Localization of a series of RNA-protein cross-link sites in the 23S and 5S ribosomal RNA from Escherichia coli, induced by treatment of 50S subunits with three different bifunctional reagents.

50S ribosomal subunits were reacted with bis-(2-chloroethyl)methylamine, 2-iminothiolane or methyl p-azidophenyl acetimidate, and RNA-protein cross-link sites on the RNA were localised using our published procedures. The degree of precision with which these sites could be determined was variable, depending on the particular protein or RNA region concerned. The following positions in the 23S RNA were identified as encompassing the individual cross-link sites (numbered from the 5'-end, with asterisks denoting sites previously reported): L1, 1864-67, 1876-78, 2119-33, 2163-72*, L2, 1819-20*; L3, 2832-34; L4, 320-25*; 613-17*; L5, 2307; L6, 2473-81*; L9, 1484-91; L11, 1060-62; L13, 547-50; L14, 1993-2002; L17, 1260-95; L18, 2307-20; L19, 1741-58; L21, 544-48*; 1198-1248; L23, 63-65, 137-41*; L24, 99-107*; L27, 2272-83, 2320-23*; 2332-37*; L28, 195-242, 368-424; L29, 101-02*; L30, 931-38; L32, 2878-90; L33, 2422-24. Cross-links to 5S RNA were observed with L5 (positions 34-41), and L18 (precise site not localised).     

Amino acid sequence of a (32P) phosphopeptide from pig liver pyruvate kinase phosphorylated by cyclic 3',5'-AMP-stimulated protein kinase and gamma-(32P)ATP

Pig liver pyruvate kinase (type L) was ³²P-labelled by incubation with (³²P)ATP and cyclic 3′,5′-AMP-stimulated protein kinase from the same source. One major (³²P)phosphopeptide was isolated from a peptic hydrolysate of the enzyme. Its amino acid sequence was Leu-Arg-Arg-Ala-(³²P)SerP-Leu.     

Characterization of the Dihydropyridine Binding Sites of Rat Neocortical Synaptosomes and Microvessels

The dihydropyridine binding sites associated with rat neocortical synaptosomes and microvessels were compared using an in vitro [3H]PN 200-110 [(+)-[methyl-3H]-isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxycarbonylpyridine-3-carboxylate] binding assay. Saturation experiments yielded similar KD values (approximately 70 pM) and Bmax values (approximately 400 fmol/mg of protein) for the two membrane preparations. Interaction experiments with [3H]PN 200-110 and various calcium-modulating substances provided further evidence for the practically identical nature of the synaptosomal and microvascular dihydropyridine binding sites. These findings predict that lipophilic dihydropyridines, simultaneously occupying the two central binding sites, have the dual effect of altering neuronal function and local blood flow.     

Localization of the rat immunoglobulin heavy chain locus to chromosome 6

We have previously used rat/mouse somatic cell hybrids to localize the rat c-myc gene to chromosome 7 (Sümegi et al. 1983) and the rat immunoglobulin kappa locus to chromosome 4 (Perlmann et al. 1985). We now report that by utilizing rat/mouse somatic cell hybrids, we have localized the rat immunoglobulin heavy chain locus to chromosome 6.     

Combined Determination of Poly-β-Hydroxyalkanoic and Cellular Fatty Acids in Starved Marine Bacteria and Sewage Sludge by Gas Chromatography with Flame Ionization or Mass Spectrometry Detection

Extraction of lipids from bacterial cells or sewage sludge samples followed by simple and rapid extraction procedures and room temperature esterification with pentafluorobenzylbromide allowed combined determinations of poly-β-hydroxyalkanoate constituents and fatty acids. Capillary gas chromatography and flame ionization or mass spectrometric detection was used. Flame ionization permitted determination with a coefficient of variation ranging from 10 to 27% at the picomolar level, whereas quantitative chemical ionization mass spectrometry afforded sensitivities for poly-β-hydroxyalkanoate constituuents in the attomolar range. The latter technique suggests the possibility of measuring such components in bacterial assemblies with as few as 10² cells. With the described technique using flame ionization detection, it was possible to study the rapid formation of poly-β-hydroxyalkanoate during feeding of a starved marine bacterium isolate with a complex medium or glucose and correlate the findings to changes in cell volumes. Mass spectrometric detection of short β-hydroxy acids in activated sewage sludge revealed the presence of 3-hydroxybutyric, 3-hydroxyhexanoic, and 3-hydroxyoctanoic acids in the relative proportions of 56, 5 and 39%, respectively. No odd-chain β-hydroxy acids were found.     

