dsg, a gene required for cell-cell interaction early in Myxococcus development.

dsg mutants of Myxococcus xanthus are conditionally defective in fruiting body development, including sporulation. Unable to develop on their own, these mutants can assemble fruiting bodies with spores if they are mixed with wild-type cells. To elucidate the developmental defect in dsg mutants by close comparison with wild type, such mutants have been backcrossed by transduction, using a closely linked insertion of transposon Tn5 for selection. Backcrossed dsg mutants form aggregates that are larger, less compact, and less symmetrical than dsg+ fruiting bodies. Also, the starvation-induced sporulation in dsg aggregates is delayed and reduced. However, dsg mutants can be induced by glycerol or dimethyl sulfoxide to sporulate at levels approaching those of wild type. dsg mutants may thus have a primary defect early in development which diminishes their capacity to aggregate and which indirectly decreases the number of fruiting body spores. The linked insertion of Tn5 also facilitated cloning the dsg gene. The cloned dsg+ allele was shown to be dominant to both the dsg-429 and dsg-439 alleles, and both mutant alleles were shown to belong to the same genetic complementation group. Subcloning of restriction fragments, deletions, and insertions of transposon Tn5 agree in locating the dsg gene to an 850-base-pair segment of the cloned region.     

Structure-function analysis of plasma cholesteryl ester transfer protein by protease digestion and expression of cDNA fragments in Escherichia coli

In an attempt to define an active domain of the protein, fragments of cholesteryl ester transfer protein (CETP) were obtained by limited digestion of the native, plasma-derived protein with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease or by expression of CETP cDNA restriction fragments in Escherichia coli. Although digestion of native CETP with these proteases resulted in extensive fragmentation of the protein and loss of the intact 74-kDa molecule as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CE transfer activity was unaffected (trypsin or chymotrypsin treatment) or only partially lost (V8 protease treatment). Analysis by molecular sieve chromatography showed that the CE transfer-active product of this proteolysis consisted of polypeptide fragments which remained associated, retaining the native molecular weight of CETP. These proteolyzed complexes were resistant to dissociation by dithiothreitol, 8 M urea, or delipidating agents. As shown by CE transfer activity, native CETP was found to possess a stable conformation which remained unchanged in buffers containing up to 4.5 M urea, or following exposure to even higher (8 M) urea concentrations. CETP polypeptides from bacterially expressed cDNA fragments were found to be catalytically inactive although they contained the epitope for an inhibitory anti-CETP monoclonal antibody and had emulsion binding properties similar to native CETP. Selected synthetic CETP peptides (including the peptide containing the inhibitory monoclonal antibody epitope) were also devoid of CE transfer activity. Thus, no evidence was found for an independently active subunit of the CETP. Together, the results indicate that the CETP possesses a distinct and highly stable tertiary structure which is required for CE transfer catalytic activity.     

The use of monoclonal antibodies to localize the low density lipoprotein receptor-binding domain of apolipoprotein B

Human apolipoprotein (apo) B-100 is composed of 4536 amino acids. It is thought that the binding of apoB to the low density lipoprotein (LDL) receptor involves an interaction between basic amino acids of the ligand and acidic residues of the receptor. Three alternative models have been proposed to describe this interaction: 1) a single region of apoB is involved in receptor binding; 2) groups of basic amino acids from throughout the apoB primary structure act in concert in apoB receptor binding; and 3) apoB contains multiple independent binding regions. We have found that monoclonal antibodies (Mabs) specific for a region that spans a thrombin cleavage site at apoB residue 3249 (T2/T3 junction) totally blocked LDL binding to the LDL receptor. Mabs specific for epitopes outside this region had either no or partial ability to block LDL binding. In order to define the region of apoB directly involved in the interaction with the LDL receptor we have tested 22 different Mabs for their ability to bind to LDL already fixed to the receptor. A Mab specific for an epitope situated between residues 2835 and 2922 could bind to its epitope on LDL fixed to its receptor whereas a second epitope between residues 2980 and 3084 is inaccessible on receptor-bound LDL. A series of epitopes near residue 3500 of apoB is totally inaccessible, and another situated between residues 4027 and 4081 is poorly accessible on receptor-bound LDL. In contrast, an epitope that is situated between residues 4154 and 4189 is fully exposed. Mabs specific for epitopes upstream and downstream of the region 3000-4000 can bind to receptor-bound LDL with a stoichiometry close to unity. Our results strongly suggest that the unique region of apoB directly involved in the LDL-receptor interaction is that of the T2/T3 junction.     

