Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality.     

Effects of an energy-dense diet and nicotinic acid supplementation on production and metabolic variables of primiparous or multiparous cows in periparturient period

It is well observed that feeding energy-dense diets in dairy cows during the dry period can cause metabolic imbalances after parturition. Especially dairy cows with high body condition score (BCS) and fed an energy-dense diet were prone to develop production diseases due to metabolic disturbances postpartum. An experiment was conducted to determine the effects of an energy-dense diet and nicotinic acid (NA) on production and metabolic variables of primiparous and multiparous cows in late pregnancy and early lactation which were not pre-selected for high BCS. Thirty-six multiparous and 20 primiparous German Holstein cows with equal body conditions were fed with energy-dense (60% concentrate/40% roughage mixture; HC group) or adequate (30% concentrate/70% roughage mixture; LC group) diets prepartum. After parturition, concentrate proportion was dropped to 30% for all HC and LC groups and was increased to 50% within 16 days for LC and within 24 days for HC cows. In addition, half of the cows per group received 24 g NA supplement per day and cow aimed to attenuate the lipid mobilisation postpartum. Feeding energy-dense diets to late-pregnant dairy cows elevated the dry matter (p < 0.001) and energy intake (p < 0.001) as well as the energy balance (p < 0.001) without affecting the BCS (p = 0.265) during this period. However, this did not result in any metabolic deviation postpartum as the effects of prepartum concentrate feeding were not carried over into postpartum period. Multiparous cows responded more profoundly to energy-dense feeding prepartum compared with primiparous cows, and parity-related differences in the transition from late pregnancy to lactation were obvious pre- and postpartum. The supplementation with 24 g NA did not reveal any effect on energy metabolism. This study clearly showed that energy-dense feeding prepartum did not result in metabolic imbalances postpartum in multiparous and primiparous cows not selected for high BCS. A genetic predisposition for an anabolic metabolic status as indicated by high BCS may be crucial for developing production diseases at the onset of lactation.     

Traumatic brain injury and bone healing: radiographic and biomechanical analyses of bone formation and stability in a combined murine trauma model

Introduction:The combination of traumatic brain injury (TBI) and long-bone fractures has previously been reported to lead to exuberant callus formation. The aim of this experimental study was to radiographically and biomechanically study the effect of TBI on bone healing in a mouse model.Materials and methods:138 female C57/Black6N mice were assigned to four groups (fracture (Fx) / TBI / combined trauma (Fx/TBI) / controls). Femoral osteotomy and TBI served as variables: osteotomies were stabilized with external fixators, TBI was induced with controlled cortical impact injury. During an observation period of four weeks, in vivo micro-CT scans of femora were performed on a weekly basis. Biomechanical testing of femora was performed ex vivo.Results:The combined-trauma group showed increased bone volume, higher mineral density, and a higher rate of gap bridging compared to the fracture group. The combined-trauma group showed increased torsional strength at four weeks.Discussion:TBI results in an increased formation of callus and mineral density compared to normal bone healing in mice. This fact combined with a tendency towards accelerated gap bridging leads to increased torsional strength. The present study underscores the empirical clinical evidence that TBI stimulates bone healing. Identification of underlying pathways could lead to new strategies for bone-stimulating approaches in fracture care.     

Vaccination with Recombinant RNA Replicon Particles Protects Chickens from H5N1 Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.     

Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.     

Fish oil affects phosphoinositide turnover and thromboxane A metabolism in cultured vascular muscle cells

