Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus

Summary The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and antineurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon, GLP1 and neurotensin are shown to be colocalized in the EG-cells.     

Role of tumour necrosis factor in the tumour-necrotizing activity of agents with diverging toxicity

Summary We investigated the ability of various tumournecrotizing agents with diverging toxicity to induce tumour necrosis factor (TNF) and cytostatic activity inPropionibacterium-acnes-primed Swiss and tumour-bearing BALB/c mice, and the capacity of anti-TNF antibodies to inhibit induction of tumour necrosis by the agents. Lipid A and especially its combination with muramyl dipeptide induced high TNF levels in Swiss mice, as measured in the serum. Lower levels were induced by detoxified lipid A and the nontoxic dsRNA, polyadenylic polyuridylic acid, either alone or combined with muramyl dipeptide. The toxic agents also appeared the strongest inducers of mediators with cytostatic activity against cultured endothelial cells and MethA tumour cells. Anti-TNF antibodies partially reduced the cytostatic activity of the sera against MethA cells. Tumour-bearing BALB/c mice produced only low levels of TNF and cytostatic factors in response to all agents. Recombinant mouse TNF hardly reduced the DNA synthesis of MethA cells, unless normal mouse serum was added. Serum fromP.-acnes-treated Swiss mice and tumour-bearing BALB/c mice, that were inhibitory on their own, failed to potentiate the action of TNF. Serum from Swiss mice treated with toxic, but not detoxified, lipid A caused extensive tumour necrosis upon injection into MethA-bearing BALB/c mice. This activity was completely abolished by pre-incubation of the serum with anti-TNF. The tumour-necrotizing activity of the agents could be partially reduced by prior injection of these antibodies. Results show that the capacity of the agents to induce TNF and cytostatic activity is not related to their antitumour potential. Although TNF is likely to be a crucial mediator of the tumour-necrotizing action of the toxic as well as the nontoxic agents, it is probably not the sole mediator. Data also indicate that induction of tumour necrosis does not require induction of high and, thus toxic, TNF levels in the serum.     

The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Localization of the cellular retinoic acid binding protein (CRABP) gene relative to the acute promyelocytic leukemia-associated breakpoint on human chromosome 15

Summary A human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17) (q22;q11) positive acute promyelocytic leukemia (APL) was investigated. Although transposition of the CRABP gene could be demonstrated, we did not observe any gross CRABP rearrangement in a series of primary APL patients, nor in the acute myeloblastic leukemia cell line HL-60. Thus, the observed lack of CRABP expression in these leukemic cells may not be caused by disruption of its gene. CRABP maps to the region 15q22-qter.     

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.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Wide distribution of a 36-megadalton plasmid among clinical and environmental Spanish isolates ofLegionella pneumophila serogroup 1

With the eventual goal of characterizingLegionella pneumophila serogroup 1 plasmids at the molecular level, we have analyzed the plasmid contents of 78 clinical and environmental Spanish isolates. After selection of a suitable alkaline lysis method, we detected plasmids with approximate molecular weights of 25, 36, 40, 61, 80, 85, 90, and 95 megadalton (MDal). Several factors (i.e., wide temporal and geographic distribution, high frequency in both clinical and environmental isolates, and apparent high copy number after subculturing) make the 36 MDal type IA plasmid an appropriate plasmid for further molecular studies.     

Purification and properties ofl-serine dehydratase fromLactobacillus fermentum ATCC 14931

l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM l-cysteine in 50 mM phosphate buffer. M_r=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (M_r=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent K_m forl-serine was 65 mM. Fe⁺⁺ was required for the enzymatic activity, and the apparent K_m value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol^–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit.