Functional and Muscular Adaptations in an Experimental Model for Isometric Strength Training in Mice

Exercise training induces muscular adaptations that are highly specific to the type of exercise. For a systematic study of the differentiated exercise adaptations on a molecular level mouse models have been used successfully. The aim of the current study was to develop a suitable mouse model of isometric strength exercise training characterized by specific adaptations known from strength training. C57BL/6 mice performed an isometric strength training (ST) for 10 weeks 5 days/week. Additionally, either a sedentary control group (CT) or a regular endurance training group (ET) groups were used as controls. Performance capacity was determined by maximum holding time (MHT) and treadmill spirometry, respectively. Furthermore, muscle fiber types and diameter, muscular concentration of phosphofructokinase 1 (PFK), succinate dehydrogenase (SDHa), and glucose transporter type 4 (GLUT4) were determined. In a further approach, the effect of ST on glucose intolerance was tested in diabetic mice. In mice of the ST group we observed an increase of MHT in isometric strength tests, a type II fiber hypertrophy, and an increased GLUT4 protein content in the membrane fraction. In contrast, in mice of the ET group an increase of VO_2max, a shift to oxidative muscle fiber type and an increase of oxidative enzyme content was measured. Furthermore strength training was effective in reducing glucose intolerance in mice fed a high fat diet. An effective murine strength training model was developed and evaluated, which revealed marked differences in adaptations known from endurance training. This approach seems also suitable to test for therapeutical effects of strength training.     

Crystallization and preliminary x-ray analysis of complexes of porcine pancreatic elastase with two natural inhibitors

Two different natural protease inhibitors, the squash inhibitor MCEI III and the third domain of turkey ovomucoid inhibitor OMTKY3, were crystallized in complexes with porcine pancreatic elastase (PPE). About 700 conditions were screened altogether. Crystals of the complex between MCEI III and PPE were grown in citrate buffer with and without ammonium acetate. X-ray diffraction data were collected to 1.9 angstroms resolution at room temperature using synchrotron radiation. The crystals belong to space group P2(1), with unit-cell parameters a = 49.17, b = 44.59, c = 67.08 angstroms, beta = 110.97 degrees. Crystals of the OMTKY3/PPE complex were obtained in the presence of ammonium sulfate, MES buffer and polyethylene glycol monomethylether (PEG). These crystals of this complex diffracted to 2.1 angstroms resolution and belongs to space group I222, with unit-cell parameters a = 84.58, b = 84.61, c = 89.92 angstroms and diffracted to 2.2 angstroms resolution. The diffraction data were collected using a conventional rotating anode X-ray generator at room temperature. In both cases the presence of inhibitor in the crystals was confirmed by crystallography.     

Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites

Calcium-dependent facilitation of L-type calcium channels has been reported to depend on the function of calmodulin kinase II. In contrast, the mechanism for voltage-dependent facilitation is not clear. In HEK 293 cells expressing Ca(v)1.2, Ca(v)beta2a, and calmodulin kinase II, the calcium current measured at +30 mV was facilitated up to 1.5-fold by a 200-ms-long prepulse to +160 mV. This voltage-dependent facilitation was prevented by the calmodulin kinase II inhibitors KN93 and the autocamtide-2-related peptide. In cells expressing the Ca(v)1.2 mutation I1649E, a residue critical for the binding of Ca2+-bound calmodulin, facilitation was also abolished. Calmodulin kinase II was coimmunoprecipitated with the Ca(v)1.2 channel from murine heart and HEK 293 cells expressing Ca(v)1.2 and calmodulinkinase II. The precipitated Ca(v)1.2 channel was phosphorylated in the presence of calmodulin and Ca2+. Fifteen putative calmodulin kinase II phosphorylation sites were identified mostly in the carboxyl-terminal tail of Ca(v)1.2. Neither truncation at amino acid 1728 nor changing the II-III loop serines 808 and 888 to alanines affected facilitation of the calcium current. In contrast, facilitation was decreased by the single mutations S1512A and S1570A and abolished by the double mutation S1512A/S1570A. These serines flank the carboxyl-terminal EF-hand motif. Immunoprecipitation of calmodulin kinase II with the Ca(v)1.2 channel was not affected by the mutation S1512A/S1570A. The phosphorylation of the Ca(v)1.2 protein was strongly decreased in the S1512A/S1570A double mutant. These results suggest that voltage-dependent facilitation of the Ca(v)1.2 channel depends on the phosphorylation of Ser1512/Ser1570 by calmodulin kinase II.     

Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage

Mesenchymal stem cells (MSC) can be obtained from human bone marrow aspirates and, thanks to their differentiation potential and excellent in vitro culture properties, represent an attractive cell line for the regeneration of mesenchymal tissue. Both in vitro and in vivo, they can differentiate into cartilage, bone, tendons and fat cells, and-in contrast to embryonic stem cells-they are not under ethical scrutiny. Cultured on three-dimensional scaffolds according to the tissue engineering concept, they have already been successfully employed for reconstruction of mesenchymal tissues in numerous studies involving both small and large animal models. Recently, immunological properties of MSC have been investigated by several groups. On the basis of the available literature, MSC have to be referred to as immune privileged, and they seem to be available for HLA-independent cell transplantation. While clinical MSC transplantation has also been successfully performed in pilot studies in humans, numerous points still remain to be clarified, underscoring the need for further intensive research before large-scale clinical application can be contemplated. Only then can it be shown whether the associated high expectations are justified.     

One-week once-daily triple therapy with esomeprazole, moxifloxacin, and rifabutin for eradication of persistent Helicobacter pylori resistant to both metronidazole and clarithromycin

Aim: To investigate a 1‐week once‐daily triple therapy with esomeprazole, moxifloxacin, and rifabutin for rescue therapy of Helicobacter pylori infection. Methods: Consecutive patients (n = 103) with at least one previous treatment failure and H. pylori infection resistant to both metronidazole and clarithromycin were treated with esomeprazole 40 mg, moxifloxacin 400 mg, and rifabutin 300 mg, given once daily for 7 days. Eradication was confirmed by histology and culture. CYP2C19 status was determined by polymerase chain reaction‐restriction fragment length polymorphism. Results: Intention‐to‐treat and per‐protocol eradication rates were 77.7% (68.4–85.3) and 83.3% (74.4–90.2). Five patients discontinued prematurely (4.8%). Eradication was achieved in 93.1% of poor/intermediate metabolizers and in 78.8% of homozygous extensive metabolizers (p = .14). Eradication rates in patients with one, two, three, and four or more previous failures were 78.3%, 89.6%, 68.6%, and 88.9%, respectively (p = .21). The regimen was effective in seven of nine patients who previously failed quadruple therapy. Post‐treatment resistance to moxifloxacin and rifabutin was detected in two (12.5%) and five (31%) patients after treatment failure. Conclusion: Once‐daily triple therapy with esomeprazole, moxifloxacin, and rifabutin is a promising, safe, and convenient regimen for rescue therapy of H. pylori infection that may serve as a valuable alternative to quadruple therapy, particularly for patients with intolerance to amoxicillin.     

PTHR1 Loss-of-Function Mutations in Familial, Nonsyndromic Primary Failure of Tooth Eruption

Tooth eruption is a complex developmental process requiring coordinated navigation through alveolar bone and oral epithelium. Primary failure of tooth eruption (PFE) is associated with several syndromes primarily affecting skeletal development, but it is also known as a nonsyndromic autosomal-dominant condition. Teeth in the posterior quadrants of the upper and lower jaw are preferentially affected and usually result in an open bite extending from anterior to posterior. In this study, we show that familial, nonsyndromic PFE is caused by heterozygous mutations in the gene encoding the G protein-coupled receptor for parathyroid hormone and parathyroid hormone-like hormone (PTHR1). Three distinct mutations, namely c.1050-3C > G, c.543+1G > A, and c.463G > T, were identified in 15 affected individuals from four multiplex pedigrees. All mutations truncate the mature protein and therefore should lead to a functionless receptor, strongly suggesting that haplo-insufficiency of PTHR1 is the underlying cause of nonsyndromic PFE. Although complete inactivation of PTHR1 is known to underlie the autosomal-recessive Blomstrand osteochondrodysplasia (BOCD), a lethal form of short-limbed dwarfism, our data now imply that dominantly acting PTHR1 mutations that lead to haplo-insufficiency of the receptor result in a nonsyndromic phenotype affecting tooth development with high penetrance and variable expressivity.     

Cytopathogenicity of classical Swine Fever virus correlates with attenuation in the natural host

For the important livestock pathogens classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), cytopathogenic (cp) and non-cp viruses are distinguished according to the induction of apoptosis in infected tissue culture cells. However, it is currently unknown whether cp CSFV differs from non-cp CSFV with regard to virulence in the acutely infected host. In this study, we generated helper virus-independent CSFV Alfort-Jiv, which encompasses sequences encoding domain Jiv-90 of cellular J-domain protein interacting with viral protein (Jiv). Expanding the knowledge of BVDV, our results suggest that Jiv acts as a regulating cofactor for the nonstructural (NS) protein NS2 autoprotease of CSFV and initiates NS2-3 cleavage in trans. For Alfort-Jiv, the resulting expression of large amounts of NS3 correlated with increased viral RNA synthesis and viral cytopathogenicity. Moreover, both cp Alfort-Jiv and the parental non-cp CSFV strain Alfort-p447 efficiently replicate in cell culture. Animal experiments demonstrated that in contrast to parental non-cp Alfort-p447, infection with cp Alfort-Jiv did not cause disease in pigs but induced high levels of neutralizing antibodies, thus elucidating that cp CSFV is highly attenuated in its natural host. In contrast to virulent Alfort-p447, the attenuated CSFV strain Alfort-Jiv induces the expression of cellular Mx protein in porcine PK-15 cells. Accordingly, the remarkable difference between cp and non-cp CSFV with regard to the ability to cause classical swine fever in pigs correlates with different effects of cp and non-cp CSFV on cellular antiviral defense mechanisms.     

