Enzyme sensor-FIA-system for on-line monitoring of glucose, lactate and glutamine in animal cell cultures.

Enzyme sensors for glucose, lactate and glutamine were connected via flow-injection analysis (FIA) devices to two different bioprocesses. They were used for on-line process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

An expression vector inhibits gene expression in Xenopus embryos by antisense RNA

Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.     

Enzyme activity in organic solvent as a function of water activity determined by membrane inlet mass spectrometry

Summary Membrane inlet mass spectrometry (MIMS) is introduced as a method for measuring water activity in nonpolar solvents, aqueous solutions and gas phase. The determination of the rate of hydrolysis of diphenyl carbonate by porcine liver esterase as a function of water activity in diisopropyl ether is presented as an example. A linear relationship is found between the enzyme activity and the water activity.     

Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

Thermogenic effect of adrenaline: interaction with insulin

The contribution of insulin (3.6 pmol.kg body mass-1.min-1) to adrenaline-induced (0.164 nmol.kg fat free mass-1.min-1) thermogenesis was studied in ten postabsorptive healthy volunteers using two sequential protocols. Variables considered were oxygen consumption as well as carbon dioxide production, heart rate, blood pressure, plasma concentrations of glucose, insulin, glycerol, free fatty acids, beta-HO-butyrate and lactate. Adrenaline increased plasma concentrations of glucose, glycerol, free fatty acids, and beta-HO-butyrate, and heart rate and metabolic rate during normo-insulinaemia [61.3 (SEM 6.6) pmol.l-1]. Similar effects were observed during hyperinsulinaemia [167.9 (SEM 18.7) pmol.l-1], but the effect of adrenaline on oxygen consumption was reduced. On average, metabolic rate increased by 12.9% during normo-insulinaemia and by 8.9% during hyperinsulinaemia. We concluded that relative hyperinsulinaemia resulted in decreased adrenaline-induced thermogenesis and therefore increased whole body anabolism.     

Inhibition of rat yolk sac tumour growth in vivo by a monoclonal antibody to the retroviral molecule P15E

Summary A monoclonal mouse IgG2b antibody 19F8, directed towards a determinant on the retroviral transmembranous molecule p15E, binds selectively to certain rat tumours, including all tested yolk sac tumours, one rat colon carcinoma, one spontaneous kidney carcinoma and an adenovirus-type-9-induced rat breast tumour, as tested by antibody-dependent cellular cytotoxicity (ADCC) and immunohistochemistry. Groups of rats receiving yolk sac tumour F56 isografts intraperitoneally (i.p.) or subperitoneally (s.p.) were treated with this monoclonal antibody (mAb), 19F8, inoculated twice a week in doses of 100 µg. Parallel control groups received analogous inoculations of an isotype-matched monoclonal antibody. A significant growth inhibitory effect was observed with 19F8. In 5/10 rats isografted i.p., tumour outgrowth was completely inhibited and in the other 5 rats the outgrowth was delayed compared to the 10 rats in the control group, which all developed tumours. All rats of the control group developed large volumes of ascites, whereas the 5 rats in the therapy group with eventual tumour outgrowth had little or no ascites. In two experiments with rats carrying subperitoneal isografts and treated with the 19F8 mAb, tumour grew out in 4/5 and 5/10 rats, though growth was delayed compared to the control groups, in which 5/5 and 9/9 rats developed tumours. The tumours grew significantly more slowly in the therapy groups compared to the controls. All rats that developed tumours in the therapy groups showed an anti-idiotypic response against mAb 19F8. The single tumour-free rat in the first experiment and 1/5 tumour-free rats in the other showed no such response. The draining lymph node cells from the tumour-free animals showed a specific proliferative response to yolk sac tumour F56 cells in a mixed lymphocyte tumour cell culture (MLTC), and the MLTC-induced cells were cytotoxic to F56 but not to the natural-killer-sensitive rat T cell lymphoma G1—Tc1. The cytotoxic cell population was more than 90% CD4⁺. It is concluded that the two test systems for identification of the epitope of p15E detected by mAb 19F8 correlated well in detection of the epitope in the cells (immunohistochemistry) and at the cell surface (ADCC). It is also concluded that mAb 19F8 has a growth-inhibitory effect on yolk sac tumour F56 and that, as a result of the treatment, T cells with specificity for F56 are appearing in draining lymph nodes of tumour-free animals.     

Assignment of the gene coding for human cytochrome c oxidase subunit VIb to chromosome 19, band q13.1, by fluorescence in situ hybridisation

Summary A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit VIb gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.     

Restricted range of ocular accommodation in barn owls (Aves:Tytonidae)

Summary In an examination of the focusing abilities of 15 species of owls, the North American barn owl, Tyto alba pratincola (Bonaparte 1838), was an outstanding accommodator, having a range of accommodation exceeding 10 diopters (Murphy and Howland 1983). Using comparable methods, we examined the accommodation of 4 specimens of the Australian barn owl, Tyto alba delicatula (Gould 1837). We failed to elicit accommodation greater than two diopters, and most stimuli failed to evoke any discernable accommodation at all. Furthermore, examination of other Australian tytonid owls, the grass owl, T. longimembris, the sooty owl, T. tenebricosa, and both the mainland and Tasmanian subspecies of the masked owl, T. novaehollandiae novaehollandiae and T. novaehollandiae castanops, also failed to reveal anything but very moderate accommodative ranges. We conclude that the outstanding accommodative ability of the American barn owl is truly an exception to the modest accommodative abilities of the tytonid owls generally.