Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process; Part II: instrumentation of integrated flow injection analysis (FIA) system for BODst estimation

An instrument with integrated flow injection analysis (FIA) system has been developed for on-line monitoring a process for conversion of biomass under field condition. The instrument consists of a newly designed biosensor for easy renewal of the bio-receptor without disassembling the sensor, a FIA controller for controlling the analysis operations, and a computer-based data acquisition system for data recording and processing. The instrument performed a sequence operations automatically including preparation of sample in the desired concentration, sample loading, sample injection, signal recording, data processing, and self-cleaning of the system. This makes the instrument being an interesting and promising device for on-line process monitoring.     

Production of fungal alpha-amylase by Saccharomyces kluyveri in glucose-limited cultivations

Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.     

Growth and enzyme production by three Penicillium species on monosaccharides

The growth and preference for utilisation of various sugar by the Penicillium species Penicillium pinophilum IBT 4186, Penicillium persicinum IBT 13226 and Penicillium brasilianum IBT 20888 was studied in batch cultivations using various monosaccharides as carbon source, either alone or in mixtures. P. pinophilum IBT 4186 and P. persicinum IBT 13226 had a micro(max) around 0.08-0.09 h(-1) using either glucose or xylose as carbon source. The micro(max) of P. brasilianum IBT 20888 was 0.16 and 0.14 h(-1) on glucose and xylose, respectively. Glucose was found to exert repression on the catabolism of mannose, galactose, xylose and arabinose. The three species were able to utilise all the tested monosaccharides, but arabinose was only slowly metabolised. Glucose was also found to repress the production of endoglucanases, endoxylanases and beta-xylosidases. After glucose depletion, the fungi started producing beta-glucosidase and endoglucanases. Xylose did not repress the enzyme production and it induced the production of endoxylanases and beta-xylosidases.     

Rat intestinal ceramidase: purification, properties, and physiological relevance

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of approximately 116 kDa. The enzyme acts on both [14)]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249-262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.     

Induction of homologous recombination in the hprt gene of V79 Chinese hamster cells in response to low- and high-LET irradiation

Dense ionization tracks from high linear energy transfer (LET) radiations form multiple damaged sites (MDS), which involve several types of DNA lesions in close vicinity. The primary DNA damage triggers sensor proteins that activate repair processes, cell cycle control or eventually apoptosis in subsequent cellular responses. The question how homologous recombination (HR) and non-homologous end joining (NHEJ) interact in the repair of radiation-induced DNA damage of MDS type has been addressed in different model systems but several questions remain to be answered. We have therefore challenged cells with treatments of ionizing radiation of different qualities to investigate whether primary DNA damages of different complexity are reflected in the processes of repair by HR as well as cell survival. We used the V79 derived SPD8 cell line to determine the induction of HR in the hprt exon 7 and clonogenic assay for survival in response to radiation. SPD8 cells were irradiated with gamma-rays (137Cs 0.5 keV/microm), boron ions (40 and 80 keV/microm) and nitrogen ions (140 keV/microm), with doses up to 5 Gy. Analysis of clonogenic survival showed that B-ions (80 keV/microm) and N-ions were more toxic than gamma-rays, 4.1 and 5.0 times respectively, while B-ions at 40 keV/microm were 2.0 times as toxic as gamma-rays. Homologous recombination in the cells exposed to B-ions (80 keV/microm) increased 2.9 times, a significant response as compared to cells exposed to gamma-rays, while for B-ions (40 keV/microm) and N-ions a nonsignificant increase in HR of 1.2 and 1.4, respectively, was observed. We hypothesize that the high-LET generated formation of MDS is responsible for the enhanced cytotoxicity as well as for the mobilization of the HR machinery.     

L’automatisation des sérologies auto-immunes

Pendant longtemps les analyses d’auto-immunité ont été réalisées de fa?on manuelle, en utilisant des techniques parfois artisanales. Plusieurs facteurs conduisent aujourd’hui les laboratoires à automatiser ces examens.     

