Construction and characterization of a region-specific microdissection library from human chromosome 2q35-q37.

A region-specific genomic library for human chromosome 2q35-q37 has been constructed using the microdissection and polymerase chain reaction-mediated linker-adaptor microcloning method. Twenty fragments from the chromosome region 2q35-q37 were dissected and a library consisting of 20,000 recombinant microclones was obtained. The insert size ranged between 50 and 800 bp, with a mean of approximately 270 bp. About 50-60% of the microclones contained unique sequences. The microdissection library has been demonstrated to derive from the dissected region 2q35-q37 by chromosome painting using the fluorescence in situ hybridization (FISH) technique. Southern blot analysis of the unique sequence microclones from the library showed that 54% (26/48) of the clones are of human origin and chromosome 2 specific. Four of these microclones have been further mapped to the 2q37 region by using a cell hybrid containing only 2q37. The unique sequence microclones have also been characterized for their insert size and the hybridizing genomic fragments cleaved with HindIII. As shown previously, these microclones will be useful in isolating corresponding yeast artificial chromosome (YAC) clones with large inserts for high-resolution physical mapping and also in screening cDNA libraries to isolate expressed gene sequences as candidate genes to facilitate search for the crucial genes underlying genetic diseases and specific forms of cancer assigned to the region.     

Induction of intercellular communications in epithelial cell cultures

A popular criterion of cell-cell communication in tissue cultures is dye coupling: the ability of the injected fluorescent dye of low molecular weight to be transferred from one cell to another. We report about a new factor which induces cell-to-cell dye coupling in previously uncoupled epithelial sheets. Paradoxically it is the standard fluorescent microscopy itself (that is, blue light of 320- to 480-nm wavelength) which induces rapid morphological alterations of cell culture followed by the transfer of fluorescent dye from one cell to another. Thus monitoring cell-cell dye coupling by fluorescent microscopy may itself induce the dye coupling in previously uncoupled epithelial cells.     

Percottus glehni Dybowski — a new colonizer in the ichthyofauna of Lake Glubokoe

Percottus glehni is a new colonizer in the ichthyofauna of Lake Glubokoe. Recently, this species, established in the European part of the USSR nearly 30 years ago, has become common in the lakes and ponds located 3–5 km from Lake Glubokoe. In Lake Glubokoe it keeps mainly to water thyme (Elodea) in shallow water with individuals up to 7 cm in size being predominant. Apparently, these fishes are not abundant due to strong predation pressure from larger predators.     

Interaction of histones with estrogens. Covalent adduct formation with 16 alpha-hydroxyestrone

Disturbed estrogen metabolism leading to increased 16 alpha-hydroxyestrone (16 alpha-OHE) has been described in patients with systemic lupus erythematosus and mammary carcinoma. Previous studies showed the formation of covalent complexes between 16 alpha-OHE and nonspecific cellular membrane proteins. The present study is concerned with the interaction of 16 alpha-OHE and histones. Covalent adduct formation between 16 alpha-OHE and individual histones was maximal with H1 histone. Other endogenous estrogens such as estrone, estradiol, and estriol did not interact with histones and form covalent adducts, nor did they interfere with the interaction of 16 alpha-OHE with these nuclear proteins. The evidence supports that the adduct formation between 16 alpha-OHE and histones proceeds via a stabilized Schiff base and subsequent rearrangement. This adduct formation which may have in vivo analogues may represent a mechanism for cellular transformation by this estrogen metabolite.     

A Yersinia enterocolitica serotype 0:3 lipopolysaccharide-specific monoclonal antibody reacts more strongly with bacteria cultured at room temperature than those cultured at 37 degrees C

It has been suggested that the O-side chains of the lipopolysaccharide (LPS) of serotype 0:3 strains of Yersinia enterocolitica vary quantitatively and qualitatively depending on whether they are cultured at 37 degrees C or 25 degrees C. It is uncertain whether this affects the expression of the serotype-specific antigens that are probably carried on the LPS. We studied this question with a serotype 0:3-specific monoclonal antibody, 2C1. This monoclonal antibody immunoprecipitated a 39K major protein from detergent-solubilized 125I-labeled Yersinia preparation. However, results of Western blot experiments demonstrated that the antigens reactive with 2C1 were not actually the 39K protein but the O-side chains of the LPS. Significantly, reactivity could be detected whether the bacteria were cultured at room temperature or at 37 degrees C. However, absorption experiments confirmed that there were less accessible antigenic determinants on the 37 degrees C-LPS. The LPS preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. These SDS-PAGE profiles showed that less O-side chains were present in the 37 degrees C-LPS. Hence, the most likely explanation for our observation is that the 37 degrees C incubation condition induced a partial smooth to rough transition of the Yersinia LPS with a decrease in the amount of 2C1-reactive antigen.     