Separation and characterization of isoforms of DT-diaphorase from rat liver cytosol.

Rats were treated with 3-methylcholanthrene (MC) and DT-diaphorase from liver was partially purified on an azodicoumarol-Sepharose 6B column and applied to an FPLC-chromatofocusing column in order to resolve isoforms. Six peaks showing significant DT-diaphorase activity were eluted from this column with a pH gradient between 7.30 to 4.80. The amino acid compositions of the two major peaks (II and VIb) were found to be nearly identical, suggesting existence of isoforms rather than isozymes of DT-diaphorase. The isoforms of DT-diaphorase showed broad substrate specificities towards four different quinones (menadione, vitamin K-1, benzo(a)pyrene 3,6-quinone and cyclized-dopamine ortho-quinone), although quantitative differences in the specific activities were also found. All isoforms are glycoproteins but contain different carbohydrates. Thus isoform II reacts with biotinylated lectins which are specific for N-acetylgalactosamine, mannose, fucose and galactosyl(beta-1,3)N-acetylgalactosamine, while isoform VIb reacts only with biotinylated lectins specific for mannose and N-acetylgalactosamine. Separation of DT-diaphorase isoforms from control rat liver cytosol using FPLC-chromatofocusing revealed that the induction of the isoforms is not uniform, since isform II was not found and the major isoform was composed of three peaks, whereas the major isoform of DT-diaphorase from liver cytosol of rats treated with 3-methylcholanthrene was composed of only two peaks.     

Lipopolysaccharide-Enhanced Expression of Interleukin-6 in Dibutyryl Cyclic AMP-Differentiated Rat C6 Glioma

Abstract: Rat C6 glioma synthesizes a low basal level of interleukin-6 (IL-6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 m M N ⁶, O ^2'-dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP-differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL-6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 m M dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL-6 mRNA concentration and IL-6 immunoreactivity, whereas titration of the biological activity indicated a 100-fold increase in the secreted IL-6 activity. The enhanced secretion of IL-6 is correlated with the induction of differentiation. Chromatography on heparin-Sepharose and on DEAE-5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL-6-like activities could be neutralized by an anti-murine IL-6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.     

EFFECTS ON FITNESS COMPONENTS OF P-ELEMENT INSERTS IN DROSOPHILA MELANOGASTER: ANALYSIS OF TRADE-OFFS

We analyzed the trade-offs between fitness components detected in four experiments in which traits were manipulated by inserting small (control) and large (treatment) P-elements into the Drosophila melanogaster genome. Treatment effects and the interactions of treatment with temperature, experiment, and line were caused by the greater length and different positions of the treatment insert. In inbred flies, the treatment decreased early and total fecundity. Whether it increased the lifespan of mated females depended upon adult density. Analysis of line-by-treatment-by-temperature interactions revealed hidden trade-offs that would have been missed by other methods. They included a significant trade-off between lifespan and early fecundity. At 25°C high early fecundity was associated with decreased reproductive rates and increased mortality rates 10–15 days later and persisting throughout life, but not at 29.5°C. Correlations with Gompertz coefficients suggested that flies that were heavier at eclosion also aged more slowly and that flies that aged more slowly had higher fecundity late in life at 25°C. The results support the view that lifespan trades off with fecundity and that late fecundity trades off with rate of aging in fruitflies. Genetic engineering is an independent method for the analysis of trade-offs that complements selection experiments.     

Nature and occurrence of sulfoxidizing bacteria in Barégine developing in sulfurated thermal waters at Barèges (France)

An Attempt is made to define the nature of the barégine, a whitish mucilaginous complex found in the thermal waters of the village of Barèges, Hautes-Pyrénées, France, and the organisms responsible for its formation. In sulfurated waters, a Thiobacterium-like micro-organism produces this substance. It uses reduced sulfur as a source of energy. Filamentous species of Thiothrix, often indicated as the main element of barégine, are only part of an accompanying flora in this medium.     

Evolutionary plasticity in prokaryotes: A panglossian view

Enzyme directed genetic mechanisms causing random DNA sequence alterations are ubiquitous in both eukaryotes and prokaryotes. A number of molecular geneticist have invoked adaptation through natural selection to account for this fact, however, alternative explanations have also flourished. The population geneticist G.C. Williams has dismissed the possibility of selection for mutator activity on a priori grounds. In this paper, I attempt a refutation of Williams' argument. In addition, I discuss some conceptual problems related to recent claims made by microbiologists on the adaptiveness of molecular variety generators in the evolution of prokaryotes. A distinction is proposed between selection for mutations caused by a mutator activity and selection for the mutator activity proper. The latter requires a concept of fitness different from the one commonly used in microbiology.     

Acquisition and assimilation of gaseous ammonia as revealed by intracellular pH changes in leaves of higher plants

Atmospheric ammonia (NH₃) from various anthropogenic sources has become a serious problem for natural vegetation. Ammonia not only causes changes in plant nitrogen metabolism, but also affects the acid-base balance of plants. Using the pH-sensitive fluorescent dyes pyranine and esculin, cytosolic and vacuolar pH changes were measured in leaves of C₃ and C₄ plants exposed for brief periods to concentrations of NH₃ in air ranging from 1.33 to 8.29 mol NH₃ · mol^-1 gas (0.94–5.86 mg · m^-3). After a lag phase, uptake of NH₃ from air at a rate of 200 nmol NH₃ · m ^{- 2} leaf area · s^{- 1} into leaves of Zea mays L. increased pyranine fluorescence indicating cytosolic alkalinisation. The increase was much larger in the dark than in the light. In illuminated leaves of the C₃ plant Pelargonium zonale L. and the C₄ plants Z. mays and Amaranthus caudatus L., NH₃-dependent cytosolic alkalinisation was particularly pronounced when CO₂ was supplied at very low levels (16 or 20 mol CO₂ · mol^{- 1} gas, containing 210 mmol O₂ · mol^{- 1} gas). An increase in esculin fluorescence, which was smaller than that of pyranine, was indicative of trapping of some of the NH₃ in the vacuoles of leaves of Spinacia oleracea L. and Z. mays. Photosynthesis and transpiration remained unchanged during exposure of illuminated leaves to NH₃, yielding an influx of 200 nmol NH₃ · m^-2 leaf area · s^-1 for up to 30 min, the longest exposure time used. Both CO₂ and O₂ influenced the extent of cytosolic alkalinisation. At 500 mol CO₂ · mol^-1 gas the cytosolic alkalinisation was suppressed more than at 16 or 20 mol CO₂ · mol^-1 gas. The suppressing effect of CO₂ on the NH₃induced alkalinisation was larger in illuminated leaves of the C₄ plants Z. mays and A. caudatus than in leaves of the C₃ plant P. zonale. A reduction of the O₂ concentration from 210 to 10 mmol O₂ · mol ^-1 gas, which inhibits photorespiration, increased the NH₃induced cytosolic alkalinisation in C₃ plants. Suppression by CO₂ or O₂ of the alkaline pH shift caused by the dissolution and protonation of NH₃ in queous leaf compartments, and possibly by the production of organic compounds synthesised from atmospheric NH₃, indicates that NH₃ which enters leaves is rapidly assimilated if photosynthesis or photorespiration provide nitrogen acceptor molecules.This work was supported by the Biotechnology and Biological Sciences Research Council and the Deutsche Forschungsgemein-schaft within the framework of the research of Sonderforschun-gsbreich 251 of the University of Würzburg. We are grateful to Dr. B. Wollenweber (The Royal Veterinary and Agricultural University, Denmark) for discussions.