Tris(methylthio)methane produced by Mortierella hyalina affects sulfur homeostasis in Arabidopsis.

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Incorporation of the sulfur from the fungal volatile into plant metabolism was shown by 34S labeling experiments. Under sulfur deficiency, TMTM down-regulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

Sclerotinia sclerotiorum Infection Triggers Changes in Primary and Secondary Metabolism in Arabidopsis thaliana.

Sclerotinia sclerotiorum is a devastating plant pathogen that causes substantial losses in various agricultural crops. Although plants have developed some well-known defense mechanisms against invasive fungi, much remains to be learned about plant responses to fungal pathogens. In this study, we investigated how S. sclerotiorum infection affects plant primary and secondary metabolism in the model plant Arabidopsis thaliana. Our results showed that soluble sugar and amino acid content changed significantly in A. thaliana leaves upon fungal colonization, with a decrease in sucrose and an increase in mannitol, attributed to fungal biosynthesis. Furthermore, the jasmonate signaling pathway was rapidly activated by S. sclerotiorum infection, and there was a striking accumulation of antifungal metabolites such as camalexin, p-coumaroyl agmatine, feruloyl agmatine, and Nδ-acetylornithine. On the other hand, the characteristic defense compounds of the Brassicaceae, the glucosinolates, were not induced in A. thaliana infected by S. sclerotiorum. Our study provides a better understanding of how A. thaliana primary and secondary metabolism is modified during infection by a fungal pathogen like S. sclerotiorum that has both hemibiotrophic and necrotrophic stages.

PRR2, a pseudo-response regulator, promotes salicylic acid and camalexin accumulation during plant immunity.

Calcium signalling mediated by Calmodulin (CaM) and calmodulin-like (CML) proteins is critical to plant immunity. CaM and CML regulate a wide range of target proteins and cellular responses. While many CaM-binding proteins have been identified, few have been characterized for their specific role in plant immunity. Here, we report new data on the biological function of a CML-interacting partner, PRR2 (PSEUDO-RESPONSE REGULATOR 2), a plant specific transcription factor. Until now, the physiological relevance of PRR2 remained largely unknown. Using a reverse genetic strategy in A. thaliana, we identified PRR2 as a positive regulator of plant immunity. We propose that PRR2 contributes to salicylic acid (SA)-dependent responses when challenged with the phytopathogenic bacterium Pseudomonas syringae. PRR2 is transcriptionally upregulated by SA and P. syringae, enhances SA biosynthesis and SA signalling responses; e.g. in response to P. syringae, PRR2 induces the production of SA and the accumulation of the defence-related protein PR1. Moreover, PRR2 overexpressing lines exhibit an enhanced production of camalexin, a phytoalexin that confers enhanced resistance against pathogens. Together, these data reveal the importance of PRR2 in plant immune responses against P. syringae and suggest a novel function for this particular plant specific transcription factor in plant physiology.

Wound and insect-induced jasmonate accumulation in carnivorous Drosera capensis: two sides of the same coin.

Carnivorous sundew plants catch and digest insect prey for their own nutrition. The sundew species Drosera capensis shows a pronounced leaf bending reaction upon prey capture in order to form an 'outer stomach'. This formation is triggered by jasmonates, phytohormones typically involved in defence reactions against herbivory and wounding. Whether jasmonates still have this function in D. capensis in addition to mediating the leaf bending reaction was investigated here. Wounded, insect prey-fed and insect-derived oral secretion-treated leaves of D. capensis were analysed for jasmonates (jasmonic acid, JA; jasmonic acid-isoleucine conjugate, JA-Ile) using LC-MS/MS. Prey-induced jasmonate accumulation in D. capensis leaves was persistent, and showed high levels of JA and JA-Ile (575 and 55.7 pmol · g · FW(-1) , respectively), whereas wounding induced a transient increase of JA (maximum 500 pmol · g · FW(-1) ) and only low (3.1 pmol · g · FW(-1) ) accumulation of JA-Ile. Herbivory, mimicked with a combined treatment of wounding plus oral secretion (W+OS) obtained from Spodoptera littoralis larvae induced both JA (4000 pmol · g · FW(-1) ) and JA-Ile (25 pmol · g · FW(-1) ) accumulation, with kinetics similar to prey treatment. Only prey and W+OS, but not wounding alone or OS, induced leaf bending. The results indicate that both mechanical and chemical stimuli trigger JA and JA-Ile synthesis. Differences in kinetics and induced jasmonate levels suggest different sensing and signalling events upon injury and insect-dependent challenge. Thus, in Drosera, jasmonates are still part of the response to wounding. Jasmonates are also employed in insect-induced reactions, including responses to herbivory and carnivory.

Myocardial glycophagy - a specific glycogen handling response to metabolic stress is accentuated in the female heart.

