The mutation p.D313Y is associated with organ manifestation in Fabry disease.

Fabry disease (FD) is a multisystem lysosomal storage disorder caused by mutations in the GLA gene. The clinical significance of the mutation p.D313Y is still under debate. Retrospective chart analysis of clinical (neurological, cardiac, renal, and ophthalmological), genetic, and biochemical (lyso-globotriaosylsphingosine, lyso-Gb3; enzyme activity) data was performed in all our patients carrying the p.D313Y mutation. Fourteen patients from 5 families (10 female, 4 male; age range 10-51) were included. Symptoms and organ manifestations compatible with FD could be identified in 10 patients. Cerebrovascular events occurred in 4 females. Seven patients reported pain or acroparaesthesia. Cornea verticillata was found in 1 patient, mild retinal vascular tortuosity in 5 patients. Lyso-Gb3 was elevated in 2 females with cerebrovascular involvement. Classical cardiac, renal or skin manifestations could not be identified. The mutation p.D313Y in the GLA gene may lead to organ manifestations and elevation of the Fabry-specific biomarker lyso-Gb3. Neurological symptoms (stroke and pain) and ocular manifestations seem to be the leading findings. Annual routine visits are recommended for patients carrying the p.D313Y mutation. Enzyme replacement therapy might be considered in symptomatic patients.

Amyloid formation: functional friend or fearful foe?

Amyloid formation has been most studied in the context of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, as well as in amyloidosis. However, it is becoming increasingly clear that amyloid is also present in the healthy setting; for example nontoxic amyloid formation is important for melanin synthesis and in innate immunity. Furthermore, bacteria have mechanisms to produce functional amyloid structures with important roles in bacterial physiology and interaction with host cells. Here, we will discuss some novel aspects of fibril-forming proteins in humans and bacteria. First, the amyloid-forming properties of the antimicrobial peptide human defensin 6 (HD6) will be considered. Intriguingly, unlike other antimicrobial peptides, HD6 does not kill bacteria. However, recent data show that HD6 can form amyloid structures at the gut mucosa with strong affinity for bacterial surfaces. These so-called nanonets block bacterial invasion by entangling the bacteria in net-like structures. Next, the role of functional amyloid fibrils in human semen will be discussed. These fibrils were discovered through their property to enhance HIV infection but they may also have other yet unknown functions. Finally, the role of amyloid formation in bacteria will be reviewed. The recent finding that bacteria can make amyloid in a controlled fashion without toxic effects is of particular interest and may have implications for human disease. The role of amyloid in health and disease is beginning to be unravelled, and here, we will review some of the most recent findings in this exciting area.

Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties.

Hemofiltrate CC chemokine (HCC)-1 is a recently described human chemokine that is constitutively expressed in numerous tissues and is present at high concentrations in normal plasma. Using a cell line expressing CC chemokine receptor (CCR)5 as a bioassay, we isolated from human hemofiltrate an HCC-1 variant lacking the first eight amino acids. HCC-1[9-74] was a potent agonist of CCR1, CCR3, and CCR5 and promoted calcium flux and chemotaxis of T lymphoblasts, monocytes, and eosinophils. It also blocked entry of HIV-1 strains using CCR5 as coreceptor. Limited tryptic digestion of HCC-1 generated the active variant. Conditioned media from several tumor cell lines activated HCC-1 with a high efficiency, and this activity could be inhibited by serine protease inhibitors. Our results indicate that HCC-1 represents a nonfunctional precursor that can be rapidly converted to the active chemokine by proteolytic processing. This process represents an additional mechanism by which tumor cells might generate chemoattractant molecules and recruit inflammatory cells. It might also affect HIV-1 replication in infected individuals and play an important role in AIDS pathogenesis.

Resonantly diode-pumped continuous-wave and Q-switched Er:YAG laser at 1645 nm.

We describe an efficient Er:YAG laser that is resonantly pumped using continuous-wave (CW) laser diodes at 1470 nm. For CW lasing, it emits 6.1 W at 1645 nm with a slope efficiency of 36%, the highest efficiency reported for an Er:YAG laser that is pumped in this manner. In Q-switched operation, the laser produces diffraction-limited pulses with an average power of 2.5 W at 2 kHz PRF. To our knowledge this is the first Q-switched Er:YAG laser resonantly pumped by CW laser diodes.

