The SEG1 element: a new DNA region promoting stable mitotic segregation of plasmids in the zygomycete Absidia glauca.

Summary A series of new vectors for the model zygomycete Absidia glauca was constructed on the basis of the structural neomycin resistance (Neo^r) gene controlled by the promoter of the gene for elongation factor 1 (TEF). In order to select for transformed colonies with a stable Neo^r phenotype, spores from primary transformants were pooled and grown for two sporulation cycles under non-selective conditions. Southern blot analysis of DNA from single spore isolates originating from independent transformant pools allowed the identification of two autonomously replicating plasmids. Retransformation of Escherichia coli and restriction analysis of the two plasmids provided evidence for spontaneous in vivo insertion of a new DNA element (SEG1) from the A. glauca genome. The inserted regions in both plasmids are essentially identical and do not represent repetitive DNA. Compared with other autonomously replicating vectors, these SEG1-containing plasmids are mitotically extremely stable and are passed on to the vegetative spore progeny of a retransformed A. glauca strain. We assume that SEG1 contains structural elements involved in partitioning and stable segregation of plasmids. For the construction of stable transformants of A. glauca, the SEG1 element may be regarded as a major breakthrough, because stabilization of transformed genetic traits by integration is difficult to achieve in all mucoraceous fungi and all known replicating plasmids are mitotically unstable.     

Chemical synthesis, purification, and characterization of two inflammatory proteins, neutrophil activating peptide 1 (interleukin-8) and neutrophil activating peptide

Two recently identified pro-inflammatory proteins, namely, neutrophil activating peptide 1 (NAP-1) [also termed interleukin-8 (IL-8)] and NAP-2, were chemically synthesized, purified, and characterized. The fully protected NAP-1/IL-8 (72 residues) and NAP-2 (70 residues) peptide chains were assembled by automated solid-phase methods with average stepwise yields of 99.5 and 99.3%, resulting in overall chain assembly yields of 70 and 62%, respectively. Deprotection resulted in crude products, which were allowed to fold by air oxidation, and were purified by two cycles of reverse-phase high-pressure liquid chromatography, yielding 27 mg of NAP-1/IL-8 and 22 mg of NAP-2. Purity was established by reverse-phase high-pressure liquid chromatography and isoelectric focusing, and the primary structures of the purified products were verified by using mass spectrometry and Edman sequencing methods. Synthetic and recombinant NAP-1/IL-8 were equally active on human neutrophil granulocytes as determined by measuring the induction of cytosolic free calcium, elastase release, and chemotaxis. Synthetic NAP-2 was equivalent to purified natural NAP-2 in the elastase release and calcium mobilization assays, but it was consistently less potent (3-5-fold) as a stimulus of chemotaxis, perhaps indicative of additional chemotactic components in the natural preparation. The results indicate that by chemical synthesis these cytokines can be obtained in purity and quantities suitable for further structural analysis, as well as functional studies both in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human alpha- to zeta-thrombin cleavage occurs with neutrophil cathepsin G or chymotrypsin while fibrinogen clotting activity is retained

Human neutrophil cathepsin G or bovine chymotrypsin proteolytically cleaved human alpha-thrombin at the B-chain Trp148-Thr149 bond generating a new form, zeta-thrombin. While incubation of alpha-thrombin with cathepsin G at pH 7.4 and 37 degrees C resulted in a partial loss of fibrinogen clotting activity, 86 +/- 13% of the clotting activity and 99 +/- 16% of the active sites titratable with p-nitrophenyl p-guanidinobenzoate were retained upon controlled passage of alpha-thrombin through chymotrypsin-Sepharose 4B at pH 6.2 or 7.4 and 24 degrees C (n = 15). Kinetic parameters for H-D-hexahydrotyrosyl-Ala-Arg p-nitroanilide were Km = 1.52 +/- 0.60 vs 1.32 +/- 0.18 microM and kcat = 51.9 +/- 2.9 vs 35.8 +/- 6.4 s-1 with alpha-thrombin vs chymotrypsin-prepared zeta-thrombin (n = 4 vs 3), respectively (I = 0.15 M, pH 7.4, and 24 degrees C). Some 95% of the clotting activity was lost when zeta-thrombin was passed through trypsin-Sepharose 4B under conditions for converting alpha- to nonclotting beta- and subsequently gamma-thrombin. The resulting gamma-like thrombins eluted bimodally with 260 and 310 mM NaCl when applied to Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)] developed with a linear salt gradient in 50 mM Tris at pH 7.4 and 24 degrees C. These elution peaks correspond to 240, 330, and 350 mM NaCl for gamma-, alpha-, and zeta-thrombin, respectfully, implying that the anion-binding exosite is partially destroyed in gamma-like thrombins but is intact in zeta-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-sequestering smooth endoplasmic reticulum in an invertebrate photoreceptor. I. Intracellular topography as revealed by OsFeCN staining and in situ Ca accumulation

