[Neonatal molding for minor deformities of auricular cartilage: a simple method]. / Modelage néonatal des déformations mineures du cartilage auriculaire : une méthode simple.

Non-surgical correction of minor ear deformities by external splinting during neonatal age is a well-known, effective technique, but not frequently used in France. We would like to popularize an established, simple method that uses cheap, available means (a wire, adhesive strips and a silicone probe). It can be performed by parents, paediatricians and nurses. Spreading this method would allow early onset of treatment and better clinical results. On the long run, it would have a certain economic aftermath on national health insurance by reducing the number of surgical procedures for deformed ears.

[Cleft lip and palate management by Pr Hosaka's team at the Showa University, Tokyo (Japan)]. / Prise en charge des fentes labiopalatines par l'équipe du Pr Hosaka à l'université de Showa, Tokyo (Japon).

We describe the particularities of cleft lip and palate treatment in the department of plastic surgery managed by Pr Hosaka at the Showa University in Tokyo. Their surgical technic inherited from Pr Onizuka, their multidisciplinary approach, and their experience with over 300 cases a year were not reported in a non-Japanese journal. Therefore, we found interesting to describe their whole management.

Identification of genes for resistance to a Digitaria isolate of Magnaporthe grisea in common wheat cultivars.

Common wheat cultivars are resistant to Magnaporthe grisea, a crabgrass (Digitaria sanguinalis)-specific species of the blast fungus. To dissect the genetic basis of this "nonhost" type of resistance, we need an exceptional cultivar that is susceptible to M. grisea. A screening under various conditions revealed that Triticum aestivum 'Chinese Spring' (CS) was susceptible to M. grisea isolate Dig41 when incubated at high temperature (26 degrees C) after inoculation. By contrast, T. aestivum 'P168', 'Shin-chunaga' (Sch), 'Norin 4' (N4), 'Norin 26' (N26), 'Norin 29' (N29), 'Red Egyptian' (RE), and 'Salmon' (Slm) and Triticum compactum 'No. 44' (Cmp) were highly resistant even at the high temperature. When F2 seedlings derived from crosses between the resistant cultivars and CS were inoculated with Dig41, they segregated in a 3:1 ratio of resistant to susceptible, suggesting that the resistance of each cultivar is controlled by one major gene. Crosses of N4 with P168, Sch, N26, N29, and Cmp yielded no susceptible F2 seedlings, suggesting that these six cultivars share the same gene. Similarly, a cross between RE and Slm yielded no susceptible F2 seedlings, suggesting that these two cultivars share the same gene. On the other hand, crosses between the N4 group and the RE group produced resistant and susceptible seedlings in a 15:1 ratio, indicating that these two groups carry different genes inherited independently. The gene in N4 was located on chromosome 4A by a monosomic analysis and designated Rmg4, while the gene in RE was located on chromosome 6D using a series of chromosome substitution lines and designated Rmg5. These results suggest that the resistance of common wheat to M. grisea, an inappropriate species of the blast fungus, is under a simple genetic control.

Identification of two genes for resistance to Triticum isolates of Magnaporthe oryzae in wheat.

A screening of common wheat cultivars revealed that Triticum aestivum 'Thatcher' was resistant to Triticum isolates of Magnaporthe oryzae, whereas T. aestivum 'Chinese Spring' was susceptible. When F2 seedlings from a cross between 'Thatcher' and 'Chinese Spring' were inoculated with the Triticum isolates, resistant and susceptible seedlings segregated in a 15:1 ratio, suggesting that the resistance of 'Thatcher' was conditioned by two major genes. An inoculation test of 'Chinese Spring' substitution lines carrying individual chromosomes from 'Thatcher' indicated that these genes, designated Rmg2 and Rmg3, were located on chromosomes 7A and 6B.

Comparative Genetic Analysis of Magnaporthe oryzae Isolates Causing Gray Leaf Spot of Perennial Ryegrass Turf in the United States and Japan.

