Linkage data suggesting allelic heterogeneity for paramyotonia congenita and hyperkalemic periodic paralysis on chromosome 17

Summary Paramyotonia congenita (PC), an autosomal dominant non-progressive muscle disorder, is characterised by cold-induced stiffness followed by muscle weakness. The weakness is caused by a dysfunction of the sodium channel in muscle fibre. Parts of the gene coding for the -subunit of the sodium channel of the adult human skeletal muscle (SCN4A) have been localised on chromosome 17. To investigate the role of this gene in the etiology of PC, a linkage analysis in 17 well-defined families was carried out. The results (z=20.61, =0.001) show that the mutant gene responsible for the disorder is indeed tightly linked to the SCN4A gene. The mutation causing hyperkalemic periodic paralysis (HyperPP) with myotonia has previously been mapped to this gene locus by the same candidate gene approach. Thus, our data suggest that PC and HyperPP are caused by allelic mutations at a single locus on chromosome 17.Dedicated to Professor P. E. Becker on the occasion of his 83rd birthday.     

Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

The gene for the alpha i1 subunit of human guanine nucleotide binding protein maps near the cystic fibrosis locus.

The gene for the alpha i1 subunit of human guanine nucleotide binding (G) protein was mapped by in situ hybridization to chromosome 7 at band q21. The regional chromosomal location of the human alpha i1 gene was confirmed using human/mouse somatic-cell hybrid lines containing portions of human chromosome 7. Because the alpha i1 gene mapped near the cystic fibrosis locus and because an abnormal G protein might be expected to contribute to the pathophysiology of this disease, the alpha i1 gene was mapped with respect to the cystic fibrosis locus as defined by the Met oncogene and anonymous DNA marker pJ3.11. The location of the alpha i1 gene proved to be distinct from that of the cystic fibrosis locus.     

Repression of Meiotic Crossing over by a Centromere (CEN3 ) in SACCHAROMYCES CEREVISIAE

The location of the centromere of chromosome III (CEN3) of Saccharomyces cerevisiae has been altered by means of transformation. The frequency of meiotic crossing over in the CEN3-PGK1 and LEU2-CEN3 intervals increases approximately 1.5- and fourfold, respectively, when CEN3 is repositioned at HIS4. The centromere-distal HIS4-LEU2 region experiences a three- to fivefold decrease in the frequency of meiotic exchange when CEN3 is repositioned at HIS4. The inhibition of meiotic crossing over is conferred by a 627-base-pair fragment of CEN3 DNA and is not dependent on the orientation of CEN3 relative to the rest of chromosome III.     

Distinguishing between Nitrification and Denitrification as Sources of Gaseous Nitrogen Production in Soil

The source of N₂O produced in soil is often uncertain because denitrification and nitrification can occur simultaneously in the same soil aggregate. A technique which exploits the differential sensitivity of these processes to C₂H₂ inhibition is proposed for distinguishing among gaseous N losses from soils. Denitrification N₂O was estimated from 24-h laboratory incubations in which nitrification was inhibited by 10-Pa C₂H₂. Nitrification N₂O was estimated from the difference between N₂O production under no C₂H₂ and that determined for denitrification. Denitrification N₂ was estimated from the difference between N₂O production under 10-kPa C₂H₂ and that under 10 Pa. Laboratory estimates of N₂O production were significantly correlated with in situ N₂O diffusion measurements made during a 10-month period in two forested watersheds. Nitrous oxide production from nitrification was most important on well-drained sites of a disturbed watershed where ambient NO₃⁻ was high. In contrast, denitrification N₂O was most important on poorly drained sites near the stream of the same watershed. Distinction between N₂O production from nitrification and denitrification was corroborated by correlations between denitrification N₂O and water-filled pore space and between nitrification N₂O and ambient NO₃⁻. This technique permits qualitative study of environmental parameters that regulate gaseous N losses via denitrification and nitrification.     

