Twelve loci form a continuous linkage map for human chromosome 18

We have constructed a primary genetic map of human chromosome 18 consisting of 11 DNA markers and one serological marker (JK). Two of these loci define highly polymorphic VNTR systems. The markers define a continuous genetic linkage map of 97 cM in males and 205 cM in females; female genetic distances in a panel of 59 three-generation families were consistently about twice those observed in males. The high odds in support of the linear order of the markers on this recombination map, and the extent of coverage of chromosome 18, indicate that this map will permit efficient linkage studies of human genetic diseases that may be segregating on chromosome 18 and will provide anchor points for development of high-resolution maps for this chromosome.     

Microbial transformation of heterocyclic molecules in deep subsurface sediments

Recently attempts have been made to establish the presence and to determine the metabolic versatility of microorganisms in the terrestrial deep subsurface at the Savannah River Plant, Aiken, SC, USA. Sediment samples obtained at 20 different depths of up to 526 m were examined to determine carbon mineralization under aerobic, sulfate-reducing, and methanogenic conditions. The evolution of¹⁴CO₂ from radiolabelled glucose was observed under aerobic conditions in all sediments, whereas pyridine was transformed in 50% of the 20 sediments and indole was metabolized in 85% of the sediments. Glucose mineralization in certain sediments was comparable to that in the surface environment. Sulfate was reduced in only five sediments, and two were carbon limited. Methane production was detected in ten sediments amended with formate only after long-term incubations. The transformation of indole and pyridine was only rarely observed under sulfate-reducing conditions and was never detected in methanogenic incubations. This study provides information concerning the metabolic capability of both aerobic and anaerobic microorganisms in the deep subsurface and may prove useful in determining the feasibility of microbial decontamination of such environments.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Hidden smectic properties of a chiral side-chain co-oligomer

We recently showed that a side-chain industrial co-oligosiloxane presents a quenchable enlarged blue phase behaviour at the cholesteric-isotropic phase transition. In this paper, we present the results of a structural study based on X-ray diffraction, differential scanning calorimetry and optical measurements. In particular, the smectic A organisation is demonstrated in the lower temperature domain, which was hitherto understood as a cholesteric phase. A structural model for this phase is proposed on the basis of the analysis of the anisotropic scattering of stretched fibers. Our results also suggest that the observed glass transition is indeed a rather complex phenomenon, which seems to involve not only the freezing of the main chains, but also smectic correlations at the side-chain level. Moreover, the calorimetric study indicates that, notwithstanding the conservation of the processed film's optical properties, low kinetic reorganisations occur at room temperature.     

Ionic transporting systems of skeletal muscle in relation with innervation and their involvement in myotonic diseases

Excitation-contraction coupling describes the series of events that begins with propagated action potential on the muscle fiber surface membrane and leads to the twitch contraction of the fiber. The generation of an action potential during excitation requires rapid sequential changes in membrane conductances of Na⁺, Ca²⁺, and K⁺ ions that depend upon the opening and closing of the respective channels. Myotonic disorders are inherited diseases whose clinical manifestations include electrophysiological signs such as increased excitability and delayed relaxation of the muscles after voluntary contraction. All these disorders appears to be due to an abnormality of the muscle itself since they persist after section or blocking of the motor nerve after curarization. Most experimental and clinical data suggest that human myotonia arises from genetically-induced structural and functional alterations of the muscle membrane. The purpose of this article is to focus on the more recent developments in the molecular and pharmacological analysis of cation transporting systems such as ionic channels and (Na⁺, K⁺) ATPase in myotonic disorders.Special issue dedicated to Dr. Lawrence Austin.     

Biochemical and pharmacologic properties of the Ca2+ channel of skeletal muscle: appearance during myogenesis and the relation between its structure and function

Two distinct and interdependent binding sites for inhibitors of voltage-dependent Ca2+ channels have been identified. They include one site for molecules of the 1,4-dihydropyridine serie such as nitrendipine, nifedipine or PN200-110 and one site for a chemically heterogenous group of compounds comprising verapamil, D600 and desmethoxyverapamil, bepridil and diltiazem. Ca2+ binds to its own coordination site which is distinct from the receptor site for organic Ca2+ channel inhibitors. The molecular size of the native [3H] nitrendipine receptor of transverse tubule membrane, brain and heart, have been determined using the radiation inactivation technique. The [3H] nitrendipine receptor is found to have a Mr of 210,000 +/- 20,000. CHAPS solubilization and purification indicate that the dihydropyridine receptor contains polypeptides of apparent molecular weights of 142,000, 32,000 and 33,000 which copurifie with (+) [3H] PN200-110 binding activity. Two stages in which there is an increased binding of [3H]nitrendipine have been observed during chick myogenesis. The first one occurs during embryonic life and has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors. This increase is accompanied by a decrease in the affinity of nitrendipine for its receptor by a factor of 4 to 10. The second stage of development is partly under innervation control and its expression is modulated by the intracellular cyclic AMP content. The two dihydropyridines Bay K8644 and CGP 28932 work preferentially on polarized membranes. 45Ca2+ flux experiments yielded results which are in good agreement with electrophysiological, contraction and binding data obtained with rat cardiac cells and skeletal muscle cells.     

In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.     

A search for an 'ouabain-like' substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids

The electric organ of Electrophorus electricus contains substances which inhibit (Na+ + K+)-ATPase activity, the specific binding of [3H]ouabain to purified (Na+ + K+)-ATPase and 86Rb+ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous 'ouabain-like' activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.