Gray leaf spot caused by Magnaporthe oryzae is a serious disease of perennial ryegrass (Lolium perenne) turf in golf course fairways in the United States and Japan. Genetic relationships among M. oryzae isolates from perennial ryegrass (prg) isolates within and between the two countries were examined using the repetitive DNA elements MGR586, Pot2, and MAGGY as DNA fingerprinting probes. In all, 82 isolates of M. oryzae, including 57 prg isolates from the United States collected from 1995 to 2001, 1 annual ryegrass (Lolium multiflorum) isolate from the United States collected in 1972, and 24 prg isolates from Japan collected from 1996 to 1999 were analyzed in this study. Hybridization with the MGR586 probe resulted in approximately 30 DNA fragments in 75 isolates (designated major MGR586 group) and less than 15 fragments in the remaining 7 isolates (designated minor MGR586 group). Both groups were represented among the 24 isolates from Japan. All isolates from the United States, with the exception of one isolate from Maryland, belonged to the major MGR586 group. Some isolates from Japan exhibited MGR586 fingerprints that were identical to several isolates collected in Pennsylvania. Similarly, fingerprinting analysis with the Pot2 probe also indicated the presence of two distinct groups: isolates in the major MGR586 group showed fingerprinting profiles comprising 20 to 25 bands, whereas the isolates in the minor MGR586 group had less than 10 fragments. When MAGGY was used as a probe, two distinct fingerprint types, one exhibiting more than 30 hybridizing bands (type I) and the other with only 2 to 4 bands (type II), were identified. Although isolates of both types were present in the major MGR586 group, only the type II isolates were identified in the minor MGR586 group. The parsimony tree obtained from combined MGR586 and Pot2 data showed that 71 of the 82 isolates belonged to a single lineage, 5 isolates formed four different lineages, and the remaining 6 (from Japan) formed a separate lineage. This study indicates that the predominant groups of M. oryzae associated with the recent outbreaks of gray leaf spot in Japan and the United States belong to the same genetic lineage.

Genetic Analysis of Host Species Specificity of Magnaporthe oryzae Isolates from Rice and Wheat.

ABSTRACT A Triticum isolate (pathogenic on wheat) of Magnaporthe oryzae was crossed with an Oryza isolate (pathogenic on rice) to elucidate mechanisms of their parasitic specificity on wheat. When the pathogenicity of their F (1) cultures (hybrids between a Triticum isolate and an Oryza isolate) was tested on wheat, avirulent and virulent cultures segregated in a 7:1 ratio. This result suggests that three loci are involved in avirulence of the Oryza isolate on wheat. One of the three loci conditioned papilla formation, whereas the others conditioned the hypersensitive reaction. Allelism tests revealed that the locus conditioning papilla formation is Pwt2 while one of the two loci conditioning the hypersensitive reaction is Pwt1. The other locus conditioning the hypersensitive reaction was different from any other known loci and, therefore, was designated as Pwt5.

Genetic Constitution and Pathogenicity of Lolium Isolates of Magnaporthe oryzae in Comparison with Host Species-Specific Pathotypes of the Blast Fungus.

ABSTRACT Fungal isolates from gray leaf spot on perennial ryegrass (prg isolates) were characterized by DNA analyses, mating tests, and pathogenicity assays. All of the prg isolates were interfertile with Triticum isolates and clustered into the crop isolate group (CC group) on a dendrogram constructed from rDNA-internal transcribed spacer 2 sequences. Since the CC group corresponded to a newly proposed species, Magnaporthe oryzae, all of the prg isolates were designated M. oryzae. However, DNA fingerprinting with MGR586, MGR583, and Pot2 showed that the prg isolates are divided into two distinct populations, i.e., TALF isolates and WK isolates. The TALF isolates were virulent only on Lolium species, whereas the WK isolates were less specific, suggesting that gray leaf spot can be caused not only by Lolium-specific isolates but also by less specific isolates. We designated the TALF isolates as Lolium pathotype. The TALF isolates showed diverse karyotypes in spite of being uniform in DNA fingerprints, suggesting that theyare unstable in genome organization.

Meiotic behavior of a supernumerary chromosome in Magnaporthe oryzae.

A 1.2-Mb DNA band from an isolate of Magnaporthe oryzae was detected in a pulsed-field gel. A chromosomal entity corresponding to this band was observed at the mitotic metaphase stage. This minichromosome, carrying many transposable elements and two telomeres, was transmitted to ascosporic F(1) cultures in a non-Mendelian manner with frequent changes in its size and number. Segregation analysis with RFLP markers indicated that the minichromosome underwent structural rearrangements, such as deletion and duplication, not only during meiosis but also after meiosis. An ectopic sister chromatid recombination may cause the size variation of the minichromosomes.

Analysis of Host Species Specificity of Magnaporthe grisea Toward Foxtail Millet Using a Genetic Cross Between Isolates from Wheat and Foxtail Millet.

