Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.Peer reviewe

Spin coated chitin films for biosensors and its analysis are dependent on chitin-surface interactions

Chitin, abundant in nature, is a renewable resource with many possible applications in bioengineering. Biosensors, capable of label-free and in-line evaluation, play an important role in the investigation of chitin synthesis, degradation and interaction with other materials. This work presents a comparative study of the usefulness of a chitin surface preparation, either on gold (Au) or on polystyrene (PS). In both cases the most common method to dissolve chitin was used, followed by a simple spin-coating procedure. Multi-parametric surface plasmon resonance (MP-SPR), modeling of the optical properties of the chitin layers, scanning electron microscopy, and contact angle goniometry were used to confirm: the thickness of the layers in air and buffer, the refractive indices of the chitin layers in air and buffer, the hydrophobicity, the binding properties of the chitin binding domain (CBD) of Bacillus circulans, and the split-intein capture process. Binding of the CBD differed between chitin on Au versus chitin on PS in terms of binding strength and binding specificity due to a less homogenous structured chitin-surface on Au in comparison to chitin on PS, despite a similar thickness of both chitin layers in air and after running buffer over the surfaces. The use of the simple method to reproduce chitin films on a thin polystyrene layer to study chitin as a biosensor and for chitin binding studies was obvious from the SPR studies and the binding studies of CBD as moiety of chitinases or as protein fusion partner. In conclusion, stable chitin layers for SPR studies can be made from chitin in a solution of dimethylacetamide (DMA) and lithium chloride (LiCl) followed by spin-coating if the gold surface is protected with PS.Peer reviewe

Urethral bulk injection therapy for female stress urinary incontinence:A multiperspective evaluation

We evaluated the efficacy, safety and effect on sexual function of a relative new urethral bulking agent (polydimethylsiloxane Urolastic® (PDMS-U)) for female stress urinary incontinence (SUI) through a high power post-market clinical follow-up study and demonstrated the value of PDMS-U economically and from the patients’ perspective. Lastly, we analyzed to what extent the physicians learning-curve influenced safety outcomes. We showed that patients want to be informed about PDMS-U as primary treatment option for SUI. Next, we showed that when patients had to choose between bulk injection therapy and mid-urethral sling surgery (MUS-surgery) personal factors play also a role and not only efficacy and procedural factors. Some patients even preferred bulk injection therapy although it was associated with a lower cure rate. In a treatment trade-off model, however, we showed that the majority of patients are not willing to give up much efficacy (0-6%) to prefer bulk injection therapy over MUS-surgery. In our comparative two-armed cohort study we reported that the objective and subjective efficacy of PDMS-U at one year follow-up is lower than MUS-surgery. PDMS-U was associated with a 20% chance of excision of the bulk material. The sexual function after both MUS-surgery and PDMS-U equally improved. In a learning curve study we showed that the experience of the physician did not influence the chance of complications. The cost-effectiveness analysis showed that, depending on the outcome measure, MUS-surgery was the most cost-effective treatment option in improving disease specific QoL, whereas PDMS-U was more cost-effective in improving generic quality of life

Proton gradient formation in early endosomes from proximal tubules

AbstractHeavy endosomes were isolated from proximal tubules using a combination of magnesium precipitation and wheat-germ agglutinin negative selection techniques. Two small GTPases (Rab4 and Rab5) known to be specifically present in early endosomes were identified in our preparations. Endosomal acidification was followed fluorimetrically using acridine orange. In presence of chloride ions and ATP, the formation of a proton gradient (ΔpH) was observed. This process is due to the activity of an electrogenic V-type ATPase present in the endosomal membrane since specific inhibitors bafilomycin and folimycin effectively prevented or eliminated endosomal acidification. In presence of chloride ions (Km = 30 mM) the formation of the proton gradient was optimal. Inhibitors of chloride channel activity such as DIDS and NPPB reduced acidification. The presence of sodium ions stimulated the dissipation of the proton gradient. This effect of sodium was abolished by amiloride derivative (MIA) but only when loaded into endosomes, indicating the presence of a physiologically oriented Na+/H+-exchanger in the endosomal membrane. Monensin restored the gradient dissipation. Thus three proteins (V-type ATPase, Cl−-channel, Na+/H+-exchanger) present in early endosomas isolated from proximal tubules may regulate the formation, maintenance and dissipation of the proton gradient

Argus-T adjustable male sling:A follow-up study on urinary incontinence and patient's satisfaction

Aims: The use of Argus-T adjustable sling may be a promising alternative option for the treatment of urinary incontinence after radical prostatectomy, however long-term data is lacking. The aim of this study is to evaluate the long-term results of the Argus-T sling on incontinence rates, patient's quality of life and tape-related complications. Methods: Patients were eligible if persistent stress incontinence was present >= 12 months after radical prostatectomy. Measurements included 24 h frequency volume micturition list, 24 h pad test, 24 h pad count and quality of life questionnaires. Argus-T adjustable sling was placed with a single perineal route incision approach. Results: Seventy-eight patients were included, 69 +/- 6 years, pre-intervention 24 h urinary loss 212 (75-385) g. Directly after surgery, 63.6% of the patients was completely dry, 79.2% of the patients reported greater than 90% improvement of their urinary loss and 92.2% > 50% improvement. Median follow-up time was 3.2 (2.5-6.1) years. After 5 years of follow-up, 53.3% of the patients were completely dry, 71.5% reported an improvement greater than 90% and 79.6% reported an improvement of greater than 50%. Patients with preoperative urinary loss less than 250 g reported significantly higher improvement of their urinary loss compared to patients with urinary loss >= 250 g (p = .02). Patients satisfaction was still increased after 5 years follow-up (70 +/- 21 vs.16 +/- 9, p < .001) and patients quality of life remained high (85 +/- 20 vs. 88 +/- 13, p = .1). Complications were mainly observed directly after surgery. Two patients (2.6%) needed reimplantation of the sling. Conclusion: These data indicate that Argus-T sling is an effective treatment option in obtaining substantial long-term incontinence relief in patients with invalidating moderate stress urinary incontinence after radical prostatectomy