Reporter genes and highly regulated promoters as tools for transformation experiments in Volvox carteri.

The multicellular alga Volvox is an attractive model for the study of developmental processes. With the recent report of successful transformation, regulated promoters as well as reporter genes working in this organism are now required. The Volvox genes encoding arylsulfatase and the extracellular glycoprotein ISG are strictly regulated. The former is transcribed only under conditions of sulfur starvation, whereas the latter operates under extreme developmental control--i.e., it is transcribed for only a few minutes in Volvox embryos at the stage of embryonic inversion. The gene encoding the sexual pheromone of Volvox carteri was placed under the control of the arylsulfatase promoter. In response to sulfur deprivation, V. carteri transformed by this construct synthesized and secreted biologically active pheromone. In addition, the gene encoding Volvox arylsulfatase was placed under the control of the ISG promoter. Transformed algae synthesized arylsulfatase mRNA only during embryonic inversion. These experiments demonstrate the usefulness of both the arylsulfatase and the sexual pheromone reporter genes. In addition, the highly regulated arylsulfatase promoter allows the construction of inducible expression vectors for cloned genes.     

Effect of dietary protein on Masheri-induced drug metabolising enzymes

The modulation of drug metabolising enzymes by Masheri extract (ME) and Benzo(a)Pyrene [B(a)P] was studied in male Sprague Dawley rats fed different dietary protein levels. Two groups of 21 days old male Sprague Dawley rats were put on a high protein diet (SHP) with 20% Casein, and a low protein diet (SLP) with 3% Casein semisynthetic based diets for 12 weeks. The SLP fed animals showed lower basal levels of the Phase I activating enzymes viz. Cytochrome P450, Benzo(a)Pyrene hydroxylase, Benzphetamine demethylase and Phase II glutathione detoxification system viz. Glutathione (GSH) and Glutathione-S-transferase. ME and B(a)P treatment significantly depleted the glutathione detoxification system in the SLP group whereas an opposite effect was observed in the SHP group. Interstingly, ME and B(a)P treated rats in the SLP group showed a higher percent increase in the hepatic and pulmonary Phase I enzyme activities than those observed in the treated ME/B(a)P treated SHP rats. Furthermore, both ME and B(a)P significantly decreased the hepatic pool of vitamin A while a concomittant increase in that of vitamin C was observed.     

CC chemokine ligand 20 partially controls adhesion of naive B cells to activated endothelial cells under shear stress

Chemokines are thought to control lymphocyte recruitment to the inflamed endothelium. To dissect chemokine-mediated adhesion, binding of ex vivo isolated splenocytes to tumor necrosis factor (TNF)-activated endothelial cells was analyzed under shear stress. We observed specific adhesion of naive follicular B cells, which could be blocked by pertussis toxin. This indicated a G protein-mediated binding and pointed at a contribution of chemokine receptors to B-cell adhesion. Analysis of chemokines expressed by TNF-activated endothelial cells showed that CC chemokine ligand 2 (CCL2), CCL17, and CCL20 were up-regulated. Only on follicular B cells was the cognate receptor for CCL20, CC chemokine receptor 6 (CCR6), expressed strongly, and a functional transmigration assay with CCR6-negative B cells demonstrated conclusively the sole signaling of CCL20 through CCR6. Desensitization of CCR6 on naive B cells with CCL20 resulted in receptor down-regulation and reduced B-cell adhesion. We conclude that CCL20 plays a vital role in B-cell adhesion to the inflamed endothelium.     

Human stem cell models of neurodegeneration: a novel approach to study mechanisms of disease development

The number of patients with neurodegenerative diseases is increasing significantly worldwide. Thus, intense research is being pursued to uncover mechanisms of disease development in an effort to identify molecular targets for therapeutic intervention. Analysis of postmortem tissue from patients has yielded important histological and biochemical markers of disease progression. However, this approach is inherently limited because it is not possible to study patient neurons prior to degeneration. As such, transgenic and knockout models of neurodegenerative diseases are commonly employed. While these animal models have yielded important insights into some molecular mechanisms of disease development, they do not provide the opportunity to study mechanisms of neurodegeneration in human neurons at risk and thus, it is often difficult or even impossible to replicate human pathogenesis with this approach. The generation of patient-specific induced pluripotent stem (iPS) cells offers a unique opportunity to overcome these obstacles. By expanding and differentiating iPS cells, it is possible to generate large numbers of functional neurons in vitro, which can then be used to study the disease of the donating patient. Here, we provide an overview of human stem cell models of neurodegeneration using iPS cells from patients with Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington’s disease, spinal muscular atrophy and other neurodegenerative diseases. In addition, we describe how further refinements of reprogramming technology resulted in the generation of patient-specific induced neurons, which have also been used to model neurodegenerative changes in vitro.     

Surface characterization of dental Y-TZP ceramic after air abrasion treatment

Objective

The aim of this study was to characterize the surface of Y-TZP after abrasion with various airborne particles.

