Fungi, hidden in soil or up in the air: light makes a difference.

Light is one of the most important environmental factors for orientation of almost all organisms on Earth. Whereas light sensing is of crucial importance in plants to optimize light-dependent energy conservation, in nonphotosynthetic organisms, the synchronization of biological clocks to the length of a day is an important function. Filamentous fungi may use the light signal as an indicator for the exposure of hyphae to air and adapt their physiology to this situation or induce morphogenetic pathways. Although a yes/no decision appears to be sufficient for the light-sensing function in fungi, most species apply a number of different, wavelength-specific receptors. The core of all receptor types is a chromophore, a low-molecular-weight organic molecule, such as flavin, retinal, or linear tetrapyrrols for blue-, green-, or red-light sensing, respectively. Whereas the blue-light response in fungi is one of the best-studied light responses, all other light-sensing mechanisms are less well studied or largely unknown. The discovery of phytochrome in bacteria and fungi in recent years not only advanced the scientific field significantly, but also had great impact on our view of the evolution of phytochrome-like photoreceptors.

Control of charge generation and recombination in ternary polymer/polymer:fullerene photovoltaic blends using amorphous and semi-crystalline copolymers as donors.

Charge generation and recombination processes occurring in ternary photoactive copolymer:copolymer:fullerene blends consisting of different mixing ratios between entirely amorphous and semi-crystalline PPE-PPV copolymers are investigated by transient absorption pump-probe and pump-push photocurrent spectroscopy. The experiments reveal that an excess of semi-crystalline polymer facilitates exciton dissociation into free charge carriers, slows down geminate recombination, and suppresses non-geminate recombination leading to increased short-circuit currents and high fill factors. In contrast, blends utilizing solely the amorphous polymer for their donor phase suffer from a large fraction of sub-nanosecond geminate recombination of interfacially bound charge-transfer states and also from fast non-geminate recombination of free charges, resulting in a significantly reduced photovoltaic performance. However, small fractions of the amorphous polymer blended into the semi-crystalline polymer increase the open-circuit voltage and the fill factor, while keeping the charge generation and recombination parameters largely unaltered in turn leading to an optimized device performance for the ternary PPE-PPV copolymer:copolymer:fullerene blends.

Light inhibits spore germination through phytochrome in Aspergillus nidulans.

Aspergillus nidulans responds to light in several aspects. The balance between sexual and asexual development as well as the amount of secondary metabolites produced is controlled by light. Here, we show that germination is largely delayed by blue (450 nm), red (700 nm), and far-red light (740 nm). The largest effect was observed with far-red light. Whereas 60 % of the conidia produced a germ tube after 20 h in the dark, less than 5 % of the conidia germinated under far-red light conditions. Because swelling of conidia was not affected, light appears to act at the stage of germ-tube formation. In the absence of nutrients, far-red light even inhibited swelling of conidia, whereas in the dark, conidia did swell and germinated after prolonged incubation. The blue-light signaling components, LreA (WC-1) and LreB (WC-2), and also the cryptochrome/photolyase CryA were not required for germination inhibition. However, in the phytochrome mutant, ∆fphA, the germination delay was released, but germination was delayed in the dark in comparison to wild type. This suggests a novel function of phytochrome as far-red light sensor and as activator of polarized growth in the dark.

Ocular fibroblast types differ in their mRNA profiles--implications for fibrosis prevention after aqueous shunt implantation.