ABSTRACT Host species specificity of Magnaporthe grisea toward foxtail millet was analyzed using F(1) cultures derived from a cross between a Triticum isolate (pathogenic on wheat) and a Setaria isolate (pathogenic on foxtail millet). On foxtail millet cvs. Beni-awa and Oke-awa, avirulent and virulent cultures segregated in a 1:1 ratio, suggesting that a single locus is involved in the specificity. This locus was designated as Pfm1. On cv. Ki-awa, two loci were involved and one of them was Pfm1. The other locus was designated as Pfm2. Interestingly, Pfm1 was not involved in the pathogenic specificity on cv. Kariwano-zairai. These results suggest that there is no "master gene" that determines the pathogenic specificity on all foxtail millet cultivars and that the species specificity of M. grisea toward foxtail millet is governed by cultivar-dependent genetic mechanisms that are similar to gene-for-gene interactions controlling race-cultivar specificity.

Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages.

AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.

A Gene-for-Gene Relationship Underlying the Species-Specific Parasitism of Avena/Triticum Isolates of Magnaporthe grisea on Wheat Cultivars.

ABSTRACT To elucidate genetic mechanisms of the species-specific parasitism of Magnaporthe grisea, a Triticum isolate (pathogenic on wheat) was crossed with an Avena isolate (pathogenic on oat), and resulting F(1) progeny were subjected to segregation analyses on wheat cvs. Norin 4 and Chinese Spring. We found two fungal loci, Pwt3 and Pwt4, which are involved in the specific parasitism on wheat. Pwt3 operated on both cultivars while Pwt4 operated only on 'Norin 4'. Using the cultivar specificity of Pwt4, its corresponding resistance gene was successfully identified in 'Norin 4' and designated as Rmg1 (Rwt4). The presence of the corresponding resistance gene indicated that Pwt4 is an avirulence locus. Pwt3 was assumed to be an avirulence locus because of its temperature sensitivity. We suggest that gene-for-gene interactions underlie the species-specific parasitism of M. grisea.

Novel evidence for apoptotic cell response and differential signals in chromatin condensation and DNA cleavage in victorin-treated oats.

Histological and cytological evidence of where and when apoptotic cells occur in Pc-2/Vb oat cells treated with victorin was obtained by observing DNA strand breaks at both light (LM) and electron microscope (EM) levels using TUNEL techniques. DNA from leaf segments that had been floated on victorin solution with the abaxial epidermis removed showed typical ladders on agarose gels. Nuclear chromatin condensation, followed by cell collapse, started in the mesophyll cells closest to the victorin solution. LM-TUNEL was positive in the non-collapsed cells but not in the collapsed cells in the treated leaves. However, the EM-TUNEL assay was positive in the nuclei of the non-collapsed as well as the collapsed cells where nuclear fragments dispersed into the cytoplasm, and the immunogold density was much higher than that in the cells killed by a high concentration of H2O2, suggesting that the victorin-treated collapsed cells are in the last stage of apoptotic cell death. The immunogold labelling in the victorin-treated non-collapsed cells was restricted to condensed heterochromatin, indicating that chromatin condensation is associated with DNA cleavage. Pharmacological studies indicated that proteases and nucleases may play a role in the apoptotic response. However, the EM-TUNEL assay indicated that EGTA co-incubated with victorin blocked DNA cleavage, but failed to prevent chromatin condensation. Moreover, protein kinases were involved in chromatin condensation, but did not affect DNA digestion, suggesting that chromatin condensation and DNA cleavage are differentially regulated in the death process in oats.

Pyret, a Ty3/Gypsy retrotransposon in Magnaporthe grisea contains an extra domain between the nucleocapsid and protease domains.

A novel Ty3/Gypsy retrotransposon, named Pyret, was identified in the plant pathogenic fungus Magnaporthe grisea (anamorph Pyricularia oryzae). Pyret-related elements were distributed in a wide range of Pyricularia isolates from various gramineous plants. The Pyret element is 7250 bp in length with a 475 bp LTR and one conceptual ORF. The ORF contains seven nonsense mutations in the reading frame, indicating that the Pyret clone is lightly degenerate. Comparative domain analysis among retroelements revealed that Pyret exhibits an extra domain (WCCH domain) beyond the basic components of LTR retrotransposons. The WCCH domain consists of approximately 300 amino acids and is located downstream of the nucleocapsid domain. The WCCH domain is so named because it contains two repeats of a characteristic amino acid sequence, W-X(2)-C-X(4)-C-X(2)-H-X(3)-K. A WCCH motif-like sequence is found in the precoat protein of some geminiviruses, viral RNA-dependent RNA polymerase and also in an Arabidopsis protein of unknown function. Interestingly, detailed sequence analysis of the gag protein revealed that Pyret, as well as some other chromodomain-containing LTR retrotransposons, displays significant sequence homology with members of the gammaretroviruses (MLV-related retroviruses) in the capsid and nucleocapsid domains. This suggests that chromodomain-containing LTR retrotransposons and gammaretroviruses may share a common ancestor with the gag protein.

