Volker R. Stoldt

Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi.

We identified a gene of the fungal pathogen Candida albicans, designated EFG1, whose high‐level expression stimulates pseudohyphal morphogenesis in the yeast Saccharomyces cerevisiae. In a central region the deduced Efg1 protein is highly homologous to the StuA and Phd1/Sok2 proteins that regulate morphogenesis of Aspergillus nidulans and S.cerevisiae, respectively. The core of the conserved region is homologous to the basic helix–loop–helix (bHLH) motif of eukaryotic transcription factors, specifically to the human Myc and Max proteins. Fungal‐specific residues in the bHLH domain include the substitution of an invariant glutamate, responsible for target (E‐box) specificity, by a threonine residue. During hyphal induction EFG1 transcript levels decline to low levels; downregulation is effected at the level of transcriptional initiation as shown by a EFG1 promoter–LAC4 fusion. A strain carrying one disrupted EFG1 allele and one EFG1 allele under the control of the glucose‐repressible PCK1 promoter forms rod‐like, pseudohyphal cells, but is unable to form true hyphae on glucose‐containing media. Overexpression of EFG1 in C.albicans leads to enhanced filamentous growth in the form of extended pseudohyphae in liquid and on solid media. The results suggest that Efg1p has a dual role as a transcriptional activator and repressor, whose balanced activity is essential for yeast, pseudohyphal and hyphal morphogenesis of C.albicans. Functional analogies between Efg1p and Myc are discussed.

Portal application of autologous CD133+ bone marrow cells to the liver : a novel concept to support hepatic regeneration

The liver has a large capacity for regeneration after resection. However, below a critical level of future liver remnant volume (FLRV), partial hepatectomy is accompanied by a significant increase of postoperative liver failure. There is accumulating evidence for the contribution of bone marrow stem cells (BMSCs) to participate in liver regeneration. Here we report on three patients subjected to intraportal administration of autologous CD133+ BMSCs subsequent to portal venous embolization of right liver segments, used to expand left lateral hepatic segments as FLRV. Computerized tomography scan volumetry revealed 2.5‐fold increased mean proliferation rates of left lateral segments compared with a group of three consecutive patients treated without application of BMSCs. This early experience with portovenous application of CD133+ BMSCs could suggest that this novel therapeutic approach bears the potential of enhancing and accelerating hepatic regeneration in a clinical setting.

Review: The Cct eukaryotic chaperonin subunits of Saccharomyces cerevisiae and other yeasts

All eight of the CCT1‐CCT8 genes encoding the subunits of the Cct chaperonin complex in Saccharomyces cerevisiae have been identified, including three that were uncovered by the systematic sequencing of the yeast genome. Although most of the properties of the eukaryotic Cct chaperonin have been elucidated with mammalian systems in vitro, studies with S. cerevisiae conditional mutants revealed that Cct is required for assembly of microtubules and actin in vivo. Cct subunits from the other yeasts, Candida albicans and Schizosaccharomyces pombe, also have been identified from partial and complete DNA sequencing of genes. Cct8p from C. albicans, the only other completely sequenced Cct protein from a fungal species other than S. cerevisiae, is 72% and 61% similar to the S. cerevisiae and mouse Cct8 proteins, respectively. The C. albicans CCT8 sequence has been assigned the Accession Number U37371 in the GenBank/EMBL database.

Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating

Decellularization is a promising option to diminish immune and inflammatory response against donor grafts. In order to accelerate the autologous in vivo recellularization of aortic conduits for an enhanced biocompatibility, we tested fibronectin surface coating in a standardized rat implantation model. Detergent-decellularized rat aortic conduits (n = 36) were surface-coated with covalently Alexa488-labeled fibronectin (50 μg/ml, 24 h) and implanted into the systemic circulation of Wistar rats for up to 8 weeks (group FN; n = 18). Uncoated implants served as controls (group C; n = 18). Fibronectin-bound fluorescence on both surfaces of the aortic conduits was persistent for at least 8 weeks. Cellular repopulation was examined by histology and immunofluorescence (n = 24). Luminal endothelialization was significantly accelerated in group FN (p = 0.006 after 8 weeks), however, local myofibroblast hyperplasia with significantly increased ratio of intima-to-media thickness occurred (p = 0.0002 after 8 weeks). Originating from the adventitial surface, alpha-smooth muscle actin and desmin positive cell invasion into the media of fibronectin-coated conduits was significantly increased as compared to group C (p < 0.0001). In these medial areas, in situ zymography revealed enhanced matrix metalloproteinase activity. In both groups, inflammatory cell markers (CD3 and CD68) and signs of thrombosis proved negative. With regard to several markers of cell adhesion, inflammation and calcification, quantitative real-time PCR (n = 12) revealed no significant inter-group differences. Fibronectin surface coating of decellularized cardiovascular implants proved feasible and persistent for at least 8 weeks in the systemic circulation. Biofunctional protein coating accelerated the autologous in vivo endothelialization and induced a significantly increased medial recellularization. Therefore, this strategy may contribute to the improvement of current clinically applied bioprostheses.