HIV-induced immune activation: pathogenesis and clinical relevance - summary of a workshop organized by the German AIDS Society (DAIG e.v.) and the ICH Hamburg, Hamburg, Germany, November 22, 2008.

This manuscript is communicated by the German AIDS Society (DAIG) (www.daignet.de). It summarizes a series of presentations and discussions during a workshop on immune activation due to HIV infection. The workshop was held on November 22nd 2008 in Hamburg, Germany. It was organized by the ICH Hamburg under the auspices of the German AIDS Society (DAIG e.V.).

Changes of diversity pattern of proteolytic bacteria over time and space in an agricultural soil.

The genetic heterogeneity of neutral metalloprotease (npr) gene fragments from soil proteolytic bacteria was investigated at a cultivated field site with four different soil types and at three different depths in April, July, and October. Terminal restriction fragment length polymorphism (T-RFLP) analyses of polymerase chain reaction-amplified npr gene fragments were applied to study the dynamic of the npr gene pool with regard to environmental conditions. The aim of this study was to relate differences in npr community structure and richness to the vertical, site, and seasonal variations naturally occurring at the field site under investigation. T-RFLP analysis revealed a noticeable seasonal variability in the community structure of npr-containing bacteria. The data suggest that the composition of the npr proteolytic bacterial population in July differed from those at the other dates. Additionally, the diversity of npr genes decreased with increasing soil depth revealing the highest values in upper layers. The reasons behind the observed patterns in the community structure might be mainly seasonal and vertical variation of the quantity and heterogeneity of available substrates as well as spatial isolation caused by a varying water amount and the connectivity of soil particles among the soil profile. Sequencing and phylogenetical analysis of 120 npr clones from the top soils collected in July revealed that most of the clones exhibit only poor homology to npr genes of isolates previously obtained from various environments, indicating the presence of until now uncharacterized npr coding proteolytic bacteria at the study site.

Diagram of the aging dynamics in laponite suspensions at low ionic strength.

We measure the dynamic structure factor (DSF) of probe particles embedded in an aging laponite suspension quenched by cessation of shear and the associated relaxation time tau as a function of wave vector q and aging time t(w). The different q dependences measured in the successive exponential and full aging regimes, respectively, tau approximately q(-2) and tau approximately q(-1.25), yield a weak positive q dependence for the aging time t(wc) corresponding to the crossover between the two regimes. This implies that the full aging behavior is first seen when investigating large length scales in the aging suspension. We propose a qualitative diagram of the aging dynamics and discuss the features of the DSF of the probes and of the matrix in the two aging regimes. Consistently with the idea that the full aging regime is first observed when probing large length scales, t(wc) is markedly shorter when the motion of the probes is tracked instead of the collective fluctuations of concentration in the matrix. The exponential aging regime is most probably related to the liquid-glass transition induced by the cessation of shear rather than to the aging of a glass.

Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems.

Ectomycorrhizas, the dominating mycorrhizal symbiosis in boreal, temperate and some tropical forests, are formed by 5000-6000 species of the asco- and basidiomycetes. This high diversity of fungal partners allows optimal foraging and mobilisation of various nitrogen and phosphorus forms from organic soil layers. In this review, two approaches to study the functioning of this multitude of symbiotic associations are presented. On selected culture models, physiological and molecular investigations have shown that the supply of hexoses has a key function in controlling the plant-fungus interaction via partner-specific regulation of gene expression. Environmental factors which affect fungal carbon supply, such as increased nitrogen availability, also affect mycorrhiza formation. Based on such laboratory results, the adaptative capability of ectomycorrhizas to changing field conditions is discussed. The second approach consists of analysing the distribution of mycorrhizas in ecosystem compartments and to relate distribution patterns to variations of ecological factors. Recent advances in identification of fungal partners in ectomycorrhizas by analysing the internal transcribed spacer of ribosomal DNA are presented, which can help to resolve sampling problems in field studies. The limits of the laboratory and the field approaches are discussed. Despite some problems, this combined approach is the most promising. Direct investigation of gene expression, which has been introduced for soil bacteria, will be difficult in the case of mycorrhizal fungi which constitute organisms with functionally varying structures.