Methods

The Y-TZP blanks were cut into 44 discs and sintered according to the manufacturer's instructions. The specimens were treated as follows: (a) control specimens, (b) abraded with 50 μm alumina, (c) abraded with 110 μm alumina, (d) abraded with 30 μm silica-coated alumina, (e) abraded with 110 μm silica-coated alumina, (f) abraded with 110 μm alumina followed by 110 μm silica-coated alumina particles. Airborne abrasion was performed at a pressure of 2.5 bar for 15 s/cm². The Y-TZP was characterized using X-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy (FESEM) and X-ray diffraction analysis (XRD).

Results

Surface morphology of Y-TZP ceramic was changed after the airborne abrasion process compared to the control specimens. The grain boundaries disappeared and part of the airborne particles are embedded and/or rested on the ceramic surfaces. The elemental composition of the Y-TZP surface after the airborne abrasion process depended on the type and size of these particles. The concentration of Si resulted higher after the airborne abrasion process with 110 μm alumina followed by 110 μm silica-coated alumina particles in comparison to the specimens abraded with 110 μm silica-coated alumina particles. The ratio of elements normalized by yttrium for these specimens was: [Zr]/[Y]/[Al]/[Si] = 15.2/1.0/26.0/73.6, respectively.

Conclusion

The change of grain topography occurred during each impact process. Silica nano-particles covered not only loosely the abraded ceramic surface after abrasion process, but the release of kinetic energy in form of thermal energy resulted in melting of the ceramic surface and in the formation of zirconium silicate.     

Das weltweite Medizinernetzwerk Aufklärung gegen Tabak – Ehrenamtliche Prävention made in Germany

Smoking is the leading preventable cause of premature death in Germany. The network “Education Against Tobacco” (EAT) is an initiative that was founded in Germany in 2012, in which more than 3500 medical students and physicians engage in volunteer work in about 80 medical faculties in 14 countries. In this article, the concept, activities, objectives and associated research studies oft he EAT initiative are introduced.On the school level, the initiative addresses 10- to 15-year-old secondary school students. In addition to a multimodal approach, school visits use modern media such as facemorphing apps, which are not only used by students (45,000 per year in 14 countries), but by a total of over 500,000 other people as well. The effectiveness of the school-based intervention is currently being investigated in randomised long-term studies with 20,000 adolescents in Germany. A first long-term study demonstrated evidence of a protective effect regarding the onset of smoking, especially among female students, students having a low level of education and students with a migratory background.The programme educates several hundred prospective physicians at 13 (of 28 participating) German medical faculties each year in science-based elective courses for the well-established smoking cessation counselling of patients and sensitises them to the tobacco epidemic. The approved members engage in dialogue with local members of the German house of representatives as “Ärzteverband Tabakprävention”.EAT motivates the prospective generation of physicians, initially through prevention in school settings, to face the challenge of national tobacco control at the university and federal level.     

Gene replacement by homologous recombination in the multicellular green alga Volvox carteri

With only two different cell types, the haploid green alga Volvox represents the simplest multicellular model system. To facilitate genetic investigations in this organism, the occurrence of homologous recombination events was investigated with the intent of developing methods for gene replacement and gene disruption. First, homologous recombination between two plasmids was demonstrated by using overlapping nonfunctional fragments of a recombinant arylsulfatase gene (tubulin promoter/arylsulfatase gene). After bombardment of Volvox reproductive cells with DNA-coated gold microprojectiles, transformants expressing arylsulfatase constitutively were recovered, indicating the presence of the machinery for homologous recombination in Volvox. Second, a well characterized loss-of-function mutation in the nuclear nitrate reductase gene (nitA) with a single G → A nucleotide exchange in a 5′-splice site was chosen as a target for gene replacement. Gene replacement by homologous recombination was observed with a reasonably high frequency only if the replacement vector containing parts of the functional nitrate reductase gene contained only a few nucleotide exchanges. The ratio of homologous to random integration events ranged between 1:10 and 1:50, i.e., homologous recombination occurs frequently enough in Volvox to apply the powerful tool of gene disruption for functional studies of novel genes.     

Melatonin enhances antioxidant action of α‐tocopherol and ascorbate against NADPH‐ and iron‐dependent lipid peroxidation in human placental mitochondria

Abstract: Human placental mitochondria might be a significant source of NADPH‐ and iron‐dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH‐ and iron‐dependent lipid peroxidation of human placental mitochondria in a concentration‐dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal‐chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and α‐tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 μm ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 μm ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro‐oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and α‐tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.     

The voltage-gated sodium channel beta2-subunit gene and idiopathic generalized epilepsy

Recent identification of ion channel gene mutations in Mendelian epilepsies suggests that genetically driven neuronal hyperexcitability plays an important role in epileptogenesis. In this study, we tested the hypothesis that genetic variation in the human SCN2B gene confers liability to common subtypes of idiopathic generalized epilepsies (IGE). A systematic search for mutations was performed in 92 IGE patients. We detected a novel single nucleotide polymorphism (SNP), however, allele frequencies did not differ between IGE patients and controls (chi2 = 0.19, df = 1, p = 0.744). Furthermore, a missense mutation in codon 209 (Asn209Pro) was identified in one patient, but was found to be absent in an affected sibling of the index patient. Thus, our results do not suggest a major role of the SCN2B gene in the etiology of common IGE subtypes.