Sequence diversity and unusual variability at the het-c locus involved in vegetative incompatibility in the fungus Podospora anserina

The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.     

Isolation of the two allelic incompatibility genes s and S of the fungus Podospora anserina

Summary The two allelic genes s and S are responsible for heterogenic incompatibility between wild type strains of the fungus Podospora anserina. The s gene has been cloned by SIB selection and expression in a strain containing a neutral allele of this locus. The S gene was isolated from a genomic library using the DNA of the s locus as a probe. The physical map of the DNA fragments carrying the two genes are highly dissimilar and restriction polymorphism exists at the s locus between s and S strains. Nevertheless, homology between the two alleles was revealed by cross-hybridization at the DNA and RNA levels.     

Non-Mendelian and skewed segregation of DNA markers in wide crosses of the bean rust fungus,Uromyces appendiculatus

The inheritance of DNA markers was investigated in 27 F₂ progeny from a single F₁ hybrid derived from a wide cross inUromyces appendiculatus. This cross was unusual because asexual spores were used to fertilize sexual fruiting structures. Sixty percent of the DNA markers failed to segregate according to simple Mendelian ratios. Segregation bias was evident, in that F₂ progeny inherited on average 91 % of maternal bands and 52% of paternal bands, which deviates significantly from the expected value for each of 75% for dominant markers. Because of these distortions, linkage mapping was not possible with this population. Evaluation of two F₁s from a second wide cross, reciprocals obtained by normal fertilization, also showed non-Mendelian inheritance of one of three co-dominant RFLPs and five of six isozyme markers, indicating that the method of crossing was probably not responsible for the abnormal segregation patterns in the first cross. Either genetic incompatibility, similar to that of an interspecific cross, or selection of particular genotypes could explain the genetic anomalies reported here.     

Molecular organisation of an A mating type factor of the basidiomycete fungus Coprinus cinereus

Summary The A3 and A3 genes, which together constitute the A42 mating type factor of Coprinus cinereus, were isolated from a cosmid genomic library by walking 50 kb, a map distance of 0.5 units, from the closely linked metabolic gene pab-1. Cosmid clones having A gene function were identified by transformation into compatible A6 (22) and A5 (11) host cells where either 3 or 3 was expected to elicit the A factor — regulated development of unfused clamp cells. DNAs were digested with various enzymes before transformation in order to identify the smallest fragments containing an active 3 or 3 gene. Two non-overlapping fragments were identified as containing the 3 and 3 genes respectively. Southern hybridisation analyses showed that these two cloned genes had no detectable sequence homology, and that there was little or no hybridisation to the and gene alleles that constitute the A5 and A6 factors. 3 and 3 were shown to be less than 2.0 kb apart and embedded in a DNA sequence extending over 9.0 kb which was unique to our A42 strain and may contain a third A factor gene.     

Mating system of the filamentous ascomycete, Glomerella cingulata

Mating in heterothallic filamentous ascomycetes is typically controlled by a single mating-type locus with two alternate alleles or idiomorphs. In this study, five self-sterile strains of Glomerella cingulata from pecan were crossed in all possible combinations. Four of the five strains could be placed into two mating-type groups, but the fifth strain was sexually compatible with all of the other strains. Single ascospore progeny were isolated from each of the successful crosses, tested for self-fertility, and backcrossed with both parents. In addition, subsets of F₁ isolates were crossed with all five of the original strains from pecan and in all possible combinations with each other. Results from the crosses showed that the ascospore progeny had stably inherited the mating pattern of one of the parental strains and that the mating type had segregated 1:1 among the F₁ isolates. Furthermore, the five strains from pecan were sexually compatible with five additional heterothallic strains in all but one combination. Data from these experiments are consistent with a mating system composed of a single mating-type locus with multiple alternate alleles. We believe that this is the first report of this type of mating system for an ascomycete species. Received: 28 October 1997 / 1 November 1998     

Identification of the <Emphasis Type="Italic">het-r</Emphasis> vegetative incompatibility gene of <Emphasis Type="Italic">Podospora anserina</Emphasis> as a member of the fast evolving <Emphasis Type="Italic">HNWD</Emphasis> gene family

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. Sequence data reported here are available in the Genbank database under accession numbers FJ269240 and FJ269239 for het-r and het-R, respectively.     