PURPOSE: For an aqueous shunt draining from the anterior chamber into the choroidal space, fibroblasts from the choroidea and/or the sclera are most likely responsible for a fibrotic response around the outflow region of such a shunt. The prevention of fibrosis should extend the operating life of the shunt. A detailed characterization of fibroblasts derived from choroidea and sclera should provide information about whether a fibrosis reaction can be inhibited by cell type-specific agents. METHODS: We generated mRNA profiles of fibroblasts from the choroidea, sclera, and Tenon's space by gene array hybridization to provide a basis on which to search for potential pharmacological targets for fibrosis prevention. Hybridization data were analyzed by the Rosetta Resolver system and Limma to obtain mRNA profiles of the three fibroblast types. RESULTS: The three fibroblast types investigated shared fibroblast-specific gene expression patterns concerning extracellular matrix proteins as collagens and fibronectin, but also showed distinct mRNA patterns. CONCLUSIONS: Individual mRNA species overexpressed in one of the fibroblast types might serve as markers for the identification of the fibroblast type in histological analyses. Future in-depth analyses of the gene expression patterns might help identify pharmacological targets for fibrosis prevention.

A prospective 10-year study of metal ceramic single crowns and fixed dental prosthesis retainers in private practice settings.

STATEMENT OF PROBLEM: Metal ceramic restorations are widely used in prosthodontics, but long-term data on their clinical performance in private practice settings based on prospective trials are sparse. PURPOSE: This clinical trial was designed to provide realistic long-term survival rates for different outcomes related to tooth loss, crown loss, and metal ceramic defect. MATERIAL AND METHODS: Ninety-five participants were provided with 190 noble metal ceramic single crowns and 138 participants with 276 fixed dental prosthesis retainer crowns on vital posterior teeth. Follow-up examinations were scheduled 2 weeks after insertion, annually up to 8 years, and after 10 years. Kaplan-Meier survival analyses, Mantel-Cox logrank tests, and Cox regression analyses were conducted. RESULTS: Because of variations in the time of the last examinations, the maximum observation period was 12.1 years. For the primary outcome 'loss of crown or tooth', the Kaplan-Meier survival rate was 94.3% ±1.8% (standard error) at 8.0 years (last outcome event) for single crowns and 94.4% ±1.5% at 11.0 years for fixed dental prosthesis retainer crowns. The difference between the survival functions was not significant (P>.05). For the secondary outcome 'metal ceramic defect', the survival rate was 88.8% ±3.2% at 11.0 years for single crowns and 81.7% ±3.5% at 11.0 years for fixed dental prosthesis retainer crowns. In Cox regression models, the only significant covariates for the outcome event 'metal ceramic defect' were bruxism in the medical history (single crowns) and signs and symptoms of bruxism (fixed dental prosthesis retainer crowns) with hazard ratios of 3.065 (95% CI 1.063 - 8.832) and 2.554 (95% CI 1.307 - 4.992). CONCLUSIONS: Metal ceramic crowns provided in private practice settings show good longevity. Bruxism appears to indicate a risk for metal ceramic defects.

A lighthearted approach to mindfulness: development and evaluation of a humor-enriched mindfulness-based program in a randomized trial.

Objectives: Recently, research on mindfulness and humor has begun to spark interest, both being essential contributors to well-being. This article proposes that forms of humor that share intention and attitudinal foundations with mindfulness, constitute the foundation of a mindful-humorous perspective and mindful humor. Once intention and attitude are in alignment, shared mechanisms underlying a mindful-humorous perspective may lead to synergetic effects (e.g., reinforcing a shift in perspective and attitudinal foundations) as summarized in the proposed mindful humor filter model (MHFM). Based on this theoretical framework, the humor-enriched mindfulness-based program (HEMBP) was developed and evaluated for validity and efficacy in a randomized controlled trial as a first step to test the newly introduced model. Methods: A total of 60 participants were randomly allocated to either the HEMBP or a wait-list control group. Participants' mindfulness, comic styles (e.g., benevolent humor, sarcasm, and cynicism), psychological well-being, life satisfaction, stress, and gelotophobia (fear of being laughed at) were assessed 1 week before and after training, and at a 1-month follow-up. Linear mixed-effects models were fitted to model changes in outcome variables over time. Results: The HEMBP was effective in increasing mindfulness, benevolent humor, and psychological well-being, and in reducing sarcasm, cynicism, stress, and gelotophobia, compared to the control group. Conclusion: Results support the validity and efficacy of the HEMBP as a promising approach for improving well-being and reducing stress. The HEMBP broadens the scope of existing mindfulness-based programs by cultivating a mindful-humorous outlook on life, facilitating mindfulness practice and insights, and fostering positive emotions and relationships with others through mindful humor. Results are also suggestive of the validity of the mindful humor filter model.