Amidination of amino groups of aldehyde reductase from human liver.

Amidination of human liver aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) with monofunctional n-alkane methylimidates increased the enzymic activity by 10--30%, whereas analogous bifunctional imidoesters caused a loss of activity of about 80%. Both effects were prevented in the presence of the coenzyme NADPH or NADP+, but not of the substrate 4-nitrobenzaldehyde. Amidination increased the apparent Michaelis constant of both the coenzyme (up to 20-fold) and the substrate (about 5-fold). Bifunctional imidoesters with at least 4 carbon atoms between the functional groups (approx. 0.7 nm) crosslinked the enzyme intramolecularly. This reaction was retarded in the presence of the coenzyme, whereas 4-nitrobenzaldehyde had no effect. The results suggest the presence of reactive amino groups at the coenzyme binding site of aldehyde reductase.

The plant's capacity in regulating resource demand.

Regulation of resource allocation in plants is the key to integrate understanding of metabolism and resource flux across the whole plant. The challenge is to understand trade-offs as plants balance allocation between different and conflicting demands, e.g., for staying competitive with neighbours and ensuring defence against parasites. Related hypothesis evaluation can, however, produce equivocal results. Overcoming deficits in understanding underlying mechanisms is achieved through integrated experimentation and modelling the various spatio-temporal scaling levels, from genetic control and cell metabolism towards resource flux at the stand level. An integrated, interdisciplinary research concept on herbaceous and woody plants and its outcome to date are used, while drawing attention to currently available knowledge. This assessment is based on resource allocation as driven through plant-pathogen and plant-mycorrhizosphere interaction, as well as competition with neighbouring plants in stands, conceiving such biotic interactions as a "unity" in the control of allocation. Biotic interaction may diminish or foster effects of abiotic stress on allocation, as changes in allocation do not necessarily result from metabolic re-adjustment but may obey allometric rules during ontogeny. Focus is required on host-pathogen interaction under variable resource supply and disturbance, including effects of competition and mycorrhization. Cost/benefit relationships in balancing resource investments versus gains turned out to be fundamental in quantifying competitiveness when related to the space, which is subject to competitive resource exploitation. A space-related view of defence as a form of prevention of decline in competitiveness may promote conversion of resource turnover across the different kinds of biotic interaction, given their capacity in jointly controlling whole plant resource allocation.

Short term effects of ozone on the plant-rhizosphere-bulk soil system of young beech trees.

Plant growth largely depends on microbial community structure and function in the rhizosphere. In turn, microbial communities in the rhizosphere rely on carbohydrates provided by the host plant. This paper presents the first study on ozone effects in the plant-rhizosphere-bulk soil system of 4-year-old beech trees using outdoor lysimeters as a research platform. The lysimeters were filled with homogenized soil from the corresponding horizons of a forest site, thus minimizing field heterogeneity. Four lysimeters were treated with ambient ozone (1 x O3) and four with double ambient ozone concentrations (2 x O3; restricted to 150 ppb). In contrast to senescence, which was almost unaffected by ozone treatment, both the photochemical quantum yield of photosystem II (PSII) and leaf gas exchange were reduced (11 - 45 %) under the elevated O3 regime. However, due to large variation between the plants, no statistically significant O3 effect was found. Even though the amount of primary metabolites, such as sugar and starch, was not influenced by elevated O3 concentrations, the reduced photosynthetic performance was reflected in leaf biochemistry in the form of a reduction in soluble phenolic metabolites. The rhizosphere microbial community also responded to the O3 treatment. Both community structure and function were affected, with a tendency towards a lower diversity and a significant reduction in the potential nutrient turnover. In contrast, litter degradation was unaffected by the fumigation, indicating that in situ microbial functionality of the bulk soil did not change.

Successive exponential and full aging regimes evidenced by tracer diffusion in a colloidal glass.