Intraoperative isolation and processing of BM-derived stem cells

To improve tissue regeneration of ischemic myocardium, autologous bone marrow-derived stem cells have been injected intramyocardially in five patients undergoing coronary artery bypass grafting and transmyocardial laser revascularization. An innovative method for the intraoperative isolation of CD133(+)-stem cells in less than 3 hours has been established. After induction of general anesthesia, approx. 60-240 ml of bone marrow were harvested from the posterior iliac crest and processed in the operating room under GMP conditions using the automated cell selection device Clini-MACS. Following standard CABG surgery, LASER channels were shot in predefined areas within the hibernating myocardium. Subsequently, autologous CD133(+)-stem cells (1.9-9.7 x 10(6) cells; purity up to 97%) were injected in a predefined pattern around the laser channels. Through the intraoperative isolation of CD133(+)-cells, this effective treatment of ischemic myocardium can be applied to patients scheduled both for elective and for emergency revascularisation procedures.

A Candida albicans chaperonin subunit (CaCct8p) as a suppressor of morphogenesis and Ras phenotypes in C. albicans and Saccharomyces cerevisiae

Saccharomyces cerevisiae and the pathogen Candida albicans can be induced to undergo morphogenesis from a yeast to a filamentous form. A C. albicans gene (CaCCT8) was identified encoding a subunit of the Cct chaperonin complex, whose expression prevents filament formation in both fungi without interfering with growth of the yeast form. In S. cerevisiae, pseudohyphal growth induced by Ras2Val19, by overproduction of Phd1p or by expression of the C. albicans EFG1 gene, was blocked by CaCct8p and its N-terminally deleted derivative CaCct8-delta1p; in contrast, pseudohyphal induction by other components (Cph1p, Cdc42p) could not be suppressed, indicating that morphogenesis per se is not inhibited. CaCCT8 expression also interfered with other Ras2pVal19 phenotypes, including heat sensitivity, lack of glycogen accumulation and lack of sporulation. In C. albicans, overproduction of CaCct8p effectively blocked hyphal morphogenesis induced by starvation conditions and by serum. The results suggest that the activity of a component in the Ras2p signal transduction pathway is suppressed by excess chaperonin subunits. This component may be a novel folding target for the Cct complex. In agreement with this hypothesis, disruption of one of the two CaCCT8 alleles in C. albicans led to defective hyphal morphogenesis.

HPA-1 polymorphism of αIIbβ3 modulates platelet adhesion onto immobilized fibrinogen in an in-vitro flow system