Multi-laboratory evaluation of the reproducibility of polymer biodegradation assessments applying standardized and modified respirometry methods.

This research evaluated the intra- and interlaboratory variability when applying OECD 301F and OECD 301B Ready Biodegradation respirometric test methods to quantify polymer biodegradation as well as the impact of method modifications including test duration, inoculum level and test substance concentration on results. This assessment synthesizes results of mineralization studies on 5 polymers of varying structural components, molecular weight, charge, and solubility, evaluated at 8 different laboratories in 4 different countries, providing significant geographic variation in inoculum source as well as lab to lab variations in test setup. Across all laboratories, intralaboratory variability was low (≤18 % absolute difference) indicating the reproducibility of results between replicates and uniformity of test setup in each laboratory. Interlaboratory variation was also low for all 5 polymers with extent of mineralization being comparable in all OECD 301F and 301B studies even when test methods were modified. Across all studies mean mineralization was 89 ± 5.5 % for polyethylene glycol 35,000, 85 ± 7.4 % for polyvinyl alcohol 18-88, 44 ± 13 % for carboxymethyl cellulose (DS 0.6), 48 ± 4.1 % for a modified guar gum, and 88 ± 6.2 % for microcrystalline cellulose (MCC) at study completion. Due to the lack of polymeric reference materials, MCC was evaluated and found to be a suitable reference material for polymers that biodegrade rapidly in screening studies. An additional respirometric study was conducted quantifying mineralization of the 5 polymers in river water to evaluate the relationship with OECD 301 results using activated sludge as the inoculum. A similar extent of mineralization was observed for all 5 polymers in the OECD 301 and river water studies but time to reach the maximum extent of mineralization was longer using river water as the inoculum source likely due to the lower microbial counts (106 CFU/L) in the test system.

Different fibroblast subpopulations of the eye: a therapeutic target to prevent postoperative fibrosis in glaucoma therapy.

The aim of this study is the characterization of fibroblasts mainly responsible for fibrosis processes associated with trabeculectomy or microstent implantation for glaucoma therapy. Therefore we isolated human primary fibroblasts from choroidea, sclera, Tenon capsule, and orbital fat tissues. These fibroblast subpopulations were analysed in vitro for expression of the extracellular matrix components which are responsible for postoperative scarring in glaucoma therapy. For scarring the proteins of the collagen family are predominant and so we focused on the expression of collagen I, collagen III and collagen VI in every fibroblast subpopulation. Also, the extracellular matrix protein fibronectin which crosslinks collagen fibres or other extracellular matrix components and cell surfaces, was analyzed. Collagen I, III and VI were prominent in every fibroblast subpopulation. The highest amounts of collagen III were found in hCF and hOF, whereas the signal in hSF and hTF was negligible. Additionally, there is a link between scarring processes and proliferating potential of fibroblasts, in case of microstent implantation triggered through the infiltration of inflammatory cells. Thus we analyzed fibroblast subpopulations for the presence of TGF-ß1 which is one of the most important cytokines involved in proliferation processes. TGF-ß1 was prominent in all fibroblast subpopulations with lowest expression in hCF cultures. To prevent postoperative fibroblast proliferation we analyzed in vitro the proliferation-inhibitors paclitaxel and mitomycin C which are potential candidates in drug eluting drainage systems on ocular fibroblast subpopulations. These inhibitors arrest fibroblast proliferation and viability, being, however, not very specific and have a cytotoxic potential also on healthy tissues surrounding the microstent outflow area. Significant differences in protein synthesis of fibroblasts subpopulations which could be specific targets for inhibition may help to find out fibroblast specific inhibitors to prevent postoperative scarring and could prevent patients from secondary surgery after microstent implantation.

Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans.

Light sensing is very important for organisms in all biological kingdoms to adapt to changing environmental conditions. It was discovered recently that plant-like phytochrome is involved in light sensing in the filamentous fungus Aspergillus nidulans[1]. Here, we show that phytochrome (FphA) is part of a protein complex containing LreA (WC-1) and LreB (WC-2) [2, 3], two central components of the Neurospora crassa blue-light-sensing system. We found that FphA represses sexual development and mycotoxin formation, whereas LreA and LreB stimulate both. Surprisingly, FphA interacted with LreB and with VeA, another regulator involved in light sensing and mycotoxin biosynthesis. LreB also interacted with LreA. All protein interactions occurred in the nucleus, despite cytoplasmic subfractions of the proteins. Whereas the FphA-VeA interaction was dependent on the presence of the linear tetrapyrrole in FphA, the interaction between FphA and LreB was chromophore independent. These results suggest that morphological and physiological differentiations in A. nidulans are mediated through a network consisting of FphA, LreA, LreB, and VeA acting in a large protein complex in the nucleus, sensing red and blue light.

Decreased renal corin expression contributes to sodium retention in proteinuric kidney diseases.

Patients with proteinuric kidney diseases often have symptoms of salt and water retention. It has been hypothesized that dysregulated sodium absorption is due to increased proteolytic cleavage of epithelial sodium channels (ENaCs) and increased Na,K-ATPase expression. Microarray analysis identified a reduction in kidney corin mRNA expression in rat models of puromycin aminonucleoside-induced nephrotic syndrome and acute anti-Thy1 glomerulonephritis (GN). As atrial natriuretic peptide (ANP) resistance is a mechanism accounting for volume retention, we analyzed the renal expression and function of corin; a type II transmembrane serine protease that converts pro-ANP to active ANP. Immunohistochemical analysis found that corin colocalized with ANP. The nephrotic and glomerulonephritic models exhibited concomitant increased pro-ANP and decreased ANP protein levels in the kidney consistent with low amounts of corin. Importantly, kidneys from corin knockout mice had increased amounts of renal ß-ENaC and its activators, phosphodiesterase (PDE) 5 and protein kinase G II, when compared to wild-type mice. A similar expression profile was also found in cell culture suggesting the increase in PDE5 and kinase G II could account for the increase in ß-ENaC seen in nephrotic syndrome and GN. Thus, we suggest that corin might be involved in the salt retention seen in glomerular diseases.

Mechanisms of tubular volume retention in immune-mediated glomerulonephritis.

Glomerulonephritis is characterized by hematuria, proteinuria, hypertension, and edema, but the mechanisms contributing to volume disorders are controversial. Here we used the rat anti-Thy1 model of mesangioproliferative glomerulonephritis to test the hypothesis that disturbed salt and water homeostasis is based on tubular epithelial changes that cause salt retention. In this model there was an early onset of pronounced proteinuria and lipiduria associated with reduced fractional sodium excretion and a lowering of the renin-angiotensin-aldosterone system. The glomerular filtration rate and creatinine clearance were decreased on day 6. There was a reduced abundance of the major salt and water transport proteins on the proximal tubular brush border membrane and which paralleled cellular protein overload, enhanced membrane cholesterol uptake and cytoskeletal changes. Alterations in thick ascending limb were moderate. Changes in the collecting ducts were characterized by an enhanced abundance and increased subunit cleavage of the epithelial sodium channel, both events consistent with increased sodium reabsorption. We suggest that irrespective of the proximal tubular changes, altered collecting duct sodium reabsorption may be crucial for volume retention in acute glomerulonephritis. We suggest that enhanced proteolytic cleavage of ion transporter subunits might be a novel mechanism of channel activation in glomerular diseases. Whether these proteases are filtered or locally secreted awaits determination.

