Evaluation of the soft tissue biocompatibility of MgCa0.8 and surgical steel 316L in vivo: a comparative study in rabbits

Background  

Recent studies have shown the potential suitability of magnesium alloys as biodegradable implants. The aim of the present study was to compare the soft tissue biocompatibility of MgCa0.8 and commonly used surgical steel in vivo.     

On insertion of pigment-associated polypeptides during membrane biogenesis in Rhodopseudomonas capsulata

Early stages in the formation of membranes and photosynthetic units were studied under growth-limiting phototrophic and chemotrophic conditions in cells of Rhodopseudomonas capsulata. The incorporation of polypeptides, forming bacteriochlorophyll-carotinoid-protein complexes in the membrane, was followed by use of pulse-labeling and immunoprecipitation techniques. The newly synthesized polypeptides were inserted into two distinct membrane fractions at both different rates and proportions. The two membrane fractions differed in sedimentation behavior, absorption spectra and activities of the respiratory chain. The individual pigment-associated proteins did not exhibit precursor-product relationship between the two membrane fractions. The data suggest that newly synthesized polypeptides were integrated both into cytoplasmic and pre-existing intracytoplasmic membranes, where the proteins and pigments were assembled to form reaction centers and light-harvesting pigment-protein complexes.Abbreviations Bchl bacteriochlorophyll - cpm counts per minute - M _r relative molecular mass - P 100 pellet of 100,000xg, 60 min - P300 pellet of 300,000xg, 90 min - pO₂ oxygen partial pressure - R Rhodopseudomonas - dodecyl sulfate sodium dodecyl sulfate. International standard units - Bq Becquerel (s^-1) - Pa Pascal (N/m²; 1 Torr=133,3 Pa)     

Redescription of the Antarctic springtail Desoria klovstadi using morphological and molecular evidence

Isotoma klovstadi Carpenter, 1902 was one of the first Collembola described from the Antarctic continent. It was first collected in November 1899 during the British Antarctic Expedition on the north coast of Victoria Land in the Ross Sea region. It is now known to occur in an extensive area of northern Victoria Land, including the offshore Possession, Coulman, and Foyn Islands. More recently, I. klovstadi was moved to the genus Gnathisotoma Cassagnau, 1957 and has been included in this genus in an unpublished checklist (online) of all described Collembola. Here, we redescribe the species and use morphological and molecular (COI and 18S genes) evidence to investigate its affinities within the Isotominae. We show that it does not belong to Gnathisotoma, or Isotoma s. str. (the viridis group) as currently conceived, but is likely to be part of the species complex of Isotoma s. lat. We discuss reasons for placing it in the genus Desoria Nicolet, 1841. Our results reinforce the already high level of endemicity in the Antarctic fauna and emphasise the value of both morphological and molecular studies in examining relict Gondwanan taxa and their evolutionary relationships with those of other Southern Hemisphere continents.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Denatured state aggregation parameters derived from concentration dependence of protein stability

Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.     

Sequential and ordered assembly of E1 initiator complexes on the papillomavirus origin of DNA replication generates progressive structural changes related to melting

Multiple binding sites for an initiator protein are a common feature of replicator sequences from various organisms. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA by largely unknown mechanisms. Here we analyze origin of DNA replication (ori) binding by the E1 initiator and show sequential binding to a set of overlapping binding sites. The assembly of these initiator complexes is controlled by a gradual reduction in the dependence of interactions between the initiator and DNA and a gradual increase in the reliance on interactions between initiator molecules, providing a mechanism for sequential and orderly assembly. Importantly, the binding of the initiator causes progressive structural alterations both in the sites and in the sequences flanking the sites, eventually generating severe structural alterations. These results indicate that the process of template melting may be incremental, where binding of each initiator molecule serves as a wedge that upon binding gradually alters the template structure. This mechanism may explain the requirement for multiple initiator binding sites that is observed in many ori's.     

Essential prerequisites for successful bioprocess development of biological CH4 production from CO2 and H2

The production and storage of energy from renewable resources steadily increases in importance. One opportunity is to utilize carbon dioxide (CO₂)-type hydrogenotrophic methanogens, which are an intriguing group of microorganisms from the domain Archaea, for conversion of hydrogen and CO₂ to methane (CH₄). This review summarizes the current state of the art of bioprocess development for biological CH₄ production (BMP) from pure cultures with pure gasses. The prerequisites for successful quantification of BMP by using closed batch, as well as fed-batch and chemostat culture cultivation, are presented. This review shows that BMP is currently a much underexplored field of bioprocess development, which mainly focuses on the application of continuously stirred tank reactors. However, some promising alternatives, such as membrane reactors have already been adapted for BMP. Moreover, industrial-based scale-up of BMP to pilot scale and larger has not been conducted. Most crucial parameters have been found to be those, which influence gas-limitation fundamentals, or parameters that contribute to the complex effects that arise during medium development for scale-up of BMP bioprocesses, highly stressing the importance of holistic BMP quantification by the application of well-defined physiological parameters. The much underexplored number of different genera, which is mainly limited to Methanothermobacter spp., offers the possibility of additional scientific and bioprocess development endeavors for the investigation of BMP. This indicates the large potential for future bioprocess development considering the possible application of bioprocessing technological aspects for renewable energy storage and power generation.     

Functional significance of brain glycogen in sustaining glutamatergic neurotransmission

The involvement of brain glycogen in sustaining neuronal activity has previously been demonstrated. However, to what extent energy derived from glycogen is consumed by astrocytes themselves or is transferred to the neurons in the form of lactate for oxidative metabolism to proceed is at present unclear. The significance of glycogen in fueling glutamate uptake into astrocytes was specifically addressed in cultured astrocytes. Moreover, the objective was to elucidate whether glycogen derived energy is important for maintaining glutamatergic neurotransmission, induced by repetitive exposure to NMDA in co-cultures of cerebellar neurons and astrocytes. In the astrocytes it was shown that uptake of the glutamate analogue d -[³H]aspartate was impaired when glycogen degradation was inhibited irrespective of the presence of glucose, signifying that energy derived from glycogen degradation is important for the astrocytic compartment. By inhibiting glycogen degradation in co-cultures it was evident that glycogen provides energy to sustain glutamatergic neurotransmission, i.e. release and uptake of glutamate. The relocation of glycogen derived lactate to the neuronal compartment was investigated by employing d -lactate, a competitive substrate for the monocarboxylate transporters. Neurotransmitter release was affected by the presence of d -lactate indicating that glycogen derived energy is important not only in the astrocytic but also in the neuronal compartment.     

Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics

Many cellular proteins assemble into macromolecular protein complexes. The identification of protein–protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein–protein interactions with an accurate determination of the stoichiometry of the identified protein–protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.