Frequency analysis of atrial fibrillation from the surface electrocardiogram

Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice. Neither the natural history of AF nor its response to therapy are sufficiently predictable by clinical and echocardiographic parameters. Atrial fibrillatory frequency (or rate) can reliably be assessed from the surface electrocardiogram (ECG) using digital signal processing (filtering, subtraction of averaged QRST complexes, and power spectral analysis) and shows large inter-individual variability. This measurement correlates well with intraatrial cycle length, a parameter which appears to have primary importance in AF domestication and response to therapy. AF with a low fibrillatory rate is more likely to terminate spontaneously, and responds better to antiarrhythmic drugs or cardioversion while high rate AF is more often persistent and refractory to therapy. In conclusion, frequency analysis of AF seems to be useful for non-invasive assessment of electrical remodeling in AF and may subsequently be helpful for guiding AF therapy.     

Major histocompatibility complex and mate choice in sand lizards

In mice and man, females prefer males with a major histocompatibility complex (MHC) genotype different to their own. We tested whether this phenomenon also occurs in the Swedish sand lizard (Lacerta agilis). Females in a laboratory experiment preferred to associate with odour samples obtained from more distantly related males at the MHC class 1 loci. Data on free-ranging lizards suggest that associations between males and females are nonrandom with respect to MHC genotype. However, male spatial distribution and mobility during the mating season suggest that the non-random pairing process in the wild may also be driven by corresponding genetic benefits to males pairing with less related females.     

Prenatal exposure to testosterone increases ectoparasite susceptibility in the common lizard (Lacerta vivipara)

High levels of testosterone can benefit individual fitness, for example by increasing growth rate or ornament size, which may result in increased reproductive success. However, testosterone induces costs, such as a suppressed immune system, thereby generating trade-offs between growth or mate acquisition, and immunity. In birds and reptiles, females allocate steroids to their eggs, which may be a mechanism whereby females can influence the phenotype of their offspring. To our knowledge, only the benefits of early androgen exposure have been experimentally investigated to date. However, to understand this phenomenon, the costs also need to be evaluated. We manipulated testosterone levels in eggs of the viviparous common lizard and monitored growth, endurance and post-parturient responses to ectoparasites of the offspring. Testosterone-treated individuals had significantly higher growth rates than controls, but suffered a significant decrease in growth rate when exposed to ticks, whereas the corresponding difference for controls was non-significant. There was no difference in observed parasite load or leucocyte count between manipulated and control offspring. Thus, our results suggest that high testosterone levels during embryonic development have detrimental effects on immune function resulting in reduced growth rate, and that this must be taken into consideration when evaluating the potential adaptive value of maternal androgen allocation to eggs.     

Suppressive DNA vaccination in myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis involves a T1-biased immune response

Vaccination with DNA encoding a myelin basic protein peptide suppresses Lewis rat experimental autoimmune encephalomyelitis (EAE) induced with the same peptide. Additional myelin proteins, such as myelin oligodendrocyte glycoprotein (MOG), may be important in multiple sclerosis. Here we demonstrate that DNA vaccination also suppresses MOG peptide-induced EAE. MOG(91-108) is encephalitogenic in DA rats and MHC-congenic LEW.1AV1 (RT1(av1)) and LEW.1N (RT1(n)) rats. We examined the effects of DNA vaccines encoding MOG(91-108) in tandem, with or without targeting of the hybrid gene product to IgG. In all investigated rat strains DNA vaccination suppressed clinical signs of EAE. There was no requirement for targeting the gene product to IgG, but T1-promoting CpG DNA motifs in the plasmid backbone of the construct were necessary for efficient DNA vaccination, similar to the case in DNA vaccination in myelin basic protein-induced EAE. We failed to detect any effects on ex vivo MOG-peptide-induced IFN-gamma, TNF-alpha, IL-6, IL-4, IL-10, and brain-derived neurotropic factor expression in splenocytes or CNS-derived lymphocytes. In CNS-derived lymphocytes, Fas ligand expression was down-regulated in DNA-vaccinated rats compared with controls. However, MOG-specific IgG2b responses were enhanced after DNA vaccination. The enhanced IgG2b responses together with the requirement for CpG DNA motifs in the vaccine suggest a protective mechanism involving induction of a T1-biased immune response.