Heat shock, copper sulfate and oxidative stress activate the retrotransposon MAGGY resident in the plant pathogenic fungus Magnaporthe grisea.

MAGGY is a gypsy-like retrotransposon isolated from the plant pathogenic fungus Magnaporthe grisea. The ability of various stresses to activate MAGGY was tested in the original and in a heterologous host (Colletotrichum lagenarium), using beta-glucuronidase (GUS) as a reporter. The MAGGY promoter was activated in M. grisea by either heat shock, copper sulfate, or oxidative stress, but not by the antifungal substance p-coumaric acid. Transcriptional up-regulation of MAGGY RNA was also observed following heat shock and oxidative stress. The MAGGY promoter remained responsive to the above-mentioned stresses when transformed into a M. grisea isolate that had no endogenous MAGGY elements. In C. lagenarium, however, the MAGGY promoter showed only basal expression of GUS and no further up-regulation was induced by any of the stress treatments, suggesting that the stress-responding cis-element(s) in the MAGGY promoter is not functional in a wider range of fungi. The relationship between the activation of MAGGY by stress and phenotypic diversification in M. grisea, including variations in pathogenicity, is discussed.

Location and length of arteriovenous fistulas around axial-pattern skin-flap pedicles.

The importance of the location of a surgically-created arteriovenous fistula around the pedicle (both distal and proximal) on the viability of rat skin flaps was investigated. The animals were randomly divided into four groups. Group 1 included bilateral standard island groin flaps. The right side flap was used as a control. On the left side, after elevation of the flap, an X-type arteriovenous fistula greater than 1 mm (up to 2 mm) in length was created distal to the pedicle, and just before the bifurcation of the common femoral vessels. In Group 2, the flap was an axial-pattern medially-based peninsular flap, including the same vessels. In this group also, two flaps were elevated bilaterally, and the right side was used as a control; on the left side, an X-type arteriovenous fistula the same length as in Group 1 was also created distal to the pedicle. In both groups, all other branches of the common femoral vessels were kept intact. In a second part of the study, two other animal groups were used to clarify the importance of the length of the arteriovenous fistula on the viability of skin flaps. In Group 3, the model was the same as in Group 1, but the fistula was 1 mm in length. In Group 4, the length of the fistula was 1 mm, and its location was on the common femoral vessels proximal to the pedicle, using the same flap model. Flow values were measured repeatedly using a laser Doppler flowmeter. Histopathologic studies were also done. There are three important points arising from these studies. 1). The location of an X-type arteriovenous fistula around an island skin flap pedicle seems to be more important than diameter. An arteriovenous fistula proximal to the pedicle is more hazardous than an arteriovenous fistula distal to the pedicle, regarding island skin-flap viability. 2). However, the length of the fistula is also important, and an arteriovenous fistula distal to the pedicle, with a sufficiently long length, is not devoid of harmful effects. It is also clear that the larger the fistula, the greater the systemic effects. 3). An island skin flap with an arteriovenous fistula distal to its pedicle might be a useful model to study the relationship between skin-flap viability and edema formation.

Induction and signaling of an apoptotic response typified by DNA laddering in the defense response of oats to infection and elicitors.

Cells in the primary leaves of oats displayed internucleosomal DNA cleavage in response to incompatible crown rust infection. DNA laddering also was evident in leaves treated with calcium ionophore A23187, nonspecific elicitors such as chitin and chitosan oligomers, and victorin, which functions as a specific elicitor in Pc-2/Vb containing oat leaves. The nuclei in a victorin-treated susceptible oat line were positive for the TUNEL assay. These elicitors clearly induced a 28-kDa nuclease (p28) in addition to three constitutive nucleases of 33, 24, and 22 kDa. Activation of p28 preceded the appearance of DNA laddering and possibly was mediated by de novo synthesis and/or cysteine protease activity. Pharmacological studies showed that the induction of DNA laddering was associated with oxidative stress, Ca2+ influx, and serine and cysteine proteases. Protein kinase and calmodulin activities did not seem to be involved in the induction of DNA laddering by victorin, whereas kinase-mediated signals were involved in DNA laddering induced by A23187. Protein kinase, calmodulin, G-protein activities, and Ca2+ influx, however, are involved in phytoalexin production. Our results imply that p28 is a possible nuclease candidate responsible for the induction of DNA laddering. The results also demonstrated that the mediators involved in the induction of apoptosis depended on the type of stimuli, whereas p28 and serine and cysteine proteases commonly are associated with each elicitor-induced apoptosis.