Seeing the rainbow: light sensing in fungi.

Light is essential for photosynthetic organisms, but also serves as an important environmental cue for non-photosynthetic species; thus, light sensing is evolutionarily conserved throughout the kingdoms, from archaea and fungi to humans. Light sensors are chromoproteins, the low-molecular weight compound of which absorbs specific wavelengths and induces a reaction from the protein. In fungi, three light-sensing systems have been described at the molecular level. Blue-light sensing is achieved by a flavin-based photoreceptor, which itself acts as a transcription factor, and red-light sensing is achieved by a phytochrome, a molecule until recently thought to be confined to plants. A retinal-based opsin-system was discovered recently, although a biological function remains to be determined. The challenge for future research will be the identification of further components of signalling cascades, the identification of light-regulated genes and the unravelling of possible functional interplays between the different light control systems.

Revelation of Interfacial Energetics in Organic Multiheterojunctions.

Efficient charge generation via exciton dissociation in organic bulk heterojunctions necessitates donor-acceptor interfaces, e.g., between a conjugated polymer and a fullerene derivative. Furthermore, aggregation and corresponding structural order of polymer and fullerene domains result in energetic relaxations of molecular energy levels toward smaller energy gaps as compared to the situation for amorphous phases existing in homogeneously intermixed polymer:fullerene blends. Here it is shown that these molecular energy level shifts are reflected in interfacial charge transfer (CT) transitions and depending on the existence of disordered or ordered interfacial domains. It can be done so by systematically controlling the order at the donor-acceptor interface via ternary blending of semicrystalline and amorphous model polymers with a fullerene acceptor. These variations in interfacial domain order are probed with luminescence spectroscopy, yielding various transition energies due to activation of different recombination channels at the interface. Finally, it is shown that via this analysis the energy landscape at the organic heterojunction interface can be obtained.

Controlling Exciton Diffusion and Fullerene Distribution in Photovoltaic Blends by Side Chain Modification.

The influence of crystallinity on exciton diffusion and fullerene distribution was investigated by blending amorphous and semicrystalline copolymers. We measured exciton diffusion and fluorescence quenching in such blends by dispersing fullerene molecules into them. We find that the diffusion length is more than two times higher in the semicrystalline copolymer than in the amorphous copolymer. We also find that fullerene preferentially mixes into disordered regions of the polymer film. This shows that relatively small differences in molecular structure are important for exciton diffusion and fullerene distribution.

Development of a novel injectable drug delivery system for subconjunctival glaucoma treatment.

In this study we present the development of an injectable polymeric drug delivery system for subconjunctival treatment of primary open angle glaucoma. The system consists of hyaluronic acid sodium salt (HA), which is commonly used in ophthalmology in anterior segment surgery, and an isocyanate-functionalized 1,2-ethylene glycol bis(dilactic acid) (ELA-NCO). The polymer mixtures with different ratios of HA to ELA-NCO (1/1, 1/4, and 1/10 (v/v)) were investigated for biocompatibility, degradation behavior and applicability as a sustained release system. For the latter, the lipophilic latanoprost ester pro-drug (LA) was incorporated into the HA/ELA-NCO system. In vitro, a sustained LA release over a period of about 60days was achieved. In cell culture experiments, the HA/ELA-NCO (1/1, (v/v)) system was proven to be biocompatible for human and rabbit Tenon's fibroblasts. Examination of in vitro degradation behavior revealed a total mass loss of more than 60% during the observation period of 26weeks. In vivo, LA was continuously released for 152days into rabbit aqueous humor and serum. Histological investigations revealed a marked leuko-lymphocytic infiltration soon after subconjunctival injection. Thereafter, the initial tissue reaction declined concomitantly with a continuous degradation of the polymer, which was completed after 10months. Our study demonstrates the suitability of the polymer resulting from the reaction of HA with ELA-NCO as an injectable local drug delivery system for glaucoma therapy, combining biocompatibility and biodegradability with prolonged drug release.

