Probing the coupling of Ca2+ and rigor activation of rabbit psoas myofibrillar ATPase with ethylene glycol

We have exploited solvent perturbation to probe the coupling of Ca²⁺ and rigor activation of the ATPase of myofibrils from rabbit psoas. Three techniques were used: overall myofibrillar ATPases by the rapid-flow quench method; kinetics of the interaction of ATP with myofibrils by fluorescence stopped-flow; and myofibrillar shortening by optical microscopy. Because of its extensive use with muscle systems, ranging from myosin subfragment-1 to muscle fibres, we chose 40% ethylene glycol as the relaxing agent. At 4°C, the glycol had little effect on the myofibrillar ATPase at low [Ca²⁺], but at high [Ca²⁺] the activity was reduced 50-fold, close to the level found under relaxing conditions, and there was no shortening. However, the ATPase of chemically cross-linked myofibrils (permanently activated even without Ca²⁺) was reduced only 3–4-fold. The lesser reduction of the ATPase of permanently activated myofibrils was also observed in single turnover experiments in which activation occurs by a few heads in the rigor state activating the remaining heads. The addition of ADP, which also promotes strong head-thin filament interactions, also activated the ATPase but only in the presence of Ca²⁺. Further experiments revealed that in 40% ethylene glycol, Ca²⁺ does initiate shortening but only with the aid of strong interactions and at temperatures above 15°C. This confirms that in the organized and intact myofibril, Ca²⁺ and rigor activation are coupled, as proposed previously for regulated actomyosin subfragment-1. This revised version was published online in September 2006 with corrections to the Cover Date.     

Sarcomeric determinants of striated muscle relaxation kinetics

Ca²⁺ is the primary regulator of force generation by cross-bridges in striated muscle activation and relaxation. Relaxation is as necessary as contraction and, while the kinetics of Ca²⁺-induced force development have been investigated extensively, those of force relaxation have been both studied and understood less well. Knowledge of the molecular mechanisms underlying relaxation kinetics is of special importance for understanding diastolic function and dysfunction of the heart. A number of experimental models, from whole muscle organs and intact muscle fibres down to single myofibrils, have been used to explore the cascade of kinetic events leading to mechanical relaxation. By using isolated myofibrils and fast solution switching techniques we can distinguish the sarcomeric mechanisms of relaxation from those of myoplasmic Ca²⁺ removal. There is strong evidence that cross-bridge mechanics and kinetics are major determinants of the time course of striated muscle relaxation whilst thin filament inactivation kinetics and cooperative activation of thin filament by cycling, force-generating cross-bridges do not significantly limit the relaxation rate. Results in myofibrils can be explained well by a simple two-state model of the cross-bridge cycle in which the apparent rate of the force generating transition is modulated by fast, Ca²⁺-dependent equilibration between off- and on-states of actin. Inter-sarcomere dynamics during the final rapid phase of full force relaxation are responsible for deviations from this simple model.     

The role of the hypothalamic paraventricular nuclei for the regulation of pineal melatonin synthesis: new aspects derived from the vasopressin-deficient Brattleboro rat

There is evidence for an involvement of the hypothalamic paraventricular nuclei (PVN) in the regulation of pineal melatonin synthesis in rats. Since electrical stimulation of the PVN or the systemic administration of arginine-vasopressin (AVP) result in a depression of the nocturnal melatonin surge, this neuropeptide appears to be pivotal for the transduction of PVN-efferent, pinealopetal signals. We therefore used an AVP-deficient animal model, the Brattleboro rat, to further investigate the mechanisms responsible for pineal regulation. Anesthetized adult male animals received 2 min of bilateral electrical stimulation of the PVN either during the day or at night. Thirty min later, pineal glands were removed and pineal N-acetyltransferase (NAT) activities and melatonin contents were determined. Stimulation resulted neither during the day nor at night in any significant alterations of pineal NAT activity or melatonin content when compared to control or sham-stimulated animals. These data further support the proposed modulatory role of AVP for the regulation of melatonin synthesis in the Epiphysis cerebri of genetically intact rats.     

Artificial-infection protocols allow immunodetection of novel Borrelia burgdorferi antigens suitable as vaccine candidates against Lyme disease

Vaccination with recombinant outer surface protein A (OspA) from Borrelia burgdorferi provides excellent antibody-mediated protection against challenge with the pathogen in animal models and in humans. However, the bactericidal antibodies are ineffective in the reservoir host, since OspA is expressed by spirochetes only in the vector, but rarely, if at all, in mammals. Using an artificially generated immune serum (anti-10(8) spirochetes) with high protective potential for prophylactic and therapeutic treatment, we have now isolated from an expression library of B. burgdorferi (strain ZS7) three novel genes, zs7.a36, zs7.a66 and zs7.a68. All three genes are located, together with ospA/B, on the linear plasmid lp54, and are expressed in vitro and in ticks. At least temporarily two of them, ZS7.A36 and ZS7.A66, are also expressed during infection. The respective natural antigens are poorly immunogenic ininfected normal mice but elicited antibodies in Lyme disease patients. We show that recombinant preparations of ZS7.A36, ZS7.A66 and ZS7.A68 induce functional antibodies in rabbits capable of protecting immunodeficient mice against subsequent experimental infection. These findings suggest that all three recombinant antigens represent potential candidates for a "second generation" vaccine to prevent and/or cure Lyme disease.     

Low vitamin D status is associated with low cord blood levels of the immunosuppressive cytokine interleukin-10

The cytokine interleukin-10 (IL-10) plays a pivotal regulatory role in tolerizing exogenous antigens. Experimental data indicate that low cellular availability of the vitamin D hormone 1,25-dihydroxyvitamin D [1,25(OH)2D] results in a down-regulation of IL-10 concentrations. The tissue production of an adequate amount of 1,25(OH)2D depends on a high circulating 25-hydroxyvitamin D (25-OHD) level. The present study was thus aimed at evaluating the associations between season of birth, vitamin D status, and the allergy risk markers IL-10 and total immunoglobulin (IgE) in newborns. Cord blood was obtained from 49 infants born during the summer half year (mid-April to mid-October, geographic latitude 51 degrees N) and from 47 infants born during the winter half year (mid-October to mid-April, geographic latitude of 51 degrees N). Serum levels of 25-OHD were 99% higher, and IL-10 levels were 43% higher in the summer half year compared with the winter half year (p < 0.001 and p = 0.018). Moreover, the ratio of IL-10 to total IgE was 124% higher in the summer half year compared with the winter half year (p = 0.039). Serum levels of 25-OHD were correlated with IL-10 levels (r = +0.22; p < 0.05). Mothers' age, gestational ages, birth weights and serum 1,25(OH)2D levels did not differ between study groups. We conclude that the low vitamin D status of infants born in winter may at least in part adversely affect biomarkers of allergy risk.     

Single-particle EM reveals plasticity of interactions between the adenovirus penton base and integrin αVβ3