Cardiac metabolic stress is a hallmark of many cardiac pathologies, including diabetes. Cardiac glycogen mis-handling is a frequent manifestation of various cardiopathologies. Diabetic females have a higher risk of heart disease than males, yet sex disparities in cardiac metabolic stress settings are not well understood. Oestrogen acts on key glycogen regulatory proteins. The goal of this study was to evaluate sex-specific metabolic stress-triggered cardiac glycogen handling responses. Male and female adult C57Bl/6J mice were fasted for 48h. Cardiac glycogen content, particle size, regulatory enzymes, signalling intermediates and autophagic processes were evaluated. Female hearts exhibited 51% lower basal glycogen content than males associated with lower AMP-activated-kinase (AMPK) activity (35% decrease in pAMPK:AMPK). With fasting, glycogen accumulated in female hearts linked with decreased particle size and upregulation of Akt and AMPK signalling, activation of glycogen synthase and inactivation of glycogen phosphorylase. Fasting did not alter glycogen content or regulatory proteins in male hearts. Expression of glycogen autophagy marker, starch-binding-protein-domain-1 (STBD1), was 63% lower in female hearts than males and increased by 69% with fasting in females only. Macro-autophagy markers, p62 and LC3BII:I ratio, increased with fasting in male and female hearts. This study identifies glycogen autophagy ('glycophagy') as a potentially important component of the response to cardiac metabolic stress. Glycogen autophagy occurs in association with a marked and selective accumulation of glycogen in the female myocardium. Our findings suggest that sex-specific differences in glycogen handling may have cardiopathologic consequences in various settings, including diabetic cardiomyopathy.

Varicella-zoster virus neurotropism in SCID mouse-human dorsal root ganglia xenografts.

Varicella-zoster virus (VZV) is a neurotropic human alphaherpesvirus and the causative agent of varicella and herpes zoster. VZV reactivation from latency in sensory nerve ganglia is a direct consequence of VZV neurotropism. Investigation of VZV neuropathogenesis by infection of human dorsal root ganglion xenografts in immunocompromised (SCID) mice has provided a novel system in which to examine VZV neurotropism. Experimental infection with recombinant VZV mutants with targeted deletions or mutations of specific genes or regulatory elements provides an opportunity to assess gene candidates that may mediate neurotropism and neurovirulence. The SCID mouse-human DRG xenograft model may aid in the development of clinical strategies in the management of herpes zoster as well as in the development of "second generation" neuroattenuated vaccines.

Transcriptional and biochemical signatures of divergence in natural populations of two species of New Zealand alpine Pachycladon.

New Zealand is diverse in alpine and subalpine environments, a consequence of Late Tertiary tectonic and climatic change. However, few studies have sought to evaluate the importance of these environments as abiotic drivers in the diversification of plant species. Of particular interest is the Late Tertiary radiation of Pachycladon, an endemic New Zealand genus of alpine cress. Here we report observations on genome-wide levels of differential expression measured in the habitats of two closely related species of Pachycladon with distinct altitudinal preferences. Using Arabidopsis microarrays, we have identified 310 predominantly hormone- and stress-response genes up-regulated in Pachycladon fastigiata and 324 genes up-regulated in Pachycladon enysii. Expression patterns for glucosinolate biosynthesis and hydrolysis genes (MAM1, MAM-I, MAM-D, AOP2, ESP, ESM1) as well as flavonoid biosynthesis genes (F3'H, FLS, FAH1) were found to be species specific. Predicted differences in flavonoid contents were partly confirmed by high performance liquid chromatography analysis. Differences in glucosinolate profiles and glucosinolate hydrolysis products obtained by high performance liquid chromatography and gas chromatography-mass spectrometry analysis, respectively, also supported inferences from expression analyses. Five glucosinolate chemotypes were matched to known Arabidopsis ecotypes, and the potential adaptive significance of these chemotypes has been discussed. Our findings, in contrast to expectations for evolution of the New Zealand flora, suggest that biotic drivers, such as plant-herbivore interactions, are likely to be as important as abiotic drivers in the diversification of Pachycladon.

Five-wave-mixing spectroscopy of ultrafast electron dynamics at a si(001) surface.

The optically induced electron dynamics at a Si(001) surface is studied using a five-wave-mixing setup which measures the diffracted second-harmonic intensity induced by three ultrashort (13 fs) laser pulses. Depending on the time ordering of the pulses, this technique is capable of monitoring the temporal evolution of photoexcited one- or two-photon coherences, or populations. For a particular pulse sequence, the experiments show a delayed rise and a decay of the diffracted signal intensity on time scales of 50 and 250 fs, respectively. This response can be described by optical Bloch equations by including rapid scattering of the photoexcited carriers in the D(down) band of Si(001).