Two ultrastructural approaches were used in photoreceptor cells of the leech, Hirudo medicinalis, to (a) investigate the intracellular topography of the smooth endoplasmic reticulum (SER) and (b) identify among the various subregions of the SER those which might function as Ca-sequestering sites. When the cells are prefixed with CaCl2-containing glutaraldehyde and postfixed with osmium tetroxide-ferricyanide (OsFeCN), only a part of the total SER is specifically stained. The stained SER cisternae include the submicrovillar cisternae (SMC), subsurface cisternae (SSC), the nuclear envelope, Golgi-associated SER, paracrystalline SER, and SER associated with glycogen areas. An extensive tubular SER cisternal system always remains unstained. When the cells are permeabilized by saponin and subsequently incubated with Ca2+, MgATP, and oxalate, the SMC (Walz, 1979, Eur. J. Cell Biol. 20:83-91), the SSC and the nuclear envelope contain electron-opaque Ca-oxalate precipitates indicating their ability to function as an effective Ca2+ sink. The results show that the very elaborate SER in this photoreceptor cell includes many functionally heterogeneous subregions. Of special physiological significance are those components (SMC and SSC) which are effective in Ca2+-buffering in the immediate vicinity of the plasma membrane.     

Metabolic products of microorganisms. 192. The anthraquinones of the Aspergillus glaucus group. II. Biological activity

From the mycelia of Aspergillus cristatus the following anthraquionic pigments were isolated: catenarin, emodin, erythroglaucin, rubrocristin, physcion, physcion-9-anthrone, questin, viocristin, and isoviocristin. The latter two do not belong to the 9, 10-anthraquinone series but to the 1,4-anthraquinones, and so far they have not been reported among naturally occurring quinones.Emodin, catenarin, viocristin, and isoviocristin snowed antibacterial activity with minimal inhibitory concentrations ranging from 1–10 g/ml. In Bacillus brevis catenarin and emodin inhibited the incorporation of uracil and leucine preferentially. At higher concentrations the incorporation of thymidine into the trichloroacetic acid-precipitable fraction of cells was also affected. In the presence of viocristin or isoviocristin all three macromolecular syntheses came to a halt. Rubrocristin, erythroglaucin, and physcion showed no significant inhibitory effects.In Ehrlich ascites carcinoma cells catenarin, emodin, and viocristin inhibited the incorporation of uridine and thymidine. The incorporation of leucine was hardly affected.In vitro, inhibition of DNA-dependent RNA polymerase from Escherichia coli by catenarin and to a lesser extent by emodin was observed, whereas rubrocristin (catenarin-8-methyl ether), physcion, and erythroglaucin were not active.Abbreviations MIC minimal inhibitory concentration - TCA trichloroacetic acid - ECA Ehrlich ascites carcinoma Metabolic Products of Microorganisms. 191. W. Keller-Schierlein und B. Joos; Über das 4-Oxohomotyrosin, ein Abbauprodukt des Echinocandins. Helv. Chim. Acta (in press)     

Base excision repair but not DNA double‐strand break repair is impaired in aged human adipose‐derived stem cells