Human adenoviruses are double-stranded DNA viruses responsible for numerous infections, some of which can be fatal. Furthermore, adenoviruses are currently used in clinical trials as vectors for gene therapy applications. Although initial binding of adenoviruses to host attachment receptors has been extensively characterized, the interactions with the entry receptor (integrins) remain poorly understood at the structural level. We characterized the interactions between the adenovirus 9 penton base subunit and α_Vβ₃ integrin using fluorescence correlation spectroscopy and single-particle electron microscopy to understand the mechanisms underlying virus internalization and infection. Our results indicate that the penton base subunit can bind integrins with high affinity and in several different orientations. These outcomes correlate with the requirement of the pentameric penton base to simultaneously bind several integrins to enable their clustering and promote virus entry into the host cell.Human adenoviruses are classified into more than 60 different serotypes and are associated with acute respiratory, gastrointestinal, and ocular infections, as well as fulminant infections among children and immunocompromised patients. Replication-defective or conditionally replicating human adenoviruses are also used in a large number of clinical trials for vaccine and therapeutic gene transfer (1). Adenoviruses are large, nonenveloped double-stranded DNA virions sharing a common capsid architecture based on T = 25 icosahedral symmetry (2–4). The hexon, penton base, and fiber proteins are the main capsid building blocks, with several cement proteins further contributing to capsid stability. The structural conservation of hexon- and penton-like proteins among several virus families infecting organisms of the three domains of life suggested that these virions are evolutionarily related and resulted in their classification in the PRD1-adenovirus lineage (2, 3, 5–11).The trimeric fiber protein and the pentameric penton base are the major players in adenovirus attachment and cell entry. Initial attachment of the virus occurs through the binding of the fiber C-terminal distal knob domain to attachment receptors such as coxsackie and adenovirus receptor (CAR), the complement receptor CD46, the GD1a glycan, or desmoglein-2 (DSG-2) (12). After initial cell attachment, a highly conserved arginine glycine aspartic acid (RGD) motif in a long loop of the penton base subunits engages α_vβ₃ or α_vβ₅ integrins, which serve as entry receptors. Binding to integrins promotes integrin clustering and in turn rapid receptor-mediated internalization of the virion (13). Partially disassembled capsids escape from the endosome and are transported along microtubules to the perinuclear envelope, where further disintegration of the capsid allows import of the viral genome into the nucleus (14).Although binding of the adenovirus trimeric fiber protein to the attachment receptors CAR, CD46, and GD1a has been extensively characterized, the interactions between the penton base and heterodimeric integrin remain less well understood at the structural level. Integrins are heterodimeric cell-surface transmembrane proteins involved in the regulation of adhesion, migration, growth, and differentiation (15). They form a stalk-like structure composed of a large “headpiece” that engages ligands and slender “legs” that anchor the heterodimer into the membrane. Integrins can assume a bent, inactive conformation, in which the legs are wrapped around the headpiece, as well as more extended, active conformations (16–18). The large multidomain extracellular moiety formed by the two integrin chains is able to bind a variety of ligands, including several viruses (e.g., adenoviruses), whereas the smaller intracellular domains interact with cytoskeletal proteins to carry out bidirectional signaling (19–23). Some integrins, such as α_vβ₃, are able to engage RGD sequences, which are found in various physiological ligands, and a crystal structure of a complex between α_vβ₃ integrin and a small peptide harboring this sequence has shown that the RGD motif binds to an exposed region in the headpiece (17). Located at the interface of the α_v and β₃ chains, the RGD sequence inserts into a cleft formed by the β-propeller domain of the α_v chain and the βA domain of the β₃ chain. All three amino acids—RGD—make specific contacts with headpiece residues. Previous studies have attempted to characterize the interactions between adenovirus and integrin using electron cryo-microscopy and biophysical techniques (24, 25). The results suggested that four integrins could simultaneously bind onto one penton base with varying orientations (due to steric hindrance). However, these interactions could not be analyzed in detail due to the large conformational flexibility of the integrin as well as the symmetry mismatch between the two partners.We set out to describe the interactions between the adenovirus penton base and integrin to understand the mechanisms underlying virus internalization and infection. Our approach differs from previous studies as we used a “minimalist” system, which is composed of the monomeric RGD loop-containing insertion domain of the adenovirus 9 penton base subunit (pb9) and the extracellular region of the α_Vβ₃ integrin. The adenovirus 9 serotype was chosen because the penton loop harboring the RGD sequence is among the shortest, and perhaps least flexible, in the adenovirus family. We characterized the binding affinity between α_Vβ₃ integrin and pb9 using fluorescence correlation spectroscopy (FCS) and demonstrated that the complex has a dissociation constant in the nanomolar range. We also analyzed the extracellular region of the α_Vβ₃ integrin, either unliganded or in complex with the penton base domain, using single-particle electron microscopy. The results unexpectedly indicate that the penton base can bind integrins in several orientations and suggest a putative mechanism for integrin clustering and virion internalization at the onset of infection.     