Downregulation of pre-rRNA processing gene Mamrd1 decreases growth, conidiation and virulence in the entomopathogenic fungus Metarhizium acridum

Mrd1 is one of the trans-acting proteins and plays an important role in precursor ribosomal RNA processing. Here we characterized the Mamrd1 gene from Metarhizium acridum and studied its function in growth, conidiation and virulence using RNA interference. The Mamrd1 gene was identified as participating in the processing of pre-rRNA in M. acridum and was highly upregulated during the infection process. A Mamrd1-RNAi strain exhibited an appearance of fluffy mycelia, a defective branching pattern and delayed conidiation compared to the wild-type strain. Downregulation of Mamrd1 in M. acridum suppressed growth both on artificial medium and inside the insect, and significantly reduced hyphal biomass, conidium production and virulence against Locusta migratoria manilensis. These results demonstrated that Mamrd1 plays an important role in growth, conidiation and virulence in M. acridum.     

Parasite manipulation of the proximate mechanisms that mediate social behavior in vertebrates

Paul MacLean was instrumental in establishing the brain regions that mediate the expression of social behaviors in vertebrates. Pathogens can exploit these central mechanisms to alter host social behaviors, including aggressive, reproductive, and parental behaviors. Although some behavioral changes after infection are mediated by the host (e.g., sickness behaviors), other behavioral modifications are mediated by the pathogen to facilitate transmission. The goal of this review is to provide examples of parasite-mediated changes in social behavior and to illustrate that parasites affect host behavior by infecting neurons, causing central nervous system (CNS) inflammation, and altering neurotransmitter and hormonal communication. Secondarily, a comparative approach will be used to demonstrate that the effects of parasites on social behavior are retained across several classes of vertebrates possibly because parasites affect the phylogenetically primitive structures of the limbic system and related neurochemical systems.     

Analysis of switching neurons within the thermoafferent system

Summary A total of 740 midbrain, thalamic, and hypothalamic neurons were tested as to their distribution and response/stimulus characteristics by means of thermal stimulation of the scrotal skin. The most frequent response type exhibited a basic discharge rate up to a threshold temperature and then switched to a maximum firing rate. The inverse behaviour was found in a low proportion. The temperature range in which the neurons switch from the low to the high state has been called the operating range of the neuron. This study reveals, in contrast to former studies, that the operating range is extremely narrow. This holds both for the individual neuron and for the populations studied. It is confirmed by simultaneous recordings and by cross correlation analysis that there is only one temperature threshold for all warm responsive neurons involved. It is concluded that according to the mean firing rate, temperature may only be discriminated as to below or above threshold. This means that a binary information is transmitted. For continuous temperature sensation and regulation either further neuronal types disposing of a continuous temperature/frequency characteristic or additional coding mechanisms have to be assumed. The major task of the switching neurons, integrating information from all over the body should be the generation of a trigger signal for warm defence.     

Topology of the fibrinolytic system within the mural thrombus of human abdominal aortic aneurysms

Development and progression of acquired abdominal aortic aneurysms (AAAs) involve proteolytic activity. In the present study, we investigate the distribution of fibrinolytic system components within mural thrombi of human AAAs. 20 mural thrombi and the remaining AAA walls were dissected. The luminal, intermediate and abluminal thrombus layers, and media and adventitia were separately incubated in cell culture medium. Conditioned media were then analysed for plasminogen activators (PAs), plasminogen activator inhibitor-1 (PAI-1), free-plasmin, plasmin alpha(2)-antiplasmin complexes (PAPs) and D-dimers release. In parallel, PA and PAI-1 mRNA expression analysis was performed by RT-PCR. The study was completed by immunohistochemical localization of these components in AAA, ex vivo functional imaging using (99m)Tc-aprotinin as a ligand and measurement of PAP and D-dimer plasma levels. All fibrinolytic system components were present in each aneurysmal layer. However, the mural thrombus was the main source of active serine-protease release. Interestingly, the luminal layer of the thrombus released greater amounts of PAPs and D-dimers. This paralleled the preferential immunolocalization of plasminogen and PAs, and the (99m)Tc-aprotinin scintigraphic signal observed in the luminal pole of the thrombus. In contrast, mRNA expression analysis showed an exclusive synthesis of tPA and PAI-1 within the wall, whereas uPA mRNA was also expressed within the thrombus. Taken together, these results suggest that the increased plasma concentrations of PAPs and D-dimers found in AAA patients are related to mural thrombus proteolytic activity, thus explaining their known link with AAA progression. Components of the fibrinolytic system could also represent a target for functional imaging of thrombus activities in AAA.     