The Arabidopsis epithiospecifier protein promotes the hydrolysis of glucosinolates to nitriles and influences Trichoplusia ni herbivory.

Glucosinolates are anionic thioglucosides that have become one of the most frequently studied groups of defensive metabolites in plants. When tissue damage occurs, the thioglucoside linkage is hydrolyzed by enzymes known as myrosinases, resulting in the formation of a variety of products that are active against herbivores and pathogens. In an effort to learn more about the molecular genetic and biochemical regulation of glucosinolate hydrolysis product formation, we analyzed leaf samples of 122 Arabidopsis ecotypes. A distinct polymorphism was observed with all ecotypes producing primarily isothiocyanates or primarily nitriles. The ecotypes Columbia (Col) and Landsberg erecta (Ler) differed in their hydrolysis products; therefore, the Col x Ler recombinant inbred lines were used for mapping the genes controlling this polymorphism. The major quantitative trait locus (QTL) affecting nitrile versus isothiocyanate formation was found very close to a gene encoding a homolog of a Brassica napus epithiospecifier protein (ESP), which causes the formation of epithionitriles instead of isothiocyanates during glucosinolate hydrolysis in the seeds of certain Brassicaceae. The heterologously expressed Arabidopsis ESP was able to convert glucosinolates both to epithionitriles and to simple nitriles in the presence of myrosinase, and thus it was more versatile than previously described ESPs. The role of ESP in plant defense is uncertain, because the generalist herbivore Trichoplusia ni (the cabbage looper) was found to feed more readily on nitrile-producing than on isothiocyanate-producing Arabidopsis. However, isothiocyanates are frequently used as recognition cues by specialist herbivores, and so the formation of nitriles instead of isothiocyanates may allow Arabidopsis to be less apparent to specialists.

Proximal arterial vasoconstriction precedes regression of the hyaloid vasculature.

PURPOSE: To determine whether constriction of proximal arterial vessels precedes involution of the distal hyaloid vasculature in the mouse, under normal conditions, and whether this vasoconstriction is less pronounced when the distal hyaloid network persists, as it does in oxygen-induced retinopathy (OIR). METHODS: Photomicrographs of the vasa hyaloidea propria were analysed from pre-term pups (1-2 days prior to birth), and on Days 1-11 post-birth. The OIR model involved exposing pups to approximately 90% O(2) from D1-5, followed by return to ambient air. At sampling times pups were anaesthetised and perfused with india ink. Retinal flatmounts were also incubated with FITC-lectin (BS-1, G. simplicifolia,); this labels all vessels, allowing identification of vessels not patent to the perfusate. RESULTS: Mean diameter of proximal hyaloid vessels in pre-term pups was 25.44 +/- 1.98 microm; +/- 1 SEM). Within 3-12 hrs of birth, significant vasoconstriction was evident (diameter:12.45 +/- 0.88 microm), and normal hyaloid regression subsequently occurred. Similar vasoconstriction occurred in the O(2)-treated group, but this was reversed upon return to room air, with significant dilation of proximal vessels by D7 (diameter: 31.75 +/- 11.99 microm) and distal hyaloid vessels subsequently became enlarged and tortuous. CONCLUSIONS: Under normal conditions, vasoconstriction of proximal hyaloid vessels occurs at birth, preceding attenuation of distal hyaloid vessels. Vasoconstriction also occurs in O(2)-treated pups during treatment, but upon return to room air, the remaining hyaloid vessels dilate proximally, and the distal vessels become dilated and tortuous. These observations support the contention that regression of the hyaloid network is dependent, in the first instance, on proximal arterial vasoconstriction.

Identification of microcystins in cyanobacteria from the Bleiloch former drinking-water reservoir (Thuringia, Germany).

The presence of microcystins in cyanobacterial samples collected from the Bleiloch reservoir, formerly an important drinking-water supply in Thuringia, Germany, was proven by application of a combination of recently developed analytical methods. The raw extracts were cleaned by size-exclusion chromatography (SEC) or solid-phase extraction (SPE). The determination of microcystins was achieved by different HPLC separation followed by the application of alternative detection methods (UV, diode array detection (DAD), and mass spectrometry (MS), respectively). Furthermore, the different results of clean-up by SPE and SEC are demonstrated. The identity of microcystins was verified by MS/MS measurements. In the cyanobacterial sample from 1998, microcystin-RR, -LR and -YR were found, whereas in 1999 only microcystin-LR and -YR were detectable. In addition to detection of cell-bound microcystins, in 1999 traces of dissolved microcystins in water from the Bleiloch reservoir were detected. It can be assumed that not only the Bleiloch reservoir is contaminated with hepatotoxins but also many similar lakes still used for drinking water supply.

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion.

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.

Gene duplication in the diversification of secondary metabolism: tandem 2-oxoglutarate-dependent dioxygenases control glucosinolate biosynthesis in Arabidopsis.

