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.     

Intranuclear distribution of SV40 large T-antigen and transformation-related protein p53 in abortively infected cells

The intranuclear localization of SV40 T-antigen (T-Ag) and the cellular protein p53 was studied in SV40 abortively infected baby mouse kidney cells using two complementary methods of ultrastructural immunocytochemistry in combination with preferential staining of nuclear RNP components and electron microscope autoradiography. Both proteins were revealed in association with peri- and interchromatin RNP fibrils containing the newly synthesized hnRNA. In addition, T-Ag and p53 remained bound, at least in part, to the residual internal nuclear matrix following nuclease and salt extractions of infected cells. The localization of T-Ag was different in SV40 lytically infected monkey kidney cells since, in addition to hnRNP fibrils, the viral protein was also associated with cellular chromatin. However, when lytic infection was performed in conditions of blocked viral DNA replication, T-Ag was no longer associated with the cellular chromatin but remained bound to the hnRNP fibrils. We conclude that the transforming and lytic functions of T-Ag can be distinguished by different subnuclear distributions. The significance of the association of T-Ag and p53 with hnRNP fibrils and the internal nuclear matrix is discussed in relation to the role of these structures in the control of cellular mRNA biogenesis.     

Molecular basis for interference of defective interfering particles of pseudorabies virus with replication of standard virus.

Serial passage of pseudorabies virus (PrV) at high multiplicity yields defective interfering particles (DIPs), but the sharp cyclical increases and decreases in titer of infectious virus that are observed upon continued passage at high multiplicity of most DIPs of other viruses are not observed with DIPs of PrV (T. Ben-Porat and A. S. Kaplan, Virology 72:471-479). We have studied the dynamics of the interactions of the virions present in a population of DIPs to assess the cis functions for which the genomes of the DIPs are enriched. The defective genomes present in one population of DIPs, [PrV(1)42], replicate preferentially over the nondefective genomes present in that virion population at early stages of infection, indicating that the DIP DNA is enriched for sequences that can serve as origins of replication at early stages of infection. This replicative advantage of the DIP DNA is transient and disappears at later stages of infection. The defective DNA does not appear to be encapsidated preferentially over the nondefective DNA present in this virion population, which might indicate that it is not enriched for cleavage-encapsidation sites. However, the nondefective DNA in the DIP virion population has become modified and has acquired reiterations of sequences originating from the end of the unique long (UL) region of the genome. Furthermore, both the infectious and defective genomes present in the DIP population compete for encapsidation more effectively than do the genomes of standard PrV. These results indicate that the defective genomes in the population of virions studied are enriched not only for an origin of replication but probably also for sequences necessary for efficient cleavage-encapsidation. Furthermore, the nondefective genomes present in this population of DIPs have also been modified and have acquired the ability to compete with the defective genomes for cleavage-encapsidation.     

The effect of alpha-interferon, cyclosporine A, and radiation-induced immune suppression on morphine-induced hypothermia and tolerance

An interconnection between the immune and the central nervous systems has been suggested by investigators studying the actions of several types of immune modifying agents and procedures upon opiate related phenomena. These studies have included the effects of altering immune system function by administration of either alpha-interferon, cyclosporine or radiation exposure upon naloxone-precipitated opiate withdrawal and upon opioid antinociceptive effects. The present study extends these earlier investigations by examining the effect of immune modulation upon opiate induced hypothermia. The results demonstrate that interferon and cyclosporine have no effects on baseline temperature or morphine induced hypothermia, while irradiation exposure elicits hyperthermia without affecting morphine-induced hypothermia. Finally, neither cyclosporine nor irradiation affect the development of tolerance to morphine induced hypothermia, while a single injection of the immune system modifier interferon was able to prevent the development of such tolerance. These observations suggest that yet another opiate-related phenomenon may be regulated at least in part by the immune system. These results together with our previous findings are further evidence of a link between the immune system and the CNS mediated through the opioid system. In addition, these studies further support our earlier hypothesis that "Interferon" is one of the endogenous substances which serves to prevent the development of tolerance and dependence to endogenous opioids.     

Kinetics of phosphate uptake, growth, and accumulation of cyclic diphosphoglycerate in a phosphate-limited continuous culture of Methanobacterium thermoautotrophicum.