We study the aging of a colloidal laponite glass by measuring the dynamic structure factor of dilute embedded tracer particles on micrometric length scales. We show that an initial aging regime, where the decay time grows exponentially with aging time t(w), tau approximately exp (gamma t(w)), is followed by a full aging regime, tau approximately t(v)(w) with v approximately 1. The dynamics of the tracers is diffusive in the exponential regime and hyperdiffusive in the full aging regime, up to micrometric length scales.

Flocculation, deflocculation, and ions migration in latex suspensions.

We have studied the dynamics of the flocculation of poly(styrene-butadiene-acrylic acid) latex suspensions. These suspensions were flocculated by the addition of Ca2+ ions at high concentrations of latex particles. Using diffusing wave spectroscopy and dynamic single light scattering after dilution, we have observed--depending on the pH and on the Ca2+ concentration--several scenarios for flocculation including successive flocculation and deflocculation. This complex behavior reveals that the Ca2+ migration within the shell of the latex is slow in acidic solvent but fast in basic solvent.

Targeting allergenic fungi in agricultural environments aids the identification of major sources and potential risks for human health.

Fungi are, after pollen, the second most important producers of outdoor airborne allergens. To identify sources of airborne fungal allergens, a workflow for qPCR quantification from environmental samples was developed, thoroughly tested, and finally applied. We concentrated on determining the levels of allergenic fungi belonging to Alternaria, Cladosporium, Fusarium, and Trichoderma in plant and soil samples from agricultural fields in which cereals were grown. Our aims were to identify the major sources of allergenic fungi and factors potentially influencing their occurrence. Plant materials were the main source of the tested fungi at and after harvest. Amounts of A. alternata and C. cladosporioides varied significantly in fields under different management conditions, but absolute levels were very high in all cases. This finding suggests that high numbers of allergenic fungi may be an inevitable side effect of farming in several crops. Applied in large-scale studies, the concept described here may help to explain the high number of sensitization to airborne fungal allergens.

Development of a self-inactivating, minimal lentivirus vector based on simian immunodeficiency virus.

In contrast to oncoviruses, lentiviruses do not require target cell division for integration into the host genome. Lentiviral vectors can therefore expand the spectrum of target cells susceptible to retroviral gene transfer. To analyze whether vectors based on simian immunodeficiency viruses (SIVs) could be used for gene transfer, a three-plasmid vector-packaging system was developed, in which Gag-Pol and the vector itself are of SIV origin, while Env is derived either from SIV, amphotropic murine leukemia virus (MuLV), or the G glycoprotein of vesicular stomatitis virus (VSV-G). To increase the safety of the SIV vector system, a self-inactivating SIV vector was constructed. After optimization of the SIV gag-pol expression plasmid, a minimal SIV vector, which contained only SIV sequences present on the multiply spliced nef transcript, could still be produced at titers of 2 x 10(5) infectious units/ml. Growth-arrested cells could be transduced with this vector even if vif, vpr, vpx, and nef had been deleted from the packaging construct and the vector.

Dynamic light scattering studies of the aggregation of lysozyme under crystallization conditions.

The intensity autocorrelation functions of light scattered by lysozyme solutions under pre-crystallization conditions in NaCl-containing media were recorded at scattering angles from 20 degrees to 90 degrees. The measurements, conducted on freshly prepared protein solutions supersaturated more than 3-fold, indicate the simultaneous presence of two scatterer populations which can be assigned to individual protein molecules and to large particles. When solutions are undersaturated, or slightly supersaturated, light scattering only reveals the presence of the small scatterers. In the supersaturated medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are medium, where aggregates were detected, lysozyme crystals grew in a time-span of 1-3 days after the scattering experiments. These results are correlated with the nucleation step during protein crystallization.

A gnotobiotic culture system with oak microcuttings to study specific effects of mycobionts on plant morphology before, and in the early phase of, ectomycorrhiza formation by Paxillus involutus and Piloderma croceum.

