r-VKORC1 expression in factor IX BHK cells increases the extent of factor IX carboxylation but is limited by saturation of another carboxylation component or by a shift in the rate-limiting step

Carboxylation of vitamin K-dependent (VKD) proteins is required for their activity and depends on reduced vitamin K generated by vitamin K oxidoreductase (VKOR) and a redox protein that regenerates VKOR activity. VKD protein carboxylation is inefficient in mammalian cells, and to understand why carboxylation becomes saturated, we developed an approach that directly measures the extent of intracellular VKD protein carboxylation. Analysis of factor IX (fIX)-expressing BHK cells indicated that slow egress of fIX from the endoplasmic reticulum and preferential secretion of the carboxylated form contribute to secreted fIX being more fully carboxylated. The analysis also revealed the first reported in vivo VKD protein turnover, which was 14-fold faster than that which occurs in vitro, suggesting facilitation of this process in vivo. r-VKORC1 expression increased the rate of fIX carboxylation and the extent of secreted carboxylated fIX approximately 2-fold, which shows that carboxylation is the rate-limiting step in fIX turnover and which was surprising because turnover in vitro is limited by release of carboxylated fIX. Interestingly, the increases were significantly smaller than the amount of VKOR overexpression (15-fold). However, when cell extracts were tested in single-turnover experiments in vitro, where redox protein is functionally substituted with dithiothreitol, VKOR overexpression increased the fIX carboxylation rate 14-fold, showing r-VKORC1 is functional for supporting fIX carboxylation. These data indicate that the effect of VKOR overexpression is limited in vivo, possibly because a carboxylation component like the redox protein becomes saturated or because another step is now rate-limiting. The studies illustrate the complexity of carboxylation and potential importance of component stoichiometry to overall efficiency.     

Determinants of musculoskeletal frailty and the risk of falls in old age

Neuromuscular parameters that describe locomotion are indispensable variables for the diagnosis and treatment of frailty, fall risk and osteoporosis. A scientifically-based standardized locomotor assessment should be an essential part of medical examinations in research and clinical practice. There has been no previous consensus regarding which test procedures should be included in a locomotor assessment. The goal of this article is to provide a rationale for the selection of appropriate locomotor tests in a comprehensive locomotor assessment for elderly patients. We propose that a locomotor assessment should comprise the parameters that have been proven predictive for both falls and impending disability. The parameters should be measured in the standard units of physics. Therefore, we propose the following tests for a standardized locomotor assessment: (1) Self-selected gait velocity as the single best measure of general locomotor status and a good predictor of age-related adverse events; (2) Chair rise test (timed 5 chair rises) which measures power on vertical movement and the hip surrounding muscles as the most important neuromuscular risk factor for falls and fall-related fractures; (3) Tandem standing and tandem walking to measure postural capacity (balance) to the side; (4) Timed up and go test as a global screening procedure; (5) Clinical gait analysis with special focus on regularity; and (6) At least on a research level, movement must be measured referring to the terms of physics by mechanography. Mechanography (Leonardo force plate system, Novotec Pforzheim, Germany) records the time course of ground reaction forces, velocity of the vertical movements of the center of mass and power during unrestricted physiological movements. In the mechanogram the eccentric and concentric phases of movements can be differentiated and the storage of energy in the elastic elements of the body can be examined. The kinetics of human movement is explained by mechanograms of a two-legged jump. The ground reaction forces resulting from a jump down from a height of 0,46 m are demonstrated as a performance that is representative for human coordination. One goal of this text is to underline the insights that arise if the rules of physics are applied to human movement. A deeper understanding enables us to create more effective treatments for disorders of the muscle-bone unit. Bringing physics and cybernetics into the field of osteoporosis is a great heritage of Harold Frost.     

Induction of cytochrome P450 (CYP)1A1, CYP1A2, and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes

Human hepatocytes cultured serum-free for up to 6 weeks were used to study expression and induction of enzymes and membrane transport proteins involved in drug metabolism. Phase I drug metabolizing enzymes cytochrome P450 (CYP)1A1, CYP1A2, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 were detected by Western blot analyses and, when appropriate, by enzymatic assays for ethoxyresorufin-O-deethylase(EROD)-activity and testosterone-6beta-hydroxylase(T6H)-activity. Expression of the membrane transporter multi-drug resistance protein (P-glycoprotein, MDR-1), multidrug resistance-associated protein (MRP-1), and lung-resistance protein (LRP) was maintained during the culture as detected by RT-PCR and Western blot analyses. Model inducers like rifampicin, phenobarbital, or 3-methylcholanthrene and beta-naphtoflavone were able to induce CYP1A or CYP3A4 as well as EROD or T6H activities for up to 30 days. CYP2C9, CYP2C19 and CYP2E1 expression was maintained but not inducible for 48 days. Also, rifampicin and phenobarbital were unable to increase MDR-1 and MRP-1 protein levels significantly.     

Auditory brainstem responses in aging dark agouti rats

The present study examined auditory function across age in the dark agouti (DA) rat strain. Auditory brainstem responses (ABRs) were measured for frequencies 8, 16, and 32 kHz in male and female DA rats from 3 to 18 months of age. Hearing thresholds and absolute and interpeak latencies (IPLs) were analyzed. Male hearing thresholds remained stable for the first year of life and then significantly increased at 18 months across all frequencies; female hearing remained stable at all tested ages out to 18 months. At 12 months, male DA rats showed significantly longer absolute latencies by age (i.e., compared with 3-month-old males) and sex (compared with 12-month-old females), with no differences in IPLs. At 18 months, female DA rats showed significantly longer absolute latencies with age (compared with 3-month-old females) and sex (compared with 18-month-old males), particularly for the later waves. Female IPLs were also significantly longer with age and by sex for the later waves. This report supports the feasibility of using male DA rats in studies to investigate age-related hearing loss (ARHL; presbycusis).     

Limited mitochondrial DNA diversity is indicative of a small number of founders of the German raccoon (Procyon lotor) population

The raccoon (Procyon lotor) has successfully invaded central Europe, despite the population apparently having been founded by a small number of individuals in two distinct populations in Germany. The ecological success of the invasion has been explained by raccoons being an adaptable, truly omnivorous species. However, the German raccoon population might have a larger number of founders and be more genetically diverse than assumed, as accidental or deliberate releases of household pets or individuals from zoos are relatively common. In the present study, we sequenced a 550-base-pair long fragment of the mitochondrial control region in 193 raccoons from Germany and neighbouring countries. We only identified six different haplotypes; of which, five were limited to Germany. Our results support the notions that the population was founded by a small number of females and that the German raccoons originate from two separate release events in central and eastern Germany. Additionally, however, we provide evidence for the presence of a distinct population in Saxony, eastern Germany. Further studies using different genetic markers are necessary to gain additional information on genetic diversity and population genetic structure.     

Photoprotective potential of lycopene, β-carotene, vitamin E, vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, β-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10–15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low μM concentrations of vitamin E, vitamin C, or carnosic acid but not with β-carotene or lycopene. Indeed, in the presence of 0.5–1.0 μM β-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5–2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, β-carotene or lycopene (0.5–1.0 μM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and β-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Jährliche schwankungen der individuenzahl in einer nordwestdeutschen trockenen heide