BackgroundPlatelet adhesion and subsequent thrombus formation on a subendothelial matrix at the site of vascular damage play a crucial role in the arrest of posttraumatic bleeding but also in different pathological thrombotic events, such as acute coronary syndrome and stroke. Recently published studies have clearly demonstrated that platelet integri αIIbβ3 is intimately involved in the occlusive thrombus formation at the site of endothelial damage. Therefore, any genetic variation in the expression of this receptor may lead to an excessive bleeding or excessive thrombus formation. In this study, we evaluated the influence of HPA-1 polymorphism of integrin αIIbβ3 on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating blood flow.MethodsPlatelets in anticoagulated whole blood [49 healthy previously genotyped blood donors) were labelled with fluorescence dye and perfused through a rectangular flow chamber (shear rates of 50 s-1, 500 s-1 and 1500 s-1). A fluorescence laser-scan microscope was used for visualisation and quantification of platelet adhesion at 15 sec, 1 and 5 minutes after start of perfusion.ResultsDuring perfusion, the platelet adhesion linearly increased with regard to exposition time and shear rate. Perfusion of blood preincubated with Abciximab over fibrinogen-coated cover-slips showed reduced platelet adherence (absolute fluorescence: 168 ± 35 U vs. 53000 ± 19000 at control experiments, p < 0.05), as well as by perfusion over BSA-coated glass coverslips. Platelet with HPA-1a/1a genotype exhibited initial better adhesion but they also exhibited higher detachment under arterial flow conditions compared to the HPA-1b/1b platelets. Analysis of stable adhesion rate indicate that the platelets carrying the HPA-1b/1b genotype have a higher reactivity threshold for initial interaction with fibrinogen but under the higher shear rate (in regard to time of perfusion) also realize more stable bonds with fibrinogen than platelets with the HPA-1a/1a genotype.ConclusionOur data support the contention that genetically determined variants of platelet integrins αIIbβ3 could play a role in arterial thrombogenesis and thus confirm the hypothesis derived from epidemiological studies.

Rheology of perfusates and fluid dynamical effects during whole organ decellularization: a perspective to individualize decellularization protocols for single organs.

The approach of whole organ decellularization is rapidly becoming more widespread within the tissue engineering community. Today it is well known that the effects of decellularization protocols may vary with the particular type of treated tissue. However, there are no methods known to individualize decellularization protocols while automatically ensuring a standard level of quality to minimize adverse effects on the resulting extracellular matrix. Here we follow this idea by introducing two novel components into the current practice. First, a non-invasive method for online monitoring of resulting fluid dynamical characteristics of the coronary system is demonstrated for application during the perfusion decellularization of whole hearts. Second, the observation of the underlying rheological characteristics of the perfusates is employed to detect ongoing progress and maturation of the decellularization process. Measured data were contrasted to the respective release of specific cellular components. We demonstrate rheological measurements to be capable of detecting cellular debris along with a discriminative capture of DNA and protein ratios. We demonstrate that this perfusate biomass is well correlated to the biomass loss in the extracellular matrix produced by decellularization. The appearance of biomass components in the perfusates could specifically reflect the appearance of fluid dynamical characteristics that we monitored during the decellularization process. As rheological measuring of perfusate samples can be done within minutes, without any time-consuming preparation steps, we predict this to be a promising novel analytic strategy to control decellularization protocols, in time, by the actual conditions of the processed organ.

Shear-related fibrillogenesis of fibronectin.

Abstract Biomechanical forces can induce the transformation of fibronectin (Fn) from its compact structure to an extended fibrillar state. Adsorption of plasma proteins onto metallic surfaces may also influence their conformation. We used a cone-plate rheometer to investigate the effect of shear and stainless steel on conformational changes of Fn. In control experiments, cones grafted once or twice with polyethylene glycol were used. Plasma Fn was added at concentrations of 50 or 100 μg/ml to bovine serum albumin (BSA)- or Fn-coated plates and subsequently exposed to dynamic shear rates stepwise increasing from 50 to 5000 s-1 within 5 min and subsequently decreasing from 5000 to 50 s-1 within 5 min. The viscosity (mPa s) of Fn solutions was recorded over 10 min. Upon exposure to shear, the viscosity in the sample increased, suggesting conformational changes in Fn. Western blotting and densitometric analyses demonstrated that conformational changes of plasma Fn depended both on shear and protein concentration. However, there was no significant difference in fibril formation between BSA- or Fn-coated plates, suggesting that physical properties of stainless steel and biomechanical forces such as shear can affect the molecular structure of Fn. Our model may provide useful information of surface- and flow-induced alterations of plasma proteins.

Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis

Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the extracellular matrix can interact with platelets and thus contribute to platelet adhesion and aggregation. It has been shown that FN can decrease platelet aggregation but enhance platelet adhesion, suggesting a dual role of FN in haemostasis. The prevalent function(s) of FN may be determined by its fibril form. To explore the suggested dual role of this adhesive protein for haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction. Platelet aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN. Addition of plasma FN reduced platelet aggregation in response to collagen or PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments. Analyses by fluorescence resonance energy transfer (FRET) demonstrated that adherent platelets but not suspended platelets were capable of unfolding FN during 3h incubation. Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets. In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension. These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN.