleotide significantly reduced HO-1 mRNA and HO-1 protein levels and enhanced angiographic vasospasm. Thus, we demonstrate that HO-1 induction may play a important role in the resolution of delayed vasospasm after subarachnoid hemorrhage.