The decline in DNA repair capacity contributes to the age‐associated decrease in genome integrity in somatic cells of different species. However, due to the lack of clinical samples and appropriate tools for studying DNA repair, whether and how age‐associated changes in DNA repair result in a loss of genome integrity of human adult stem cells remains incompletely characterized. Here, we isolated 20 eyelid adipose‐derived stem cell (ADSC) lines from healthy individuals (young: 10 donors with ages ranging 17–25 years; old: 10 donors with ages ranging 50–59 years). Using these cell lines, we systematically compared the efficiency of base excision repair (BER) and two DNA double‐strand break (DSB) repair pathways—nonhomologous end joining (NHEJ) and homologous recombination (HR)—between the young and old groups. Surprisingly, we found that the efficiency of BER but not NHEJ or HR is impaired in aged human ADSCs, which is in contrast to previous findings that DSB repair declines with age in human fibroblasts. We also demonstrated that BER efficiency is negatively associated with tail moment, which reflects a loss of genome integrity in human ADSCs. Mechanistic studies indicated that at the protein level XRCC1, but not other BER factors, exhibited age‐associated decline. Overexpression of XRCC1 reversed the decline of BER efficiency and genome integrity, indicating that XRCC1 is a potential therapeutic target for stabilizing genomes in aged ADSCs.     

Supply versus use designs of environmental extensions in input–output analysis: Conceptual and empirical implications for the case of energy

Input–output analysis is one of the central methodological pillars of industrial ecology. However, the literature that discusses different structures of environmental extensions (EEs), that is, the scope of physical flows and their attribution to sectors in the monetary input–output table (MIOT), remains fragmented. This article investigates the conceptual and empirical implications of applying two different but frequently used designs of EEs, using the case of energy accounting, where one represents energy supply while the other energy use in the economy. We derive both extensions from an official energy supply–use dataset and apply them to the same single‐region input–output (SRIO) model of Austria, thereby isolating the effect that stems from the decision for the extension design. We also crosscheck the SRIO results with energy footprints from the global multi‐regional input–output (GMRIO) dataset EXIOBASE. Our results show that the ranking of footprints of final demand categories (e.g., household and export) is sensitive to the extension design and that product‐level results can vary by several orders of magnitude. The GMRIO‐based comparison further reveals that for a few countries the supply‐extension result can be twice the size of the use‐extension footprint (e.g., Australia and Norway). We propose a graph approach to provide a generalized framework to disclosing the design of EEs. We discuss the conceptual differences between the two extension designs by applying analogies to hybrid life‐cycle assessment and conclude that our findings are relevant for monitoring of energy efficiency and emission reduction targets and corporate footprint accounting.     

A framework for modelling soil structure dynamics induced by biological activity

Soil degradation is a worsening global phenomenon driven by socio‐economic pressures, poor land management practices and climate change. A deterioration of soil structure at timescales ranging from seconds to centuries is implicated in most forms of soil degradation including the depletion of nutrients and organic matter, erosion and compaction. New soil–crop models that could account for soil structure dynamics at decadal to centennial timescales would provide insights into the relative importance of the various underlying physical (e.g. tillage, traffic compaction, swell/shrink and freeze/thaw) and biological (e.g. plant root growth, soil microbial and faunal activity) mechanisms, their impacts on soil hydrological processes and plant growth, as well as the relevant timescales of soil degradation and recovery. However, the development of such a model remains a challenge due to the enormous complexity of the interactions in the soil–plant system. In this paper, we focus on the impacts of biological processes on soil structure dynamics, especially the growth of plant roots and the activity of soil fauna and microorganisms. We first define what we mean by soil structure and then review current understanding of how these biological agents impact soil structure. We then develop a new framework for modelling soil structure dynamics, which is designed to be compatible with soil–crop models that operate at the soil profile scale and for long temporal scales (i.e. decades, centuries). We illustrate the modelling concept with a case study on the role of root growth and earthworm bioturbation in restoring the structure of a severely compacted soil.     

Evolution and maintenance of microbe‐mediated protection under occasional pathogen infection

Every host is colonized by a variety of microbes, some of which can protect their hosts from pathogen infection. However, pathogen presence naturally varies over time in nature, such as in the case of seasonal epidemics. We experimentally coevolved populations of Caenorhabditis elegans worm hosts with bacteria possessing protective traits (Enterococcus faecalis), in treatments varying the infection frequency with pathogenic Staphylococcus aureus every host generation, alternating host generations, every fifth host generation, or never. We additionally investigated the effect of initial pathogen presence at the formation of the defensive symbiosis. Our results show that enhanced microbe‐mediated protection evolved during host‐protective microbe coevolution when faced with rare infections by a pathogen. Initial pathogen presence had no effect on the evolutionary outcome of microbe‐mediated protection. We also found that protection was only effective at preventing mortality during the time of pathogen infection. Overall, our results suggest that resident microbes can be a form of transgenerational immunity against rare pathogen infection.