Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.     

Time course of neutral red-induced autophagy in murine pancreatic acinar cells. A morphometrical and cytochemical study.

The knowledge of the time course of the influences of chemicals on autophagy is of great importance in the study of their modes of action and hence provides information relating the mechanism and dynamics of this catabolic process. Neutral red (NR) treatment has long been used to produce an accumulation of autolysosomes in different cell types. In the present study early (AV1), advanced (AV2) and late (AV3), as well as complex (fused) AVs (AVc) were distinguished. In our morphometrical measurements, we found all these AV subcompartments significantly expanded as early as 30 min after the injection of NR (0.4 mg/g b.wt.), i.e. a large number of AVs accumulated in the cells. Since cytoplasmic volume fraction (CVF) of AV increased 3-fold during this early period we conclude that, unlike vinblastine, NR is not a fusion inhibitor. Accumulation of AV1 (3-fold) in the presence of fusions possibly indicates that NR stimulates formation of AVs in this early period, after the accumulation of AVs continued. The maximal CVF of AVs were measured at 4 h, when 7.6% of the cytoplasmic volume was sequestered into the AV compartment, two third of which came from AV3. This finding indicates that NR is probably an inhibitor of intravacuolar degradation. However, the high rate of accumulation of AV2, AV3, and total AVs including a slower but still pronounced accumulation of AV1 cannot be explained solely from inhibition of degradation, but indicates a stimulated segregation (AV formation). Our results therefore argue for a possible coupling of the regulation of autophagic segregation and degradation since vinblastine and possibly some other degradation inhibitors were also found to stimulate AV formation in other studies. Another goal of this study was to follow the time course of changes in distribution of certain lysosomal enzymes after NR treatment. According to our enzyme cytochemical studies, acid phosphatase (AP) of untreated cells is mainly located in large and small lysosomal elements of the Golgi zone, aryl sulfatase B (AS) in trans-Golgi elements including pre-secretory granules and trimetaphosphatase (TP) in basal lysosomes. After NR injection TP seemed to appear first in AV1 whereas AP activity was characteristic of more advanced AVs. AS activity only occasionally appeared in AV3 and exclusively at late times after NR injection.     

Active‐Site Imprinting: Preparation of Fe–N–C Catalysts from Zinc Ion–Templated Ionothermal Nitrogen‐Doped Carbons

Atomically dispersed Fe–N–C catalysts are considered the most promising precious‐metal‐free alternative to state‐of‐the‐art Pt‐based oxygen reduction electrocatalysts for proton‐exchange membrane fuel cells. The exceptional progress in the field of research in the last ≈30 years is currently limited by the moderate active site density that can be obtained. Behind this stands the dilemma of metastability of the desired FeN₄ sites at the high temperatures that are believed to be a requirement for their formation. It is herein shown that Zn²⁺ ions can be utilized in the novel concept of active‐site imprinting based on a pyrolytic template ion reaction throughout the formation of nitrogen‐doped carbons. As obtained atomically dispersed Zn–N–Cs comprising ZnN₄ sites as well as metal‐free N₄ sites can be utilized for the coordination of Fe²⁺ and Fe³⁺ ions to form atomically dispersed Fe–N–C with Fe loadings as high as 3.12 wt%. The Fe–N–Cs are active electocatalysts for the oxygen reduction reaction in acidic media with an onset potential of E₀ = 0.85 V versus RHE in 0.1 m HClO₄. Identical location atomic resolution transmission electron microscopy imaging, as well as in situ electrochemical flow cell coupled to inductively coupled plasma mass spectrometry measurements, is employed to directly prove the concept of the active‐site imprinting approach.     

Opening of Bottleneck Pores for the Improvement of Nitrogen Doped Carbon Electrocatalysts

A facile synthesis strategy to control the porosity of ionothermal nitrogen doped carbons is demonstrated. Adenine is used as cheap and biomass based precursor and a mixture of NaCl/ZnCl₂ as combined solvent‐porogen. Variation of the ratio between the two salt influences the pore structure over a wide range. The eutectic mixture leads to micro‐ and mesoporous material with high total pore volume (TPV) of 3.0 cm³ g^?1 and very high surface area of 2900 m² g^?1 essentially rendering the product an “all‐surface‐area” nitrogen doped carbon. Increasing NaCl contents cause a continuous increase of the mesopore size and the formation of additional macropores resulting in a very high maximal TPV of 5.2 cm³ g^?1, showing 2540 m² g^?1 specific surface area using 60 mol% NaCl. Interestingly, the electrocatalytic activity of the samples toward oxygen reduction is strongly affected by the detailed pore structure. The different—however, chemically equivalent—catalysts vary up to 70 mV in their half wave potentials (E _1/2).The sample with optimized pore system shows a high selectivity toward the favored four electron process and an outstanding E _1/2 of ≈880 mV versus reversible hydrogen electrode (RHE), which is one of the best values reported for nitrogen doped carbons so far.     

