Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

The human fibroblast growth factor receptor genes: a common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain.

To determine the mechanisms by which multiple forms of fibroblast growth factor (FGF) receptors are generated, we have mapped the arrangement of exons and introns in the human FGF receptor 1 (FGFR 1) gene (flg). We found three alternative exons encoding a portion of the third immunoglobulin (Ig)-like domain of the receptor. One of these alternatives encodes a sequence that is part of a secreted form of FGFR 1. The other two encode sequences that are likely part of transmembrane forms of FGFR 1. One of these forms has not been previously reported in published cDNAs. Also, we have determined the structural organization of a portion of the human FGFR 2 gene (bek) and found a similar arrangement of alternative exons for the third Ig-like domain. The arrangement of these genes suggests that there are conserved mechanisms governing the expression of secreted FGF receptors as well as the expression of at least two distinct membrane-spanning forms of the FGF receptors. The diverse forms appear to be generated by alternative splicing of mRNA and selective use of polyadenylation signals.     

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.     

Mapping sequenced E.coli genes by computer: software, strategies and examples.

Methods are presented for organizing and integrating DNA sequence data, restriction maps, and genetic maps for the same organism but from a variety of sources (databases, publications, personal communications). Proper software tools are essential for successful organization of such diverse data into an ordered, cohesive body of information, and a suite of novel software to support this endeavor is described. Though these tools automate much of the task, a variety of strategies is needed to cope with recalcitrant cases. We describe such strategies and illustrate their application with numerous examples. These strategies have allowed us to order, analyze, and display over one megabase of E. coli DNA sequence information. The integration task often exposes inconsistencies in the available data, perhaps caused by strain polymorphisms or human oversight, necessitating the application of sound biological judgment. The examples illustrate both the level of expertise required of the database curator and the knowledge gained as apparent inconsistencies are resolved. The software and mapping methods are applicable to the study of any genome for which a high resolution restriction map is available. They were developed to support a weakly coordinated sequencing effort involving many laboratories, but would also be useful for highly orchestrated sequencing projects.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Ribotyping of 16S and 23S rRNA genes and organization of rrn operons in members of the bacterial genera Gemmata,Planctomyces, Thermotoga,Thermus, and Verrucomicrobium

The number of organization of rrn genes of two members of the order Planctomycetales, Planctomyces limnophilus and Gemmata obscuriglobus, as well as three species from other bacterial phyla, namely Thermotoga maritima, Thermus aquaticus and Verrucomicrobium spinosum were examined by Southern blot hybridization analysis of restricted DNA with labeled 16S- and 23S rRNAs. Ribotyping analysis revealed that two species contain unlinked 16S- and 23S rRNA genes. Planctomyces limnophilus possessed two unlinked rrn genes which were separated from each other by at least 4.3 kb, and Thermus aquaticus had to unlinked 16S and 23S rRNA genes, separated from each other by at least 2.5 kb. Gemmata obscuriglobus exhibited five genes for which the organization could as yet not be determined because of the complex hybridization patterns. In the other two species, rrn genes clustered in operons. Thermotoga maritima had a single gene for each rRNA species which were separated by not more than 1.5 kb, while Verrucomicrobium spinosum had four copies of probably linked 16S and 23S rRNA genes with a maximal distance between 16S and 23S rRNA genes of 1.3 kb.     

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.     

The course control system of beetles walking in an air-current field

The wind-orientated walk of carrion beetles Necrophorus humator F. was analysed under closed-loop conditions with a walking compensator and under openloop conditions with a paired tread wheel (Fig. 1).