Mechanismen der pathologischen Pigmentbleichung bei Pflanzen

Viele pathologische Pigmentbleichungen sind oxidative Prozesse. In einer Reihe von Simulationsversuchen und Modellreaktionen wurden die Grundbedingungen für die Auslösung dieser Prozesse studiert und mit ?in vivo” Meßdaten verglichen. Bei den verwendeten oxidativen Bedingungen, unter denen Pigmente gebleicht werden, handelt es sich um chemische, enzymkatalysierte und lichtabhängige Oxidationen, wobei der Singulettsauerstoff, neben weiteren reaktiven Sauerstoffspezies eine herausragende Rolle spielt. Ausgelöst werden solche Pigmentbleichungen unabhängig davon, ob der ?aktivierte Sauerstoff” außerhalb oder innerhalb der Thylakoidmembranen erzeugt wird, wobei jedoch die zugrunde liegenden Mechanismen unterschiedlich sind. Parallel zur Pigmentbleichung wird grundsätzlich eine Oxidation von Lipiden gemessen; wird primär eine Lipidperoxidation induziert, so ist die Pigmentbleichung als Kooxidation von Lipiden zu sehen. Mit Hilfe der empfindlichen HPLC-Technik wurden Kinetiken der Bleichraten von Chlorophyll a, Chlorophyll b, β-Karotin, Neoxanthin, Violaxanthin und Lutein gemessen. Die schnellsten Bleichungsraten werden unter Bedingungen erzielt, die einen stark limitierten Elektronenfluß hervorrufen. Die erzielten Ergebnisse erlauben den Vergleich mit ?in vivo” Beobachtungen. Es können Rückschlüsse über die basalen Mechanismen der virus- bzw. pilzbedingten Vergilbung von Zuckerrüben sowie der Vergilbung von Fichtennadeln gezogen werden.     

Heterogeneity in antigenic expression and radiosensitivity in human colon carcinoma cell lines

Summary A panel of human colon carcinoma cell lines were characterized regarding both antigenic heterogeneity and variations in radiosensitivity. Monoclonal antibodies were used to study the expression of carcinoembryonic antigen (CEA), gastrointestinal cancer antigen (GICA or CA 19-9) and carcinoma-associated antigen (CA-50). Radiosensitivity was studied with the clonogenic survival technique. Three cell lines, LS 174T, HCTC, and SW 1116 stained positive for all three antigens. HT-29 was positive for CA 19-9 and CA-50 whereas Caco-2 was positive for CEA and CA 19-9. The cell lines SW 620 and LIM 1215 only stained positive for one of the antigens, CA-50 and CEA, respectively. In nearly all positive cases the stainings were very heterogeneous with mixtures of positive and negative cells. One exception was the HCTC cells which stained homogeneously for the CA 19-9 and CA-50 antigens. The neuroendocrinelike COLO 320 cells were negative in all cases. The radiosensitivity varied strongly between the cell lines with D_q-values between 0.8 and 1.9, extrapolation numbers between 2.0 and 4.7, D_o-values between 1.1 and 2.8. The surviving fraction at 2 Gy varied between 0.3 and 0.7 with HCTC as the most radiosensitive and HT-29 as the most radioresistant cell line. Thus, there were differences in antigenic expression and intrinsic radiosensitivity between the cell-lines and antigenic heterogeneities within each cell line. The analyzed panel of cell lines will be valuable in studies of dose-effect relations for monoclonal antibodies labeled with toxic radionuclides simulating both antigenic heterogeneity and variations in radiosensitivity.     

Production of nerve growth-stimulating factor(s) from chick embryo heart cells : Use of Cytodex 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.