异丙酚诱导大鼠中枢FOS核蛋白表达的研究

目的　通过检测异丙酚静脉全麻诱导大鼠中枢FOS核蛋白的表达 ,明确静脉全身麻醉的中枢作用位点。方法　 2 1只Wistar大鼠随机分成 3组 :对照组 (C组 )腹腔注入生理盐水 2ml,异丙酚组 (P组 )腹腔注入异丙酚 10 0mg kg ,异丙酚作用后断尾刺激组 (S组 )。 1h后应用FOS蛋白免疫组织化学法 (ABC法 ) ,检测FOS免疫反应 (FOS IR ,immunityreactionofFOS)阳性神经元在大脑的分布。结果　C组可见部分散在FOS IR阳性神经元分布 ,P组FOS IR阳性神经元数比C组明显增多 (P <0 .0 1) ,并呈核特异性分布 ,S组在杏仁基底外侧核、丘脑室旁核、外侧缰核及海马回嗅觉小岛等核团发现FOS IR阳性神经元较P组分布增多 (P <0 .0 1)。结论　异丙酚在大鼠中枢神经系统有与其静脉麻醉作用相关的神经核团。     

The coexistence of cancer cells of different origin within the same lymph nodes

Summary The coexistence of two differently originating cancer cells within the same lymph node is reported. An 83-year-old male patient died from severe carcinomatous lymphangitis of the lungs five months after gastrectomy for gastric cancer. At autopsy, prostatic cancer metastasis to lymph node was found. Histologically, the prostatic carcinoma cells had a cribriform pattern and the gastric cells showed papillary and tubular features. Immunohistochemically, the former was immunoreactive to prostate specific antigen (PSA) and the latter was stained positively for carcinoembryonic antigen (CEA). Fifty-one of the retroperitoneal and intrapelvic lymph nodes were histologically examined; the coexistence of both types of cancer cells was found in two lymph nodes. Both types were adjoined at the hilum, as was also confirmed by an immunohistochemical double staining technique. These two lymph nodes were located in the pre-aortic and left lateral aortic areas, and they belonged to the terminal lymph node group of both the intra-pelvic and intra-abdominal intestinal organs.

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Coexistence dans les mêmes noeuds lymphatiques de cellules provenant de deux cancers différents

Résumé Les auteurs décrivent la coexistence dans un même noeud lymphatique de cellules tumorales provenant de deux cancers différents. L'observation est celle d'un sujet masculin de 83 ans décédé d'une lymphangite carcinomateuse pulmonaire cinq mois après gastrectomie pour cancer. L'autopsie montra l'existence de métastases lymphonodales d'un cancer prostatique. Histologiquement, les métastases du cancer prostatique présentaient un aspect cribriforme tandis que les cellules d'origine gastrique dessinaient des formations papillaires et tubulaires. En immunohistochimie les premières se révélaient positives à l'antigène prostatique spécifique (PSA), et les autres à l'antigène carcino-embryonnaire (ACE). Cinquante et un noeuds rétropéritonéaux et pelviens furent examinés : la coexistence des deux types de cellules métastatiques fut observée dans deux noeuds. Les deux types se retrouvaient associés au niveau du hile, fait confirmé par les réactions immunohistochimiques. Les deux noeuds intéressés étaient situés dans la région préaortique et latéro-aortiques gauche et appartenaient au groupe des noeuds proximaux qui collectent à la fois la lymphe des organes pelviens et celle des organes digestifs intra-abdominaux.