Wounding Inhibits Protein Synthesis yet Stimulates Polysome Formation in Aged, Excised Pea Epicotyls

Wounding of aged, previously-excised pea epicotyl segments byremoval of the basal 1–2 mm resulted in a rapid (beginningwithin 15 min) recruitment of monosomes on to polysomes andan even more rapid (maximal between 6–12 min) inhibitionof protein synthesis in the remaining tissue. This inhibitionof protein synthesis in vivo did not appear to be an artefactcaused by the removal of highly active tissue (e.g., callus,contaminating bacteria), since wounds inflicted at a site distantfrom the region analyzed still elicited the response, and proteinsynthesis in the 1–2 mm slices (normally discarded) wasinhibited even more strongly than it was in the remaining tissue.The proportion of radioactive methionine in nascent chains (boundto polysomes) increased, while the production of completed polypeptidesdecreased, after wounding. Cycloheximide, a known inhibitorof the ribosome translocation/release process mimicked someof the effects of wounding. We interpret the results to indicatethat the initial effect of wounding is to inhibit translationby inhibiting the ribosome translocation/release process, whereasthe subsequent recovery in protein synthesis is brought aboutpartly by a recovery in ribosome translocation/release and partlyby enhanced initiation. ¹ Present address: Harvard-MIT Division of Health Science andTechnology, MIT, Cambridge, Massachusetts 02139, U.S.A. ² Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received May 26, 1986; Accepted August 4, 1986)     

Rapid Isolation of the 7S-Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva

7S-Nerve growth factor (NGF) and its alpha, beta-NGF, and gamma subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material. Beta-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures

Using immunocytochemical methods we have studied the distribution of vinculin in the anterior and posterior latissimus dorsi skeletal (ALD and PLD, respectively) muscles of the adult chicken. The ALD muscle is made up of both tonic (85%) and twitch (15%) myofibers, and the PLD muscle is made up entirely of twitch myofibers. In indirect immunofluorescence, antivinculin antibodies stained specific regions adjacent to the sarcolemma of the ALD and PLD muscles. In the central and myotendinous regions of the ALD, staining of the tonic fibers was intense all around the fiber periphery. Staining of the twitch fibers of both ALD and PLD muscles was intense only at neuromuscular junctions and myotendinous regions. Electron microscopy revealed subsarcolemmal, electron-dense plaques associated with the membrane only in those regions where vinculin was localized by immunofluorescence. Using antivinculin antibody and protein A conjugated to colloidal gold, we found that the electron-dense subsarcolemmal densities in the tonic fibers of the ALD contain vinculin; no other structures were labeled. The basal lamina overlying the densities appeared to be connected to the sarcolemma by fine, filamentous structures, more enriched at these sites than elsewhere along the muscle fiber. Increased amounts of endomysial connective tissue were often found just outside the basal lamina near the densities. In tonic ALD muscle fibers, the subsarcolemmal densities were present preferentially over the I-bands. In partially contracted ALD muscle, subsarcolemmal densities adjacent to the Z-disk appeared to be connected to that structure by short filaments. We propose that in the ALD muscle, through their association with the extracellular matrix, the densities stabilize the muscle membrane and perhaps assist in force transmission.     

Quantitative Isolation of Undegraded Polysomes from Aged Pea Tissue in the Absence of Contaminants and Artefacts

Undegraded polysomes were isolated successfully from aged peaepicotyls by grinding frozen tissue in at least 10 volumes ofbuffer A (0.2 M Tris-HCl, pH 8.5; 0.2 M sucrose; 60 mM KCl;30 mM MgCl₂), taking care to prevent the tissue from thawingprior to homogenization. Supposedly pure polysomes, derivedfrom the membrane-bound polysome fraction, were apparently contaminatedwith membranes, and contained polysomes clumped together vianascent chains. Problems with contaminants and artefacts werepartially alleviated by the use of polyoxyethylene tridecylether as a detergent replacing Triton X-100; further alleviatedby the use of large volumes of detergent-containing buffer toresuspend the membrane-bound polysome; and almost completelyeliminated by brief treatment of resuspended polysomes withprotease K. Optimal conditions for isolating polysomes fromaged tissue are given. ¹ Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received April 24, 1985; Accepted September 2, 1985)