Bone metabolism during the perimenopausal transition: a prospective study.

OBJECTIVE: Changes in biochemical markers of bone formation and resorption were followed over the course of 1 year in premenopausal, perimenopausal and early postmenopausal women. METHODS: Sixty-four subjects were analyzed, grouped according to their menstrual pattern, menopausal complaints and endocrinological parameters to be premenopausal (n=20), perimenopausal (n=24) or early postmenopausal (n=20). The parameters studied at four visits during the 12-month study period were the urinary pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD), and N-terminal telopeptide (NTX) as bone resorption markers, as well as osteocalcin (OC) and bone-specific alkaline phosphatase (BAP) in serum, representing bone formation. The longitudinal changes over time as well as intergroup differences were analyzed using generalized estimating equations (GEE) in connection with Wald statistics. RESULTS: Over the course of 1 year BAP levels decreased in the late premenopausal group (P<0.05). The perimenopausal group exhibited significant changes of PYD, DPD and OC (P<0.01), NTX levels were higher than in premenopause. Postmenopausal subjects had elevated NTX values, while PYD and DPD levels remained close to the perimenopausal range. Only for OC a time effect was seen during postmenopause. CONCLUSIONS: Changes in bone turnover already begin in late premenopause, when decreased bone formation may precede increased bone resorption. The rise of NTX from late premenopause through early postmenopause indicates diagnostic sensitivity of this parameter to changes in bone metabolism induced by estrogen withdrawal. PYD and DPD do not follow this pattern, but change significantly with time during perimenopause to then remain largely unchanged in early postmenopause.

Permeability of the anterior lens capsule for large molecules and small drugs.

PURPOSE: For developing injectable lenses the retention properties of the capsular bag are important. Therefore the apparent permeability coefficients of sodium fluorescein and fluorescent dextrans of different sizes were determined for the human anterior lens capsule to calculate a molecular weight cutoff from these data. In addition, permeability coefficients of drugs helpful for the suppression of secondary cataract were determined. MATERIALS AND METHODS: Capsulorhexis specimens were fixed in a specially designed two compartment diffusion chamber to investigate the permeation of sodium fluorescein and fluorescent dextrans of different sizes (10, 40, 70 and 150 kDa) for 24 h (n ≥ 3) and of the antiproliferative drugs actinomycin D and methotrexate for 0.5, 24, 48 and 72 h (n ≥ 3). RESULTS: The molecular weight cutoff of the anterior lens capsule was found to be 166 ± 82 kDa. After 0.5 h, no passage of actinomycin D and methotrexate was detectable through the lens capsule. The apparent permeability coefficients for actinomycin D and methotrexate were calculated to 0.71 ± 0.02 µm/s and to 0.80 ± 0.13 µm/s, respectively. CONCLUSIONS: The capsular bag retains fluorescent dextrans with a molecular weight of >166 kDa. Hence, prepolymers are required to polymerize rapidly to be retained inside of the capsular bag. In addition, low-molecular substances intended as antiproliferative drugs for secondary cataract prevention should be applied within a time frame of five minutes in such a way that cells adjacent to the capsular bag will not be damaged.

New concepts for glaucoma implants--controlled aqueous humor drainage, encapsulation prevention and local drug delivery.