Isolation and Characterization of Vacuoles from the Ergot Fungus Claviceps purpurea

Protoplasts of Claviceps purpurea were prepared by treatment of mycelium with a lytic mixture of snail gut enzyme and cellulase from Trichoderma viride. Such protoplasts could be efficiently lysed by Triton X-100 treatment at high osmotic pressure without Ca²⁺ or Mg²⁺, allowing the release of intact vacuoles in high yields. Vacuoles obtained from cells grown in modified Vogel medium (vegetative-type cells not producing alkaloids) were isolated and purified by centrifugation from a 5% Ficoll 400 (wt/vol) phase into the interphase between two layers, one containing 0.25 M each of mannitol and sucrose, and one containing 0.5 M mannitol. Vacuoles derived from cells grown in a medium favoring ergot alkaloid synthesis (sclerotia-like cells) were isolated by gentle centrifugation of filtered protoplast lysates without addition of Ficoll 400. Biochemical analyses of the vacuole fraction isolated from either kind of cell revealed their function as compartments harboring several hydrolytic enzymes. However, the enrichment of free amino acids in vacuoles of sclerotia-like cells was less pronounced than that in vacuoles of vegetative-type cells, indicating a difference in metabolic compartmentation in the two types of cells.     

Control of mitochondrial respiration. The contribution of the adenine nucleotide translocator depends on the ATP- and ADP-consuming enzymes

The consequence of the complexity of the metabolic network on the amount of control strength of adenine nucleotide translocator was investigated with isolated rat liver mitochondria. Two experimental systems were compared: (i) mitochondria in the presence of yeast hexokinase (hexokinase system) and (ii) the same system plus additional pyruvate kinase (pyruvate kinase system). In both systems the control strength was analysed for the adenine nucleotide translocator by inhibitor titration studies with carboxyatractyloside and for the hexokinase or pyruvate kinase by changing their relative activities. Experimental results were compared with computer simulation of these systems and that of a third one, where the extramitochondrial ATP / ADP ratio was held constant by perifusion (perifusion system). The results demonstrate quite different flux-dependent control strength of the translocator in the three systems. In the hexokinase system the control strength of the translocator on mitochondrial respiration was zero up to respiration rates of about 60 nmol O₂/mg protein per min. For higher rates, the control strength increased until the maximum value (0.45) was reached in the fully active state. Here, the same value was also found in the pyruvate kinase system. In all other states of respiration the translocator exerts a higher control strength in the pyruvate kinase system than in the hexokinase system. This different behaviour was attributed to the various changes in the adenine nucleotide pattern caused by partial inhibition of the translocator in the hexokinase and pyruvate kinase system. The data clearly show that the sharing of control strength depends not only on the respiration rate but also on the complexity of the metabolic system.     

Repeated exposure to benzalkonium chloride in the Ex Vivo Eye Irritation Test (EVEIT): observation of isolated corneal damage and healing

The prediction of side-effects is a key issue in the REACH initiative on chemicals, in the production of cosmetics and in the preclinical testing of drugs. A new ex vivo test for repeated substance application is presented, that is able to identify corrosive and irritant effects on the eye by using crucial endpoints, such as cellular and morphological damage, and healing characteristics. The test is intended to replace the Draize eye test and to improve the preclinical testing of drugs and chemicals that are likely to come into direct contact with the cornea. The Ex Vivo Eye Irritation Test (EVEIT) is a self-healing system, involving living corneas obtained from abattoir rabbit eyes. The corneas are cultured in a similar way to the method used during the transplantation of corneal grafts. The corneas are exposed to multiple small, mechanical abrasions, and then test substances are repeatedly dropped onto the centres of the corneas. The test substances applied in this study were citrate-buffered hyaluronate eye drops and an artificial tear replacement, with increasing concentrations of up to 0.1% benzalkonium chloride. A dose-dependent inhibition of recovery and impairment of the lactate production mechanism in the cornea was observed with benzalkonium chloride treatment.