Molecular identification of the ten subunits of cytochrome-c reductase from potato mitochondria

Cytochrome-c reductase (EC 1.10.2.2.) from Solanum tuberosum L. comprises ten subunits with apparent molecular sizes of 55, 53, 51, 35, 33, 25, 14, 12, 11 and 10 kDa on 14% SDS-PAGE. The identity of the subunits was analysed by direct amino-acid sequencing via cyclic Edman degradation. A large-scale purification procedure for the enzyme complex based on affinity chromatography and gelfiltraton is described. All subunits were enzymatically fragmented and the generated peptides were separated by reverse-phase HPLC. Complete or partial sequence determination of 33 peptides comprising a total of nearly 500 amino acids showed, that cytochrome-c reductase from potato contains three respiratory proteins (cytochrome b, cytochrome c ₁ and the Rieske iron-sulfur protein), four small proteins with molecular sizes below 15 kDa (so-called Q-binding, hinge, cytochrome-c ₁-linked and core-linked proteins) and three proteins in the 50-kDa range which show similarity to members of the core/PEP/MPP protein family (core/processing enhancing protein/mitochondrial processing peptidase). In fact these subunits show highest sequence identity either to MPP or PEP, which is in line with earlier findings, that isolated cytochrome-c reductase from potato exhibits processing activity towards mitochondrial precursor proteins.Abbreviations MPP mitochondrial processing peptidase - PEP processing enhancing protein This research was supported by the Deutsche Forschungsgemeinschaft.     

Enantiomerization of 2,2′-diisopropylbiphenyl during chiral inclusion gas chromatography: Determination of the rotational energy barrier by computer simulation of dynamic chromatographic elution profiles

By computer simulation of experimental dynamic gas chromatographic elution profiles, the rotational energy barrier ΔG⁼ of racemic 2,2′-diisopropylbiphenyl has been determined as 114.6–115.0 kJ/mol (75–100°C). These data are in good agreement with a value that was determined previously by measuring the racemization kinetics of an enriched sample. This indicates that there is no measurable catalytic or inhibitory effect of the stationary phase. © 1994 Wiley-Liss, Inc.     

Biomarker indications for microbial contribution to Recent and Late Jurassic carbonate deposits

Summary Biomarker investigations were applied to the hydrocarbon fractions of three Recent (cyanobacterial mat, Lake Van microbialite and Lake Satonda microbialite) and two Late Jurassic carbonate samples obtained from sponge bioherms. The relative concentrations ofn-alkanes, monomethyl alkanes, acyclic isoprenoids, steroids and hopanoids in these samples are studied and their probable biological precursors are discussed. Normal alkanes with carbon chain lengths ranging from C₁₅ to C₃₄ and monomethyl alkanes ranging from C₁₇ to C₂₁ with a varying methyl branching pattern are found. The major hydrocarbons are low molecular weight (LMW)n-alkanes (C₁₅–C₂₁) with a slight to strong predominance ofn-heptadecane (C₁₇). High molecular weight (HMW)n-alkanes occur in low to moderate relative concentrations showing a preference of odd-carbon numbered compounds with a maximum at C₂₉. Within the acyclic isoprenoids, pristane, phytane/phytene, pentamethyl-eicosane, squalane and lycopane could be identified. Polycyclic terpenoids of the sterane and/or hopane type are present in all carbonate samples. The carbon numbers of these components range from 27 to 29 and 27 to 32, respectively. These organic compounds identified can be attributed to various source organisms such as cyanobacteria, archaebacteria, algae and vascular plants. All hydrocarbon fractions of the samples are characterized by moderate to high relative concentrations of compounds derived from cyanobacteria, signifying the role of these organisms as contributors to the Recent as well as to the Late Jurassic carbonate deposits.     

Cloning of proline-specific endopeptidase gene from Flavobacterium meningosepticum: expression in Escherichia coli and purification of the heterologous protein

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described. Correspondence to: G. Reipen     

Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

Electrical responses of the marine ciliate Euplotes vannus (Hypotrichia) to mechanical stimulation at the posterior cell end

Electrical responses upon mechanostimulation at the posterior cell end were investigated in the marine hypotrichous ciliate Euplotes vannus. A new mechanostimulator was developed to mimic stimuli that are identical with those involved in cell-cell collisions. The receptor potential hyperpolarized by 18–35 mV within 12–25 msec, reached a peak value of -62 mV with a delay of 4–9 msec after membrane deformation, and was deactivated after 50–70 msec. Cirri were stimulated to beat accelerated backward. The corresponding receptor current exerted a similar time course with a peak of 2.4 nA. The shift of the reversal potential by 57.6 mV at a tenfold increase of [K⁺] ₀ identifies potassium ions as current carriers within the development of the receptor potential. An intracellular K concentration of 355 mmol/liter was calculated for cells in a medium that was composed similar to sea-water. The mechanically activated potassium current was totally inhibited by extracellular TEA and intracellular Cs⁺, and partially inhibited by extracellular 4-AP. The total inhibition of the current by injected EGTA points to a Ca dependence of the posterior mechanosensitivity. It was confirmed by the increase of the peak current amplitude with rising [Ca²⁺] ₀ . Sodium presumably repolarizes the receptor potential because the repolarization was delayed and after-depolarizations were eliminated in media without sodium. Since deciliation did not affect mechano-sensitivity, the corresponding ion channels reside within the soma membrane.The authors wish to thank Mr. Norbert Spreckelmeier from the electronics workshop and Mr. Herbert Lutter from the fine-mechanical workshop of the department for their excellent work, Mrs. G. Key and Mr. H. Mikoleit for skillful technical assistance and for preparing the figures. This work was supported by Deutsche Forschungsgemeinschaft, SFB 171, C7.     

The embryonic development of the Drosophila visual system

We have used electron-microscopic studies, bromodeoxyuridine (BrdU) incorporation and antibody labeling to characterize the development of the Drosophila larval photoreceptor (or Bolwig's) organ and the optic lobe, and have investigated the role of Notch in the development of both. The optic lobe and Bolwig's organ develop by invagination from the posterior procephalic region. After cells in this region undergo four postblastoderm divisions, a total of approximately 85 cells invaginate. The optic lobe invagination loses contact with the outer surface of the embryo and forms an epithelial vesicle attached to the brain. Bolwig's organ arises from the ventralmost portion of the optic lobe invagination, but does not become incorporated in the optic lobe; instead, its 12 cells remain in the head epidermis until late in embryogenesis when they move in conjunction with head involution to reach their final position alongside the pharynx. Early, before head involution, the cells of Bolwig's organ form a superficial group of 7 cells arranged in a rosette pattern and a deep group of 5 cells. Later, all neurons move out of the surface epithelium. Unlike adult photoreceptors, they do not form rhabdomeres; instead, they produce multiple, branched processes, which presumably carry the photopigment. Notch is essential for two aspects of the early development of the visual system. First, it delimits the number of cells incorporated into Bolwig's organ. Second, it is required for the maintenance of the epithelial character of the optic lobe cells during and after its invagination.     

The Tol-Pal system of Acinetobacter baumannii is important for cell morphology,antibiotic resistance and virulence

International Microbiology - Acinetobacter baumannii is an opportunistic human pathogen that has become a global threat to healthcare institutions. This Gram-negative bacterium is one of the most...     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.