Glaucoma is a common cause of blindness in industrialized countries and is the most frequent cause of irreversible blindness worldwide. Since raised intraocular pressure (IOP) has been implicated as the major risk factor, the main goal of all glaucoma treatment is to reduce IOP sufficiently to prevent continuous irreversible retinal ganglion cell damage and progression of visual field loss. Pharmacological reduction of IOP is first-line therapy, followed by laser treatment of the trabecular meshwork and filtering glaucoma surgery, and cyclophotocoagulation of the ciliary body or allogenic implants. The most important glaucoma implants are presented (MOLTENO, AHMED, BAERVELDT, KRUPIN) together with more recent developments (Ex-Press, Eyepass, iStent, Gold micro shunt). Drainage into the suprachoroidal space is a promising option, but is also limited by scarring of the new created outflow route due to proliferation and adhesion of fibroblasts. A deeper understanding of fibroblasts in the related eye compartments is required. Characterization of scleral, choroidal, and, as a reference, Tenon fibroblast subtypes, is possible based on gene expression patterns. Alongside mitomycin C and 5-fluorouracil, newer drugs to prevent fibrosis have been proposed, offering effects that are more specific and more physiological. Effectors involved in wound healing phases and signaling pathways are potential targets for pharmaceutical intervention. Downregulation of growth factors like TGF-ß and their downstream effectors may suppress proliferation and differentiation of fibroblasts, extracellular matrix deposition, wound contraction, and neovascularization. Furthermore, current approaches to local drug delivery in glaucoma implant technology are briefly summarized.

Effects of receptor-mediated endocytosis and tubular protein composition on volume retention in experimental glomerulonephritis.

Human glomerulonephritis (GN) is characterized by sustained proteinuria, sodium retention, hypertension, and edema formation. Increasing quantities of filtered protein enter the renal tubule, where they may alter epithelial transport functions. Exaggerated endocytosis and consequent protein overload may affect proximal tubules, but intrinsic malfunction of distal epithelia has also been reported. A straightforward assignment to a particular tubule segment causing salt retention in GN is still controversial. We hypothesized that 1) trafficking and surface expression of major transporters and channels involved in volume regulation were altered in GN, and 2) proximal tubular endocytosis may influence locally as well as downstream expressed tubular transporters and channels. Effects of anti-glomerular basement membrane GN were studied in controls and megalin-deficient mice with blunted proximal endocytosis. Mice displayed salt retention and elevated systolic blood pressure when proteinuria had reached 10-15 mg/24 h. Surface expression of proximal Na(+)-coupled transporters and water channels was in part [Na(+)-P(i) cotransporter IIa (NaPi-IIa) and aquaporin-1 (AQP1)] increased by megalin deficiency alone, but unchanged (Na(+)/H(+) exchanger 3) or reduced (NaPi-IIa and AQP1) in GN irrespective of the endocytosis defect. In distal epithelia, significant increases in proteolytic cleavage products of alpha-epithelial Na(+) channel (ENaC) and gamma-ENaC were observed, suggesting enhanced tubular sodium reabsorption. The effects of glomerular proteinuria dominated over those of blunted proximal endocytosis in contributing to ENaC cleavage. Our data indicate that ENaC-mediated sodium entry may be the rate-limiting step in proteinuric sodium retention. Enhanced proteolytic cleavage of ENaC points to a novel mechanism of channel activation which may involve the action of filtered plasma proteases.

An Acanthamoeba sp. containing two phylogenetically different bacterial endosymbionts.

Acanthamoebae are ubiquitous free-living amoebae and important predators of microbial communities. They frequently contain obligate intracellular bacterial symbionts, which show a worldwide distribution. All Acanthamoeba spp. described so far harboured no or only a single specific endosymbiont phylotype, and in some cases evidence for coevolution between the symbiotic bacteria and the amoeba host has been reported. In this study we have isolated and characterized an Acanthamoeba sp. (strain OEW1) showing a stable symbiotic relationship with two morphologically different endosymbionts. 16S rRNA sequence analysis assigned these symbionts to the candidate genus Procabacter (Betaproteobacteria) and the genus Parachlamydia (Chlamydiae) respectively. Fluorescence in situ hybridization and transmission electron microscopy confirmed the affiliation of the endosymbionts and showed their co-occurrence in the amoeba host cells and their intracellular location within separate compartments enclosed by host-derived membranes. Further analysis of this stable relationship should provide novel insights into the complex interactions of intracellular multiple-partner associations.