     

骨内植入式药物缓释系统载体材料研究现状及进展

植入式药物缓释系统在治疗骨科感染、肿瘤等方面具有良好的作用。随着组织工程学的进展,研制了不同的材料作为骨内植入式药物缓释系统的载体,拟对骨内植入式药物缓释系统载体材料研究现况及进展进行综述。     

Neurotensin and cholecystokinin coexistence within neurons of the ventral mesencephalon: projections to forebrain

Summary The colocalization of neurotensin- and cholecystokinin-like immunoreactivities was demonstrated in neurons of the ventral mesencephalon of the rat by using a double-labeling indirect immunofluorescence procedure for the simultaneous detection of two antigens in the same tissue section. Greater than 90% of the neurotensin-positive perikarya distributed throughout the ventral mid-brain (primarily located in the ventral tegmental area, medial substantia nigra, and rostral and caudal linear raphe nuclei) were found to also contain cholecystokinin immunoreactivity. Neurons single-labeled for either peptide were also present, with those immunoreactive for cholecystokinin alone far outnumbering those containing only neurotensin. By combining the double-labeling colocalization technique with fluorescence retrograde tracing, some of the forebrain projections of these neurons were determined. Ventral mesencephalic neurons containing both neurotensin and cholecystokinin were found to project to the nucleus accumbens, prefrontal cortex, or amygdala. The present results, combined with those of previous studies, suggest that there are complex heterogeneous subpopulations of presumed dopaminergic ventral mesencephalic neurons which give rise to the ascending mesotelencephalic systems and which may contain both neurotensin and cholecystokinin, either peptide alone, or neither of these two peptides.     

Somatotopic distribution of trigeminal nociceptive specific neurons within the caudal somatosensory thalamus of cat

Serotonin (5-HT)-containing nerve cells in the rabbit brain were visualized immunohistochemically and were mapped on photographs of coronal brain sections. The 5-HT cells extended from the caudal medulla oblongata to the rostral midbrain and were mostly clustered in groups resembling those found in the rat brainstem. However, there were several notable differences between the rabbit and the rat in the distribution of 5-HT nerve cells. In particular, there were fewer pontine 5-HT cells in the rabbit and the major 5-HT cell groups of the midbrain and the ventral medulla oblongata were spread further laterally than in the rat brain.     

An electrophysiological study of the projections of putative sensory neurons within the myenteric plexus of the guinea pig ileum

Conduction of action potentials in the processes of AH (afterhyperpolarizing) neurons has been examined in the myenteric plexus of the guinea-pig small intestine. AH neurons are a morphologically distinct class of myenteric neurons in which the action potentials are followed by long lasting afterhyperpolarizations and which usually lack fast synaptic inputs. These neurons have large smooth cell bodies and several long processes. We have used electrophysiological methods, combined with intracellular injection of the fluorescent dye 5(6)-carboxyfluorescein, to examine the directions of projection and lengths of axons of AH neurons. AH neurons of the myenteric plexus projected circumferentially in both directions from the cell soma for electrophysiologically determined average distances of 0.74 ± 0.05 mm. Thus, the neurons span about 1.5 mm of the circumference of the intestine. About one quarter of the AH neurons had one, or rarely two, processes that ran anally after initially projecting circumferentially. All processes conducted action potentials, with average conduction velocities of 0.23 ± 0.02 ms⁻¹.     

Location and functional organization of trigeminal wide dynamic range neurons within the nucleus ventralis posteromedials of the cat

In urethane-chloralose-anesthetized cats, trigeminal wide dynamic range (WDR) neurons were explored in the posterior thalamus. They were found within a narrow zone (about 300 μm wide) of the shell region of the nucleus ventralis posteromedialis (VPM), just rostral to the region where trigeminal nociceptive specific (NS) neurons were located. This narrow zone was somatotopically organized with respect to the center of the receptive field. The mandibular division was represented ventromedially, the ophthalamic division were represented dorsolaterally, and the maxillary division fell in between. Neither NS nor WDR trigeminal units were found within the medial part of the posterior complex of the thalamus (POm).     

Function of non-NMDA receptors and NMDA receptors in synaptic responses to natural somatosensory stimulation in the ventrobasal thalamus

Summary Sensory synaptic responses of rat ventrobasal thalamus neurones were challenged with iontophoretic applications of the excitatory amino acid antagonists CNQX and CPP. CNQX, applied with currents which were selective for non-NMDA receptors, antagonised responses of VB neurones to both 10 ms and 2000 ms air jet stimulation of the peripheral receptive field. In contrast, CPP only antagonised the latter type of response. These results suggest a differential involvement of excitatory amino acid receptors in sensory synaptic transmission to the ventrobasal thalamus, with an initial synaptic component being mediated by non-NMDA receptors (including kainate receptors), and a further NMDA receptor-mediated component being manifested upon maintained sensory stimulation. The expression of this latter component appears to be largely dependent upon the integrity of the non-NMDA receptor-mediated component.