Methylation is not the main force repressing the retrotransposon MAGGY in Magnaporthe grisea.

We have introduced the LTR-retrotransposon MAGGY into a naive genome of Magnaporthe grisea and estimated the copy number of MAGGY in a cell by serial isolation of fungal protoplasts at certain time intervals. The number of MAGGY elements rapidly increased for a short period following introduction. However, it did not increase geometrically and reached equilibrium at 20-30 copies per genome, indicating that MAGGY was repressed or silenced during proliferation. De novo methylation of MAGGY occurred immediately following invasion into the genome but the degree of methylation was constant and did not correlate with the repression of MAGGY. 5-Azacytidine treatment demethylated and transcriptionally activated the MAGGY element in regenerants but did not affect transpositional frequency, suggesting that post-transcriptional suppression, not methylation, is the main force that represses MAGGY proliferation in M.grisea. Support for this conclusion was also obtained by examining the methylation status of MAGGY sequences in field isolates of M.grisea with active or inactive MAGGY elements. Methylation of the MAGGY sequences was detected in some isolates but not in others. However, the methylation status did not correlate with the copy numbers and activity of the elements.

Comparative analyses of the distribution of various transposable elements in Pyricularia and their activity during and after the sexual cycle.

We examined the distribution and activity of six transposable elements found in the blast fungus, Pyricularia spp. Sixty-eight isolates from various gramineous plants were used for the survey, and the elements were plotted on a dendrogram constructed on the basis of their rDNA-ITS2 sequences. MGR586 and Pot2 (Class II elements), Mg-SINE (SINE-like element) and MGR583 (LINE-like retrotransposon) were widely distributed among the Pyricularia isolates, suggesting that they are old elements which arose in, or invaded, the Pyricularia population at very early stages in its evolution. By contrast, the distribution of the LTR-retrotransposons MAGGY and Grasshopper was limited or sporadic, suggesting that they are relatively new elements which recently invaded the Pyricularia population by means of horizontal transfer events. The activity of these elements was evaluated by Southern analysis in progenies derived from a cross between a Setaria isolate and a Triticum isolate. Many new MAGGY signals were observed, which were absent in the parental isolates, at various stages of the sexual cycle and following vegetative growth. In contrast, the other elements yielded few, if any, such signals. Analysis of the sequences flanking the new MAGGY insertions revealed that they were each associated with a 5-bp target-site duplication at both ends of the insertion. These data suggested that MAGGY was the most active of the elements tested for transposition in Pyricularia.

OARE-1, a Ty1-copia retrotransposon in oat activated by abiotic and biotic stresses.

Transcriptionally active Ty1-copia LTR-retrotransposons were found in oat using RT-PCR for amplifying the reverse transcriptase domain. Sequence analysis of the RT-PCR clones suggested that oat LTR-retrotransposons consist of at least seven groups, which were tentatively designated as Oatrt1 to Oatrt7. A full length copy of Oatrt1 was isolated from an oat genomic library, and was designated OARE-1. OARE-1 was 8,665 bp long and a member of the BARE-1 subgroup. The oat genome carried it in multiple copies (at least 10,000 copies / a hexaploid genome). The expression of OARE-1 was intensively induced by wounding, UV light, jasmonic acid and salicylic acid, and its pattern was very similar to that of the PAL (phenylalanin ammonia lyase) gene. Furthermore, OARE-1 was highly activated by infection with an incompatible race of the crown rust fungus, Puccinia coronata. These results suggest that OARE-1 is highly sensitive to various abiotic and biotic stimuli leading to plant defense responses.

Analysis of Host Species Specificity of Magnaporthe grisea Toward Wheat Using a Genetic Cross Between Isolates from Wheat and Foxtail Millet.

ABSTRACT A genetic cross was performed between a Setaria isolate (pathogenic on foxtail millet) and a Triticum isolate (pathogenic on wheat) of Magnaporthe grisea to elucidate genetic mechanisms of its specific parasitism toward wheat. A total of 80 F(1) progenies were obtained from 10 mature asci containing 8 ascospores. Lesions on wheat leaves produced by the F(1) progenies were classified into four types, which segregated in a 1:1:1:1 ratio. This result suggested that the pathogenicity of the F(1) population on wheat was controlled by two genes located at different loci. This idea was supported by backcross analyses. We designated these loci as Pwt1 and Pwt2. Cytological analyses revealed that Pwt1 and Pwt2 were mainly associated with the hypersensitive reaction and papilla formation, respectively.