Homogeneously developed oak (Quercus robur L.) microcuttings were challenged in a Petri-dish system with the mycobionts Piloderma croceum J. Erikss. & Hjortst. and Paxillus involutus (Batsch) Fr. Non-destructive observations over 10 wk followed by d. wt measurements at the end of the assays served to precisely characterize root and shoot development, dynamics of mycorrhizal colonization and morphological ratio. In the system, plant development, and especially root morphogenesis, had more similarities to those of stump cuttings or of older seedlings than to those of 3-month-old seedlings. Whereas Paxillus involutus displayed early mycorrhizal colonization and had no significant morphological effects on the host Piloderma croceum modified markedly the entire plant development before a delayed mycorrhiza formation. The latter mycobiont stimulated elongation and production of the lateral root system and also increased the leaf surface. However, no corresponding weight increases were noted, which was reflected by significant increase of both specific root length and specific leaf area. These differential effects are discussed in relation to data concerning carbon requirement and auxin production of the mycobionts. The developed system was shown to be highly suitable for comparative studies with diverse mycobionts on recognition and physiological balance between partners before, and in the early stage of, formation of mycorrhizas.

Characterization and identification of black alder ectomycorrhizas by PCR/RFLP analyses of the rDNA internal transcribed spacer (ITS).

The identity of black alder (Alnus glutinosa (L.) Gaertn.) ectomycorrhizas was investigated using PCR/RFLP analysis of the ITS region from 16 morphotypes sampled at a 60-yr-old black alder stand. A comparison was made with restriction patterns from sporocarps of 28 mycobionts, of which 16 originated from the same stand, the remaining 12 came from two geographically distant alder stands. Eight of the mycorrhizal types could thus be identified, whereas eight mycorrhizal types remained unidentified. The identified mycorrhizas belonged to the genera Russula, Lactarius, Naucoria and Cortinarius. Four of the identified ectomycorrhizal types had identical PCR/RFLP profiles to corresponding fruit bodies from all investigated stands with no detectable intraspecific variation, despite the geographical distance of c.300 km between the sampling locations. By contrast, intraspecific variation between sporocarps from the different locations was detected in Paxillus rubicundulus, mycorrhizas of which were not found. The diversity of fruiting alder mycobionts at the main experimental plot only partly matched the diversity observed from mycorrhizas when comparing their PCR/RFLP profiles. The results are discussed regarding sampling techniques, PCR/RFLP analyses and ecological aspects.

Mutagenic by-products from chlorination of humic acid.

Chlorination of humic and fulvic acid results in the formation of direct-acting mutagenicity, detectable in the Salmonella/microsome assay (Ames test). This mutagenicity is being characterized as part of an overall effort aimed at evaluating potential health risks associated with the presence of mutagenic chemicals in drinking water. A number of chlorinated organic compounds, including several known mutagens, have been identified and quantified in diethyl ether extracts of chlorinated humic acid solutions. However, the total mutagenicity of these compounds accounts for only about 7% of the original mutagenicity. Synergistic or antagonistic interactions among the identified components have been ruled out as possible explanations for the failure to account for a higher percentage of the activity. Recent progress has been made to separate the activity into neutral and strong acid fractions. Further isolation of the strong acids by high-pressure liquid chromatography (HPLC) has resulted in the purification of the mutagenicity into a major peak of activity with a specific mutagenicity of about 20,000 TA100 revertants per milligram. Several trichlorohydroxyfuranone isomers have been tentatively identified in this fraction. The contribution of these types of compounds to the mutagenicity of chlorinated humic acid is under investigation.

RNA fingerprinting of microbial community in the rhizosphere soil of grain legumes.

Microbial structural and expression profiles of the rhizospheres of three legumes, faba beans, peas and white lupin, were compared by RNA-arbitrarily primed PCR technique. Two different primers, M13 reverse and 10-mer primers, were used in the amplification and products resolved on non-denaturing polyacrylamide gel. With both DNA and RNA profiles Lupinus and Pisum rhizospheres were more similar to each other than to Vicia rhizosphere. The RAP-PCR products were also dot blotted and probed for bacterial peptidase transcripts. Plant-dependent rhizosphere effect was evident by the marked absence of transcripts for bacterial neutral metallopeptidase in Lupinus rhizosphere. The results of dot blot were further confirmed by RT-PCR for the expression of bacterial neutral metallopeptidase in the three rhizospheres.