Mechanism and dynamics of macroautophagy in murine exocrine pancreatic cells. A review of vinblastine-induced changes.

Autophagy is a major pathway of lysosomal degradation of cellular macromolecules. The paper summarizes the results obtained in the studies on macroautophagy using the exocrine pancreatic acinar cell as model system and vinblastine as inducer. Current knowledge about the origin and properties of the limiting membranes of autophagic vacuoles, and the results of quantitative morphological studies into the dynamics and kinetics of vinblastine-induced autophagocytosis, as well as recent achievements in isolation and characterization of subclasses of autophagic vacuoles (autophagosomes and autolysosomes) are reviewed.     

Prevalence of Epstein-Barr virus (EBV) antibodies in primate stocks of zoological gardens

Abstract: Examination of 387 serum samples from 41 primate species with two different ELISAs for the presence of IgG-antibodies against Epstein-Barr virus. Antibodies were detected in 15 out of 32 species of Old World primates and none in six species of New World primates by screening ELISA (Enzygnost, Behringwerke AG, Marburg), a testkit for human diagnostics. To avoid species-dependent factors which could influence the sensitivity of the Enzygnost assay, a competition ELISA was established. The modified test assessed antibodies in all species of Old World primates and three species of the New World primates.     

Facile Metal Coordination of Active Site Imprinted Nitrogen Doped Carbons for the Conservative Preparation of Non‐Noble Metal Oxygen Reduction Electrocatalysts

Iron‐ or cobalt‐coordinated heteroatom doped carbons are promising alternatives for Pt‐based cathode catalysts in polymer‐electrolyte fuel cells. Currently, these catalysts are obtained at high temperatures. The reaction conditions complicate the selective and concentrated formation of metal–nitrogen active sites. Herein a mild procedure is introduced, which is conservative toward the carbon support and leads to active‐site formation at low temperatures in a wet‐chemical metal‐coordination step. Active‐site imprinted nitrogen doped carbons are synthesized via ionothermal carbonization employing Lewis‐acidic Mg²⁺ salt. The obtained carbons with large tubular porosity and imprinted N₄ sites lead to very active catalysts with a half‐wave potential (E_1/2) of up to 0.76 V versus RHE in acidic electrolyte after coordination with iron. The catalyst shows 4e^? selectivity and exceptional stability with a half‐wave potential shift of only 5 mV after 1000 cycles. The X‐ray absorption fine structure as well as the X‐ray absorption near edge structure profiles of the most active catalyst closely match that of iron(II)phthalocyanine, proving the formation of active and stable FeN₄ sites at 80 °C. Metal‐coordination with other transition metals reveals that Zn–N_x sites are inactive, while cobalt gives rise to a strong performance increase even at very low concentrations.     

Factors affecting the fermentative production of a lysozyme-binding antibody fragment in Escherichia coli

Fed-batch fermentation for production of a single-chain Fv antibody fragment (scFv) expressed as a recombinant periplastic protein from Escherichia coli was investigated. A high cell density of 50 g dry cell weight per liter was routinely achieved in a 14-L vessel by controlled exponential feeding of glucose to impose a constant specific growth rate. Following biomass accumulation, induction of the tac promoter by addition of IPTG was accompaied by a linear feed of yeast extract. The concentration of yeast extract feed was found to be highly influential upon both concentration and location of active product. Although scFv fragments were specifically targeted to the periplasmic space, at yeast extract feed rates of 0.72 g/h the final location was largely extracellular (68% to 79%). Total concentrations (extracellular + periplasmic) were of the order of 5 to 8 mg/L. A ten-fold increase in yeast extract supply increased total scFv concentration to almost 200 mg/L and 78% of this yield was retained in the periplasm. Control of such leakage of the recombinant product is fundamental to process design of downstream operations for product recovery. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 611-622, 1997.