Large-scale parallel 454 sequencing reveals host ecological group specificity of arbuscular mycorrhizal fungi in a boreonemoral forest.

* Knowledge of the diversity of arbuscular mycorrhizal fungi (AMF) in natural ecosystems is a major bottleneck in mycorrhizal ecology. Here, we aimed to apply 454 sequencing--providing a new level of descriptive power--to assess the AMF diversity in a boreonemoral forest. * 454 sequencing reads of the small subunit ribosomal RNA (SSU rRNA) gene of Glomeromycota were assigned to sequence groups by blast searches against a custom-made annotated sequence database. * We detected 47 AMF taxa in the roots of 10 plant species in a 10 x 10 m plot, which is almost the same as the number of plant species in the whole studied forest. There was a significant difference between AMF communities in the roots of forest specialist plant species and in the roots of habitat generalist plant species. Forest plant species hosted 22 specialist AMF taxa, and the generalist plants shared all but one AMF taxon with forest plants, including globally distributed generalist fungi. These AMF taxa that have been globally recorded only in forest ecosystems were significantly over-represented in the roots of forest plant species. * Our findings suggest that partner specificity in AM symbiosis may occur at the level of ecological groups, rather than at the species level, of both plant and fungal partners.

Identification of brain-derived neurotrophic factor promoter regions mediating tissue-specific, axotomy-, and neuronal activity-induced expression in transgenic mice.

The structure of rat brain-derived neurotrophic factor (BDNF) gene is complex; four 5' exons are linked to separate promoters and one 3' exon is encoding the BDNF protein. To analyze the relative importance of the regulatory regions in vivo, we have generated transgenic mice with six different promoter constructs of the BDNF gene fused to the chloramphenicol acetyl transferase reporter gene. High level and neuronal expression of the reporter gene, that in many respects recapitulated BDNF gene expression, was achieved by using 9 kb of genomic sequences covering the promoter regions that lie adjacent to each other in the genome (promoters I and II and promoters III and IV, respectively) and by including sequences of BDNF intron-exon splice junctions and 3' untranslated region in the constructs. The genomic regions responsible for the in vivo upregulation of BDNF expression in the axotomized sciatic nerve and in the brain after kainic acid-induced seizures and KCl-induced spreading depression were mapped. These data show that regulation of the different aspects of BDNF expression is controlled by different regions in vivo, and they suggest that these promoter constructs may be useful for targeted expression of heterologous genes to specific regions of the central and peripheral nervous systems in an inducible manner.

Expression of beta-nerve growth factor receptor mRNA in Sertoli cells downregulated by testosterone.

Nerve growth factor (NGF) is synthesized in male germ cells. The NGF receptor (NGFR) mRNA was found in the Sertoli cells of rat testis. Hypophysectomy increased both NGFR mRNA in testis and the number of NGFR hybridizing cells in seminiferous tubules. This was suppressed by treatment with chorionic gonadotropin or testosterone, but not with follicle-stimulating hormone. The NGFR mRNA also increased after destruction of Leydig cells or blocking of the androgen receptor. This suggests that NGF produced by male germ cells regulates testicular function in an androgen-modulated fashion by mediating an interaction germ and Sertoli cells.

Multiple promoters direct tissue-specific expression of the rat BDNF gene.

Brain-derived neurotrophic factor (BDNF) supports the survival of a specific set of neurons in the vertebrate nervous system. Here we show that the rat BDNF gene consists of four short 5' exons and one 3' exon encoding the mature BDNF protein. Eight different BDNF mRNAs with four different 5' ends and two alternative polyadenylation sites are transcribed from this gene. BDNF mRNAs containing exons I, II, and III are expressed predominantly in the brain, whereas exon IV transcripts predominate in the lung and heart. mRNAs containing exons I, II, and III increase markedly in the brain after kainic acid-induced seizures, whereas exon IV mRNA increases only slightly. Several transcription initiation sites were mapped upstream of the four 5' exons, and transfection of promoter-reporter gene constructs confirmed that these sequences act as promoters. Combined, the data demonstrate that alternative usage of four promoters within the BDNF gene and differential splicing control tissue-specific and seizure-induced expression of BDNF mRNA.

Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression.

Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility.

Corticosterone actions on the hippocampal brain-derived neurotrophic factor expression are mediated by exon IV promoter.