Characterization of purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26

A highly purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26 was isolated by a procedure involving Triton X-100 solubilization, calcium phosphate column chromatography, and ammonium sulfate fractionation. The purified enzyme complex contains, in nanomoles/mg of protein, cytochrome b, 8.3; cytochrome c1, 8.3; iron-sulfur protein, 15; phospholipids, 182; and ubiquinone, 5. Four major polypeptides with apparent molecular weights of 48,000, 30,000, 24,000, and 12,000 were detected in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr = 48,000 and 30,000 proteins are cytochromes b and c1, respectively. The enzyme complex catalyzes electron transfer from ubiquinol to cytochrome c with a specific activity of 12.6 mumol of cytochrome c reduced per min/mg of protein at 23 degrees C. This is lower than that of the mitochondrial enzyme, although both systems have similar essential redox components and a similar Km for ubiquinol. The activity is fully sensitive to antimycin A and 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole. The enzyme complex is stable at neutral pH and at lower temperatures, but became less stable when the incubation temperature was raised. At 37 degrees C, the half-life is 15 min. The enzymatic activity was insensitive to treatment with N',N'-dicyclohexylcarbodiimide. No p-chloromercuriphenylsulfonate-alkylable sulfhydryl groups were detected. The major phospholipids associated with the purified enzyme complex are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol with molar per cent distributions of 25, 21, and 35, respectively. About 60% of the enzymatic activity was abolished upon treatment with phospholipase A2. The phospholipase A2-inactivated activity can be partially restored by the addition of EDTA followed with phospholipids prepared from either the cytochrome b-c1 complex of the same source or a mixture of phosphatidylglycerol and asolectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of stimulating the superior colliculus on motoneurons of the neck muscle in the cat

Postsynaptic potentials produced by stimulating three sites of the midbrain superior colliculus were examined in motoneurons innervating the sternocleidomastoid, the trapezius, and the platysma cervical muscles in anesthetized cats. Stimulating the ipsilateral colliculus produced EPSP in the motoneurons as well as action potentials with a latency of 1.5–3.5 msec, averaging 2.6 ± 0.1 msec. Stimulation of the contralateral colliculus evoked EPSP with a latency of 1.5–3.2 msec and averaging 2.1 ± 0.1 msec together with IPSP with latency ranging from 2.6 to 5.0 msec. It is postulated that these postsynaptic responses are both monosynpatic and bisynaptic in nature. This type of synaptic action is assumed to be one of the mechanisms responsible for coordinated head movements produced by tectofugal impulses.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 197–202, March–April, 1986.     

Spatial perception and vestibulomotor responses in man

A study was made on normal human subjects, using a stabilograph to investigate changes in posture produced in response to transcutaneous galvanic stimulation of the right labyrinth. Results were obtained for different head positions and under the illusion of head and trunk rotation produced by stimulating (vibrating) the gulteus maximus muscle. In the absence of illusion of movement, the direction of the vestibulomotor response was determined by the position of the head in relation to the feed: with the normal head position, the body swayed on a frontal plane, and on a sagittal plane when the heat turned through 90°. Vestibulomotor responses were sagittally oriented, as with real head turning, when illusory head and trunk turning through 90° was produced by vibration. When the illusion of head rotation (in relation to the feet) was not produced by this stimulus, the direction of the postural response was not produced by this stimulus, the direction of the postural response was determined by the real orientation of the head. It is concluded that the spatial perception system plays a major part in controlling spatially oriented vestibulomotor responses.Institute for Research into Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 6, pp. 779–787, November–December, 1986.     

Analysis of characters coded for by T-DNA using hybridization between tumorous and normal plant cells

In somatic hybrids between tumourous Nicotiana tabacum (B6S3) and normal mesophyll Atropa belladonna cells, the following traits, directly or indirectly connected with T-DNA gene expression and tumourous growth, were analysed: lysopine dehydrogenase activity (LpDH), shoot suppression, root suppression, ability to grow on media with D-lactose as a sole carbon source and resistance to 2-aminoethylcysteine, 5-bromodeoxyuridine and 5-methyltryptophan. Dominant (semidominant) expression was observed for all but one trait studied, e.g. shoot suppression which behaved as a recessive character.Abbreviations LS-H Linsmaier and Skoog (1965) hormone-free medium - NAA Naphtaleneacetic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - 5-BUdR 5-Bromodeoxyuridine - 2-AEC 2-Aminoethylcysteine - 5-MT 5-Methyltryptophan     

A modified method of UDS detection in vitro suitable for screening the DNA-damaging effects of chemicals

The UDS induced in cultured FL cells by exposure to chemicals was measured as hydroxyurea-resistant incorporation of 3H-TdR in the acid-insoluble fraction of the 14C-TdR-prelabelled cells synchronized by the combination of arginine starvation and pretreatment with hydroxyurea. The level of UDS is represented by the ratios of 3H/14C radioactivities which are measures of specific activities of 3H. Two direct-acting alkylating agents, MMS and MNNG, a cross-linking agent, mitomycin C, and 3 procarcinogens, B(a)P, AFB1 and cyclophosphamide elicited UDS in the absence or presence of the liver-metabolizing system. Three chemicals of unknown carcinogenicity were also able to induce UDS in this assay system, i.e., bis-(O,O-diethylphosphinothioyl)-disulphide, 4-chlorophenoxy acetic acid (sodium salt) and caramelized malt sugar. With the exception of 4-chlorophenoxy acetic acid, they were also active in the Ames test.