Secondary metabolites are a diverse set of plant compounds believed to have numerous functions in plant-environment interactions. The large chemical diversity of secondary metabolites undoubtedly arises from an equally diverse set of enzymes responsible for their biosynthesis. However, little is known about the evolution of enzymes involved in secondary metabolism. We are studying the biosynthesis of glucosinolates, a large group of secondary metabolites, in Arabidopsis to investigate the evolution of enzymes involved in secondary metabolism. Arabidopsis contains natural variations in the presence of methylsulfinylalkyl, alkenyl, and hydroxyalkyl glucosinolates. In this article, we report the identification of genes encoding two 2-oxoglutarate--dependent dioxygenases that are responsible for this variation. These genes, AOP2 and AOP3, which map to the same position on chromosome IV, result from an apparent gene duplication and control the conversion of methylsulfinylalkyl glucosinolate to either the alkenyl or the hydroxyalkyl form. By heterologous expression in Escherichia and the correlation of gene expression patterns to the glucosinolate phenotype, we show that AOP2 catalyzes the conversion of methylsulfinylalkyl glucosinolates to alkenyl glucosinolates. Conversely, AOP3 directs the formation of hydroxyalkyl glucosinolates from methylsulfinylalkyl glucosinolates. No ecotype coexpressed both genes. Furthermore, the absence of functional AOP2 and AOP3 leads to the accumulation of the precursor methylsulfinylalkyl glucosinolates. A third member of this gene family, AOP1, is present in at least two forms and found in all ecotypes examined. However, its catalytic role is still uncertain.

Bus, a bushy Arabidopsis CYP79F1 knockout mutant with abolished synthesis of short-chain aliphatic glucosinolates.

A new mutant of Arabidopsis designated bus1-1 (for bushy), which exhibited a bushy phenotype with crinkled leaves and retarded vascularization, was characterized. The phenotype was caused by an En-1 insertion in the gene CYP79F1. The deduced protein belongs to the cytochrome P450 superfamily. Because members of the CYP79 subfamily are believed to catalyze the oxidation of amino acids to aldoximes, the initial step in glucosinolate biosynthesis, we analyzed the level of glucosinolates in a CYP79F1 null mutant (bus1-1f) and in an overexpressing plant. Short-chain glucosinolates derived from methionine were completely lacking in the null mutant and showed increased levels in the overexpressing plant, indicating that CYP79F1 uses short-chain methionine derivatives as substrates. In addition, the concentrations of indole-3-ylmethyl-glucosinolate and the content of the auxin indole-3-acetic acid and its precursor indole-3-acetonitrile were increased in the bus1-1f mutant. Our results demonstrate for the first time that the formation of glucosinolates derived from methionine is mediated by CYP79F1 and that knocking out this cytochrome P450 has profound effects on plant growth and development.

New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters.

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.

The yeast two-hybrid system reveals no interaction between p73 alpha and SV40 large T-antigen.

SV40 and/or DNA sequences indistinguishable from SV40 have been detected in several types of human tumours. The oncoprotein of Simian virus 40, SV40 large T-antigen (Tag), is known to bind and inactivate tumour suppressor proteins, such as members of the retinoblastoma family and p53, thereby promoting cell transformation. In this study, we used the yeast two-hybrid system to investigate whether the Simian virus 40 (SV40) large T-antigen is able to interact with p73, a noval discovered putative tumour suppressor, that is homologous both structurally and functionally to p53. The yeast two-hybrid system is a genetic method to detect protein-protein-interactions in vivo. Our results suggest that the SV40 large T-antigen is not able to bind p73 in yeast although both proteins are expressed in the transformed yeast strain as was shown by western blot analysis.

[Improved flow through aortocoronary venous bypasses after anticoagulant therapy. A prospective randomized study]. / Verbesserte Durchgängigkeit aorto-koronarer Venen-Bypasses nach Antikoagulantientherapie. Eine prospektive randomisierte Studie.

To evaluate the effect of oral anticoagulant therapy on graft patency rate during the first 2 months after bypass surgery 174 patients were randomly assigned to treatment with phenprocoumon (89) or to a control group (85) starting on day 7 after bypass surgery. Until day 7 all patients received low dose heparin. There was no significant difference between the two groups with respect to age, sex distribution, number of vessels diseased, left ventricular enddiastolic pressure, preoperative exercise tolerance or number of grafts constructed per patient. All patients underwent angiographic evaluation 8 weeks after bypass surgery. Graft patency rate was 90.4% in the treatment group versus 83.6% in the control group (p less than 0.015). None of the grafts with a flow rate of greater than 90 ml/min was occluded 8 weeks after surgery. Oral anticoagulation improved the patency rate of grafts with a flow of less then 90 ml/min.