Brain-derived neurotrophic factor (BDNF) expression is strongly regulated by adrenocorticosteroids via activated gluco- and mineralocorticoid receptors. Four separate promoters are located upstream of the BDNF noncoding exons I to IV and may thus be involved in adrenocorticosteroid-mediated gene regulation. In adrenalectomised rats, corticosterone (10 mg/kg s.c.) induces a robust down-regulation of both BDNF mRNA and protein levels in the hippocampus peaking at 2-8 h. To study the role of the individual promoters in the corticosterone response, we employed exon-specific riboprobe in situ hybridisation as well as real-time polymerase chain reaction (PCR) in the dentate gyrus. We found a down-regulation, mainly of exon IV and the protein-coding exon V, in nearby all hippocampal subregions, but exon II was only down-regulated in the dentate gyrus. Exon I and exon III transcripts were not affected by corticosterone treatment. The results could be confirmed with real-time PCR in the dentate gyrus. It appears as if the exon IV promoter is the major target for corticosterone-mediated transcriptional regulation of BDNF in the hippocampus.

Co-operative domains in fibronectin.

The melting of human plasma fibronectin and its proteolytic fragments has been studied by scanning microcalorimetry to reveal co-operative structural domains in the molecule. It has been established that each of the two similar polypeptide chains of fibronectin has at least 12 structural domains, which differ in stability, size and function. Many of the domains in the N-terminal half of the polypeptide chains appear to be composed of two homologous repeat modules that co-operate to form a single co-operative unit. In the intact fibronectin molecule, the C-terminal regions of both chains seem to interact forming a stable co-operative block.

Regulatory elements and transcriptional regulation by testosterone and retinoic acid of the rat nerve growth factor receptor promoter.

The low-affinity nerve growth factor receptor (LNGFR) is a membrane-associated glycoprotein which is thought to participate in some of the biological activities of nerve growth factor (NGF). Expression of the LNGFR gene is known to be regulated both during development and in response to various agents in cell culture. However, molecular mechanisms responsible for the regulation have not been described. We report here an analysis of a 4.8-kb sequence from the 5'-flanking region of the rat LNGFR gene. Several regulatory elements were identified in this region by transfection of plasmid constructs containing sequences from LNGFR fused to a bacterial cat reporter gene. The proximal part of the promoter region (0.4-kb) was shown to be sufficient to support cat expression in all cell types used. A silencer element located between -1.5 kb and -1.8 kb from the start of translation, as well as an enhancer element in more upstream regions of the promoter, were identified in the phaeochromocytoma cell line, PC12, and in the Sertoli cell line, TM4, that express the LNGFR gene. Treatment of TM4 cells with retinoic acid (RA) increases the level of LNGFR mRNA twofold, while testosterone treatment results in a tenfold decrease. Regions of the promoter responsive to testosterone and RA in TM4 cells were found at -610 to -860 bp and -1840 to -4800 bp upstream from the translation start codon, respectively. A RA-responsive element active in PC12 cells is located between bp -610 to -860 from the start codon.

Nerve growth factor induces rapid redistribution of F-actin in PC12 cells.

Nerve growth factor (NGF) induces the redistribution of F-actin in rat pheochromocytoma PC12 cells within 2-10 min, whereas epidermal growth factor (EGF) has no effect on microfilament organization. This redistribution of F-actin in PC12 cells is not protein synthesis dependent, but can be blocked by methyltransferase inhibitors.

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments.

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.

Alcohol dehydrogenase of class I: kiwi liver enzyme, parallel evolution in separate vertebrate lines, and correlation with 12S rRNA patterns.

Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low Km with ethanol and a branched-chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala-141 and a higher Km). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding.

A comparative study of structural properties of fibronectin and its 180 kDa fragment.

Fibronectin from human plasma and its 180 kDa fragment which retained collagen-binding, cell-attachment and heparin-binding activities, were studied by velocity centrifugation and 1H-NMR methods. The fibronectin hydrodynamic radius strongly increased at pH 11 while the hydrodynamic properties of the fragment did not change noticeably. 1H-NMR spectroscopy also showed differences in the molecular properties of fibronectin and its 180 kDa fragment. Under physiological conditions the structure of fibronectin differs from that of its 180 kDa fragment. At pH 11 and in 4 M urea no differences in their structures are observed. It is suggested that interdomain and intersubunit interactions play an important role in maintaining the native conformation of intact fibronectin.

Spatiotemporal selective effects on brain-derived neurotrophic factor and trkB messenger RNA in rat hippocampus by electroconvulsive shock.

Electroconvulsive therapy is used in the treatment of affective disorders and schizophrenia and experimental electroconvulsive shock may serve as an animal model for this treatment. The aim of this study was to investigate a possible role for neurotrophins in the mechanism of action of experimental electroconvulsive shock and thus in clinical electroconvulsive therapy. The effect of electroconvulsive shock on levels of messenger RNAs encoding the neurotrophin brain-derived neurotrophic factor and the receptor trkB in rat hippocampus was determined by in situ hybridization with RNA probes 1, 3, 9 and 27 h following the shock. Brain-derived neurotrophic factor messenger RNA levels were increased at 1, 3 and 9 h following the shock and normalized after 27 h. Granule cells of the dentate gyrus showed a more rapid response as compared to hilar cells and pyramidal cells of CA1. Total trkB messenger RNA levels, including the transcripts for both the truncated and full length trkB receptor protein (gp95trkB and gp145trkB, respectively), showed a pattern of increase very similar to that of the brain-derived neurotrophic factor messenger RNA. However, using a probe selective for the full length (gp145trkB) trkB messenger RNA, we determined a delayed pattern of activation with significant increase only at 3 and 9 h after the shock. In hippocampus total trkB messenger RNA was found to consist of approximately one-quarter of mRNA encoding gp145trkB and three-quarters encoding gp95trkB as revealed by RNAase protection. While brain-derived neurotrophic factor and the truncated trkB messenger RNAs appear to increase with a similar pattern, suggesting a similar mechanism of activation by electroconvulsive shock, full length receptor trkB messenger RNA appears to increase with a delayed pattern suggesting a separate mechanism of activation. Electroconvulsive shock-induced seizures seem to include activation of a brain neurotrophin known to be important for neuronal plasticity.

Entorhinal cortex regulation of multiple brain-derived neurotrophic factor promoters in the rat hippocampus.

Developmental or degenerative damage of the neuronal architecture in the entorhinal cortex may disintegrate a functional part of hippocampal input since the entorhinal cortex provides a major source of neocortical and subcortical input to the hippocampus. These alterations, such as seen in Alzheimer's disease, schizophrenia and temporal lobe epilepsy are likely to be associated with cognitive deficits. To understand the basis for pathological changes in the corticohippocampal loop it is important to study mechanisms involved in neuronal plasticity. Brain-derived neurotrophic factor provides a possible substrate to mediate such plasticity. We have previously provided evidence that stimulation of hippocampal afferents transynaptically increase the level of brain-derived neurotrophic factor messenger RNA within the hippocampus. In the present study we have investigated whether different brain-derived neurotrophic factor messenger RNAs are specifically regulated in the hippocampus. We provide evidence for a differential and dose-dependent regulation of the different brain-derived neurotrophic factor promoters in the hippocampus by afferents in the entorhinal cortex. Our finding of a graded regulation is in contrast to earlier evidence of an "all-or-none" type of regulation.

Developmental regulation of brain-derived neurotrophic factor messenger RNAs transcribed from different promoters in the rat brain.

During the development of the nervous system many types of neurons are produced in excess and die because they fail to obtain sufficient amounts of target-derived neurotrophic factors (for review, see Refs 1, 16). Brain-derived neurotrophic factor is a protein that belongs to the nerve growth factor family of neurotrophins (for review, see Ref. 17) which promotes the survival and differentiation of distinct neuronal populations that partially overlap with those affected by the other members of the family (for review, see Ref. 11). We have recently shown that four promoters direct tissue specific expression of the rat brain-derived neurotrophic factor gene. In the present study we have quantified the levels of brain-derived neurotrophic factor messenger RNAs transcribed from different promoters during the rat brain development. Our data show that different promoters have specific regulation patterns during embryonic development but are regulated coordinately in several regions of rat brain during postnatal development, suggesting that the four transcription units have both distinct and common regulatory elements. Quantification of absolute levels of brain-derived neurotrophic factor messenger RNA revealed that it is expressed significantly higher in the rat brain than previously estimated.

Effects of cholinergic denervation on seizure development and neurotrophin messenger RNA regulation in rapid hippocampal kindling.

Intraventricular 192 IgG-saporin was used to induce a selective lesion of basal forebrain cholinergic neurons in rats. When subjected to 40 rapid hippocampal kindling stimulations with 5-min intervals, these animals exhibited increased number of generalized seizures and a higher mean seizure grade in response to the first five stimulations, and required fewer stimuli to develop focal behavioural seizures, as compared to non-lesioned rats. In contrast, both groups showed similarly enhanced responsiveness when test stimulated four weeks later. Using in situ hybridization, cholinergic denervation was found to cause a significant decrease of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex, whereas gene expression for nerve growth factor, neurotrophin-3, and TrkB and TrkC was unchanged. Four weeks after rapid kindling stimulations, basal levels of brain-derived neurotrophic factor messenger RNA in the dentate granule cells were restored to normal in the lesioned rats, whereas neurotrophin-3 messenger RNA levels were decreased. No differences in the seizure-evoked levels of neurotrophin and Trk messenger RNAs were detected, except in the dentate granule cell layer, which had significantly higher brain-derived neurotrophic factor messenger RNA expression in the lesioned animals at 2 h. In conclusion, the basal forebrain cholinergic system (i) dampens the severity of recurring seizures induced by rapid hippocampal kindling stimulations, but has no effect on the subsequent delayed phase of epileptogenesis; and (ii) exerts a tonic stimulation of basal brain-derived neurotrophic factor messenger RNA levels in the hippocampal formation and piriform cortex. The findings also indicate that the cholinergic lesion does not affect neurotrophin and Trk gene expression after recurring seizures, and that the kindling process leads to long-term changes in basal brain-derived neurotrophic factor and neurotrophin-3 messenger RNA levels in the denervated animals.

Immunolesioning of basal forebrain cholinergic neurons facilitates hippocampal kindling and perturbs neurotrophin messenger RNA regulation.

The immunotoxin 192 IgG-saporin induces an efficient and specific lesion of low-affinity nerve growth factor receptor-bearing cholinergic neurons in the basal forebrain. Intraventricular injection of 192 IgG-saporin, which caused a complete loss of cholinergic afferents to the hippocampus and neocortex and a partial denervation of amygdala and piriform cortex, was found to markedly facilitate the initial stages of seizure development in hippocampal kindling. In contrast, the progression of kindling process from focal to generalized seizures was not affected. In situ hybridization demonstrated that basal levels of brain-derived neutrotrophic factor messenger RNA in the hippocampal formation and piriform cortex were significantly decreased by the lesion, which also attenuated the seizure-induced increase of brain-derived neurotrophic factor messenger RNA expression in the hippocampus and frontal cortex. In the dentate gyrus, the 192 IgG-saporin lesion selectively reduced the upregulation of messenger RNAs for brain-derived neurotrophic factor exons I and III after a generalized seizure, whereas the increase of exon II messenger RNA was unchanged. The lesion abolished the seizure-evoked increase of nerve growth factor and TrkC messenger RNA levels and decrease of neutrophin-3 messenger RNA expression in dentate granule cells, while TrkB messenger RNA levels were not affected. We conclude that the basal forebrain cholinergic system (1) suppresses kindling epileptogenesis in the hippocampus, and (2) enhances both basal and seizure-evoked brain-derived neurotrophic factor synthesis in the hippocampal formation and some cortical areas through a specific pattern of activation of promoters within the brain-derived neurotrophic factor gene.

Expression of neurotrophin receptors in rat testis. Upregulation of TrkA mRNA with hCG treatment.

We report the expression of TrkA, TrkB and TrkC mRNAs in adult rat testis. With in situ hybridisation a low signal for TrkB and TrkC could be seen in postmeiotic cells of the seminiferous epithelium, whereas no signal for TrkA could be observed in untreated animals. Animals treated with hCG showed an induction of TrkA mRNA in premeiotic cells 12 h after the treatment, whereas an injection with EDS had no effect on the expression of Trk mRNAs. With the RNAse protection assay a low signal for TrkA was seen in whole testis of hCG treated animals. In staged tubules low expression was seen at stages VII-XI of untreated animals. Animals injected with hCG revealed that TrkA induction was highest during stages VIIcd and VIII of the cycle. The distinct expression pattern of these high-affinity neurotrophin receptors suggests different roles for neurotrophins during spermatogenesis. Induction of TrkA mRNA by hCG suggests that high-affinity binding of NGF during stages VIIcd-VIII in premeiotic cells is under control of the hypothalamic-pituitary-testicular axis.

Seizures increase trkC mRNA expression in the dentate gyrus of rat hippocampus. Role of glutamate receptor activation.

In this study we have shown, by in situ hybridization and RNase protection assay, a significant trkC mRNA increase confined to the dentate gyrus of hippocampus, both after seizures induced by intracerebroventricular injection of kainic acid and bicuculline. Moreover, after bicuculline treatment we observed an earlier increase of trkC mRNA level, which peaked after 3 h and returned back to normal levels by 12 h. In contrast, the kainic acid treatment produced a delayed increase of trkC mRNA, which initiated after 6 h, peaked at 12 h, and returned to normal levels at 24 h. This increase, which involves also trkC mRNA receptor with tyrosine kinase activity, was mediated by non-NMDA receptors and counteracted by GABA potentiating agent diazepam. Using embryonic neuronal cultures from cerebral hemispheres, including hippocampus, we found that glutamate receptor agonists, including glutamate, kainate, NMDA, and t-ACPD, increase trkC mRNA levels with the following rank order of efficacy: NMDA > t-ACPD > kainic acid > glutamate. In conclusion, our data show that trkC mRNA expression in granule cells of the hippocampus dentate gyrus is increased during seizure activity and that it is mediated by non-NMDA receptors.