Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.     

Continuum simulations of acetylcholine diffusion with reaction-determined boundaries in neuromuscular junction models

The reaction-diffusion system of the neuromuscular junction has been modeled in 3D using the finite element package FEtk. The numerical solution of the dynamics of acetylcholine with the detailed reaction processes of acetylcholinesterases and nicotinic acetylcholine receptors has been discussed with the reaction-determined boundary conditions. The simulation results describe the detailed acetylcholine hydrolysis process, and reveal the time-dependent interconversion of the closed and open states of the acetylcholine receptors as well as the percentages of unliganded/monoliganded/diliganded states during the neuro-transmission. The finite element method has demonstrated its flexibility and robustness in modeling large biological systems.     

Brain kinin B1 receptor is upregulated by the oxidative stress and its activation leads to stereotypic nociceptive behavior in insulin-resistant rats

Kinin B1 receptor (B1R) is virtually absent under physiological condition, yet it is highly expressed in models of diabetes mellitus. This study aims at determining: (1) whether B1R is induced in the brain of insulin-resistant rat through the oxidative stress; (2) the consequence of B1R activation on stereotypic nocifensive behavior; (3) the role of downstream putative mediators in B1R-induced behavioral activity. Sprague-Dawley rats were fed with 10% d-glucose in their drinking water or tap water (controls) for 4 or 12 weeks, combined either with a standard chow diet or a diet enriched with α-lipoic acid (1 g/kg feed) for 4 weeks. The distribution and density of brain B1R binding sites were assessed by autoradiography. Behavioral activity evoked by i.c.v. injection of the B1R agonist Sar-[D-Phe⁸]-des-Arg⁹-BK (10 μg) was measured before and after i.c.v. treatments with selective antagonists (10 μg) for kinin B1 (R-715, SSR240612), tachykinin NK1 (RP-67580) and glutamate NMDA (DL-AP5) receptors or with the inhibitor of NOS (L-NNA). Results showed significant increases of B1R binding sites in various brain areas of glucose-fed rats that could be prevented by the diet containing α-lipoic acid. The B1R agonist elicited head scratching, grooming, sniffing, rearing, digging, licking, face washing, wet dog shake, teeth chattering and biting in glucose-fed rats, which were absent after treatment with α-lipoic acid or antagonists/inhibitors. Data suggest that kinin B1R is upregulated by the oxidative stress in the brain of insulin-resistant rats and its activation causes stereotypic nocifensive behavior through the release of substance P, glutamate and NO.     

Hypocretins: The Timing of Sleep and Waking

The appropriate time and place for sleep and waking are important factors for survival. Sleep and waking, rest and activity, flight and fight, feeding, and reproduction are all organized in relation to the day and night. A biological clock, the suprachiasmatic nucleus (SCN), synchronized by photic influences and other environmental cues, provides an endogenous timing signal that entrains circadian body rhythms and is complemented by a homeostatic sleep pressure factor. Cholinergic, catecholaminergic, serotonergic, and histaminergic nuclei control wakefulness and mutually interact with the SCN as well as sleep- and wake-promoting neurons in the hypothalamus to form a bistable switch that controlls the timing of behavioral state transitions. Hypocretin neurons integrate circadian-photic and nutritional-metabolic influences and act as a conductor in the aminergic orchestra. Their loss causes narcolepsy, a disease conferring the inability to separate sleep and waking. Their role in appetitive behavior, stress, and memory functions is important to our understanding of addiction and compulsion.     

Synthesis of thromboxane B2 in incubates of dog platelet-rich plasma with arachidonic acid and its inhibition by different drugs

Dog platelets in citrated plasma fail to aggregate upon addition of AA, even though, as demonstrated by bioassay procedures and now by the radioimmunoassay, TxA2 is formed as in case of aggregating human platelets. Imidazole inhibited formation of TxB2 and increased the amounts of PGE2 formed, indicating specific inhibition of thromboxane synthetase. Other drugs tested (benzimidazolamine, compound L8027, indomethacin and isoprenaline) inhibited either cyclo-oxygenase alone, or together with thromboxane synthetase.     

A New Single Chamber Implantable Defibrillator with Atrial Sensing: A Practical Demonstration of Sensing and Ease of Implantation

Implantable cardioverter-defibrillators (ICDs) terminate ventricular tachycardia (VT) and ventricular fibrillation (VF) with high efficacy and can protect patients from sudden cardiac death (SCD). However, inappropriate shocks may occur if tachycardias are misdiagnosed. Inappropriate shocks are harmful and impair patient quality of life. The risk of inappropriate therapy increases with lower detection rates programmed in the ICD. Single-chamber detection poses greater risks for misdiagnosis when compared with dual-chamber devices that have the benefit of additional atrial information. However, using a dual-chamber device merely for the sake of detection is generally not accepted, since the risks associated with the second electrode may outweigh the benefits of detection. Therefore, BIOTRONIK developed a ventricular lead called the Linox^SMART S DX, which allows for the detection of atrial signals from two electrodes positioned at the atrial part of the ventricular electrode. This device contains two ring electrodes; one that contacts the atrial wall at the junction of the superior vena cava (SVC) and one positioned at the free floating part of the electrode in the atrium. The excellent signal quality can only be achieved by a special filter setting in the ICD (Lumax 540 and 740 VR-T DX, BIOTRONIK). Here, the ease of implantation of the system will be demonstrated.     

Deletion of Smad3 protects against C-reactive protein-induced renal fibrosis and inflammation in obstructive nephropathy

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism.Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E).Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80⁺ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling.Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.