Oral High-Dose Vitamin D Dissolved in Oil Raised Serum 25-Hydroxy-Vitamin D to Physiological Levels in Obese Patients After Sleeve Gastrectomy—A Double-Blind,Randomized, and Placebo-Controlled Trial

Background

Osteomalacia and cardiometabolic disorders are favored in morbidly obese patients due to an inadequate vitamin D (VD) status. Former trials supplementing orally VD (20–50 μg/day) in crystalline form after sleeve gastrectomy (SG) could not stabilize serum 25-hydroxycholecalciferol levels at predefined concentrations (≥50 nmol/l). We hypothesized that VD in an oily suspension would increase its bioavailability resulting in normal serum VD levels minimizing markers of cardiometabolic risk.

Methods

Morbidly obese patients (n?=?94, BMI 51.8?±?11.5 kg/m²) received orally 80 μg/day VD₃ dissolved in oil or placebo (pure oil) in a randomized, double-blind, parallel-group study for 12 weeks after SG. 25-hydroxycholecalciferol, parathyroid hormone, albumin, alkaline phosphatase, phosphate, magnesium, calcium, creatinine, C-reactive protein, lipids, glucose, and glycated hemoglobin were determined in serum/plasma before surgery and after 4 and 12 weeks of supplementation. Intake of energy, fat, and VD were monitored using a 3-day food record.

Results

Seventy-nine patients were included in statistical analysis. Preoperatively, 77.2 and 40.5 % presented 25-hydroxycholecalciferol levels <75 and <50 nmol/l, respectively. After 12 weeks of supplementation, significantly more patients in the VD group exhibited levels >50 nmol/l (92 %) and >75 nmol/l (68 %) compared to the placebo group (54 and 22 %, respectively). Parameters of mineral metabolism and cardiometabolic risk were not modulated by intervention.

Conclusion

Supplementation of 80 μg/day VD₃ by oil is an effective and safe measure to prevent VD deficiency and to treat a preexisting undersupply in patients after SG. Cardiometabolic risk factors were, however, not affected; probably, higher VD doses might be necessary.

Clinical Trial Registration

This trial was registered retrospectively on November 14, 2014, at the German Clinical Trials Register as DRKS00007143.

     

Kinetics of cardiac sarcomeric processes and rate-limiting steps in contraction and relaxation

The sarcomere is the core structure responsible for active mechanical heart function. It is formed primarily by myosin, actin, and titin filaments. Cyclic interactions occur between the cross-bridges of the myosin filaments and the actin filaments. The forces generated by these cyclic interactions provide the molecular basis for cardiac pressure, while the motion produced by these interactions provides the basis for ejection. The cross-bridge cycle is controlled by upstream mechanisms located in the membrane and by downstream mechanisms inside the sarcomere itself. These downstream mechanisms involve the Ca²⁺-controlled conformational change of the regulatory proteins troponin and tropomyosin and strong cooperative interactions between neighboring troponin-tropomyosin units along the actin filament. The kinetics of upstream and downstream processes have been measured in intact and demembranated myocardial preparations. This review outlines a conceptual model of the timing of these processes during the individual mechanical heart phases. Particular focus is given to kinetic data from studies on contraction-relaxation cycles under mechanical loads. Evidence is discussed that the dynamics of cardiac contraction and relaxation are determined mainly by sarcomeric downstream mechanisms, in particular by the kinetics of the cross-bridge cycle. The rate and extent of ventricular pressure development is essentially subjected to the mechanistic principles of cross-bridge action and its upstream and downstream regulation. Sarcomere relengthening during myocardial relaxation plays a key role in the rapid decay of ventricular pressure and in early diastolic filling.