Ohne Zusammenfassung     

Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis

Cytokinesis is crucial for integrating genome inheritance and cell functions. In multicellular organisms, Rho-guanine nucleotide exchange factors (GEFs) and Rho GTPases are key regulators of division-plane specification and contractile-ring formation during cytokinesis, but how they regulate early steps of cytokinesis in fission yeast remains largely unknown. Here we show that putative Rho-GEF Gef2 and Polo kinase Plo1 coordinate to control the medial cortical localization and function of anillin-related protein Mid1. The division-site positioning defects of gef2Δ plo1-ts18 double mutant can be partially rescued by increasing Mid1 levels. We find that Gef2 physically interacts with the Mid1 N-terminus and modulates Mid1 cortical binding. Gef2 localization to cortical nodes and the contractile ring depends on its last 145 residues, and the DBL-homology domain is important for its function in cytokinesis. Our data suggest the interaction between Rho-GEFs and anillins is an important step in the signaling pathways during cytokinesis. In addition, Gef2 also regulates contractile-ring function late in cytokinesis and may negatively regulate the septation initiation network. Collectively, we propose that Gef2 facilitates and stabilizes Mid1 binding to the medial cortex, where the localized Mid1 specifies the division site and induces contractile-ring assembly.     

Metaproteomic analysis of a winter to spring succession in coastal northwest Atlantic Ocean microbial plankton

In this study, we used comparative metaproteomics to investigate the metabolic activity of microbial plankton inhabiting a seasonally hypoxic basin in the Northwest Atlantic Ocean (Bedford Basin). From winter to spring, we observed a seasonal increase in high-affinity membrane transport proteins involved in scavenging of organic substrates; Rhodobacterales transporters were strongly associated with the spring phytoplankton bloom, whereas SAR11 transporters were abundant in the underlying waters. A diverse array of transporters for organic compounds were similar to the SAR324 clade, revealing an active heterotrophic lifestyle in coastal waters. Proteins involved in methanol oxidation (from the OM43 clade) and carbon monoxide (from a wide variety of bacteria) were identified throughout Bedford Basin. Metabolic niche partitioning between the SUP05 and ARCTIC96BD-19 clades, which together comprise the Gamma-proteobacterial sulfur oxidizers group was apparent. ARCTIC96BD-19 proteins involved in the transport of organic compounds indicated that in productive coastal waters this lineage tends toward a heterotrophic metabolism. In contrast, the identification of sulfur oxidation proteins from SUP05 indicated the use of reduced sulfur as an energy source in hypoxic bottom water. We identified an abundance of Marine Group I Thaumarchaeota proteins in the hypoxic deep layer, including proteins for nitrification and carbon fixation. No transporters for organic compounds were detected among the thaumarchaeal proteins, suggesting a reliance on autotrophic carbon assimilation. In summary, our analyses revealed the spatiotemporal structure of numerous metabolic activities in the coastal ocean that are central to carbon, nitrogen and sulfur cycling in the sea.     

Evidence for high degrees of specialisation, evolutionary diversity, and morphological distinctiveness in the genus Bremia

Bremia lactucae is one of the most important pathogens in lettuce production. Recent molecular?studies revealed considerable genetic variation in this species complex. However, only few accessions from the same host have been examined for most species and no study investigating the morphological distinctiveness of phylogenetic lineages of Bremia has so far been reported. Thus it is believed that morphological species delimitation in Bremia is not feasible. In the present study, multiple accessions of neglected species, which had been described decades ago, but have not been widely accepted, were investigated, considering both multi-gene phylogenies and morphological characters. All previously described species from host genera other than Lactuca investigated, Bremia microspora, Bremia ovata, Bremia saussureae, and Bremia sonchicola, could be confirmed as distinct, host-specific entities. Also, morphological characteristics of their conidiophores and conidia allowed delimitation of these species. Therefore, not only the wide species concept to merge all Bremia species on the Asteraceae under B. lactucae is inappropriate but also their delimitation on the basis of morphological characters seems feasible. In addition, it has been shown that Bremia elliptica is phylogenetically distinct from the other species infecting the genus Lactuca, B. lactucae. It is therefore concluded that B. lactucae is most likely limited to Lactuca sativa and closely-related species, and that most species of Bremia are highly host specific. This finding might stimulate the search for durable resistance genes in genera closely related to the genus Lactuca and in divergent species of the genus itself.