Fish oil has been reported as having beneficial effects on cardiovascular diseases. Elevated serum lipoproteins, prostaglandins and intracellular free calcium concentrations [( Ca2+]i) of the vasculature and thus the phosphoinositide (PI) turnover may be involved in the pathogenesis of these disorders. Therefore, the effect of fish oil on the potency of both low-density lipoprotein (LDL) and angiotensin II (AII) to stimulate the PI turnover in cultured rat vascular smooth muscle cells (VSMC) has been studied. Furthermore, a possible link between PI turnover activity and thromboxane A2 (TXA2) metabolism in these cells has been investigated. In VSMC cultured for up to 7 weeks with either fish oil or n-3 eicosapentaenoic acid (EPA) a decrease to 5-48% of the LDL-induced inositol trisphosphate (IP3) formation (= 100%) was found. A similar range of decreased IP3 synthesis was observed, when AII was used instead of LDL. Both LDL- and AII-stimulated TXA2 synthesis was suppressed concomitantly within the range 34-60%. Blockade of VSMC TXA2 biosynthesis with either indomethacin or TXA2 synthetase blocker (SQ-80338) inhibited LDL-induced formation of IP3 in a dose-dependent manner. Similar results were obtained, when TXA2 receptor coupling antagonists (SQ-27427 or BM-13177) were used. However, blockers of TXA2 synthesis and of TXA2 receptor binding failed to affect AII-induced formation of IP3.     

Temperature-induced transitions in the conformation of intermediates in the hydrolytic cycle of myosin.

The conformations of the transitory intermediates of the myosin ATPase occurring during the hydrolytic cycle, enzyme without ligand, enzyme-substrate complex and two different forms of enzyme-product complex, have been characterized in terms of numbers and classes of reactive thiol groups based on incorporation of radioactively labeled alkylation reagent. The techniques employed allowed this to be done under steady-state conditions in the presence of high ligand concentrations on intact myosin from rabbit fast skeletal muscles at low ionic strength where the protein is in the gel state as it is in muscle. The binding of a divalent cation (Mg2+ or Ca2+) nucleotide complex exposes thiol-1 as well as thiol-2 groups. The long-lived ATPase intermediate occurring at temperatures above 10 degrees C adopts the same conformation with Mg2+ and Ca2+ ions. This intermediate does not protect the thiol-1 and thiol-2 groups but exposes a number of thiol-3 groups which seem to be located distant from the active site. The conformation of the intermediate prevailing in the presence of ATP changes with lowering temperature below 10 degrees C and is identical with that found in the presence of ADP at 0 degree C indicating a change in the rate-limiting step of the hydrolytic cycle. In the absence of divalent cations no such temperature-dependent change in conformation was observed. Evaluation of the activation entropies shows that the structure of the long-lived intermediate occurring above 10 degrees C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20 degrees C. In the case of the monovalent-cation-stimulated ATPase a constant activation energy of around 70 kJ/mol, typical of many enzyme reactions, was found over the entire temperature range from 0--35 degrees C.     

In vitro folding and assembly of the Escherichia coli ATP-binding cassette transporter, BtuCD

Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B(12) (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits.     

Distinct gate conformations of the ABC transporter BtuCD revealed by electron spin resonance spectroscopy and chemical cross-linking

BtuCD is a type II ABC importer that catalyzes the translocation of vitamin B12 from the periplasm into the cytoplasm of Escherichia coli. Crystal structures of BtuCD and the related HiF (or Hi1470/71) protein from Haemophilus influenzae have revealed distinct conformations of the transmembrane domains that form inner and outer gates. We used electron spin resonance spectroscopy to study the reaction cycle of BtuCD after labeling the protein at residues located at these gates. The results suggest that BtuCD as a prototype type II ABC importer may have a mechanism that is distinct from that of ABC exporters such as Sav1866 or type I ABC importers such as those specific for molybdate (ModBC) or maltose (MalFGK).

Structured summary

MINT-6803800: btuF (uniprotkb: P37028), btuC (uniprotkb:P06609) and btuD (uniprotkb:P06611) physically interact (MI:0218) by molecular sieving (MI:0071)     

Structure-activity relationship in the oxazolidinone-quinolone hybrid series: influence of the central spacer on the antibacterial activity and the mode of action

Oxazolidinone-quinolone hybrids, which combine the pharmacophores of a quinolone and an oxazolidinone, were synthesised and shown to be active against a variety of susceptible and resistant Gram-positive and Gram-negative bacteria. The nature of the spacer greatly influences the antibacterial activity by directing the mode of action, that is quinolone- and/or oxazolidinone-like activity. The best compounds in this series have a balanced dual mode of action and overcome all types of resistance, including resistance to quinolones and linezolid, in clinically relevant Gram-positive pathogens.