The archaebacterium Methanobacterium thermoautotrophicum was grown in continuous culture at 65 degrees C in a phosphate-limited medium at specific growth rates from 0.06 to 0.28 h-1 (maximum growth rate [mu max] = 0.36 h-1). Cyclic-2,3-diphosphoglycerate (cyclic DPG) levels ranged from 2 to 20 mM in Pi-limited cells, compared with about 30 mM in batch-grown cells. The Monod constant for Pi-limited growth was 5 nM. Pi uptake rates were determined by following the disappearance of 32Pi from the medium. Interrupting the H2 supply stopped the uptake of Pi and the release of organic phosphates. Little or no efflux of Pi occurred in the presence or absence of H2. Pi uptake of cells adapted to nanomolar Pi concentrations could be accounted for by the operation of one uptake system with an apparent Km of about 25 nM and a Vmax of 58 nmol of Pi per min per g (dry weight). Uptake curves at 30 microM Pi or above were biphasic due to a sevenfold decrease in Vmax after an initial phase of rapid movement of Pi into the cell. Under these conditions the growth rate slowed to zero and the cyclic DPG pool expanded before growth resumed. Thus, three properties of M. thermoautotrophicum make it well adapted to live in a low-P environment: the presence of a low-Km, high-Vmax uptake system for Pi; the ability to accumulate cyclic DPG rapidly; and a growth strategy in which accumulation of Pi and cyclic DPG takes precedence over a shift-up in growth rate when excess Pi becomes available.     

Biochemical Characterization of Soybean Mutants Lacking Constitutive NADH:Nitrate Reductase

Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M₂ seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH₂-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c₁NR), constitutive NADH:NR (c₂NR), and nitrate-inducible NR (iNR).     

Concentration dependence of proteoglycan diffusion

The mutual diffusion coefficient of the bovine nasal cartilage proteoglycan subunit is found to increase rapidly with increasing concentration and decreasing ionic strength. These results have been obtained by analysis of the boundary relaxation of concentration gradients in the analytical ultracentrifuge by schlieren optics. The diffusion behavior can be understood in terms of the nonideality of the proteoglycan. The magnitude of the nonideality is dominated by charge interactions, whereas the influence of molecular size and associated excluded-volume interactions is small. The concentration dependence of the apparent diffusion coefficient of the proteoglycan subunit from dynamic light scattering was found, in contrast, to decrease with increasing concentration. Computer simulation of the dynamic light scattering suggests that the presence of a small population of aggregates may account for the difference in the two types of diffusion measurement due to their marked influence on the scattering.     

Nitrate Reductases from Wild-Type and nr(1)-Mutant Soybean (Glycine max [L.] Merr.) Leaves : I. Purification, Kinetics, and Physical Properties

NADH:nitrate reductase (EC 1.6.6.1) and NAD(P)H:nitrate reductase (EC 1.6.6.2) were purified from wild-type soybean (Glycine max [L.] Merr., cv Williams) and nr₁-mutant soybean plants. Purification included Blue Sepharose- and hydroxylapatite-column chromatography using acetone powders from fully expanded unifoliolate leaves as the enzyme source.

Two forms of constitutive nitrate reductase were sequentially eluted with NADPH and NADH from Blue Sepharose loaded with extract from wild-type plants grown on urea as sole nitrogen source. The form eluted with NADPH was designated c₁NR, and the form eluted with NADH was designated c₂NR. Nitrate-grown nr₁ mutant soybean plants yielded a NADH:nitrate reductase (designated iNR) when Blue Sepharose columns were eluted with NADH; NADPH failed to elute any NR form from Blue Sepharose loaded with this extract. Both c₁NR and c₂NR had similar pH optima of 6.5, sedimentation behavior (s_20,w of 5.5-6.0), and electrophoretic mobility. However, c₁NR was more active with NADPH than with NADH, while c₂NR preferred NADH as electron donor. Apparent Michaelis constants for nitrate were 5 millimolar (c₁NR) and 0.19 millimolar (c₂NR). The iNR from the mutant had a pH optimum of 7.5, s_20,w of 7.6, and was less mobile on polyacrylamide gels than c₁NR and c₂NR. The iNR preferred NADH over NADPH and had an apparent Michaelis constant of 0.13 millimolar for nitrate.

Thus, wild-type soybean contains two forms of constitutive nitrate reductase, both differing in their physical properties from nitrate reductases common in higher plants. The inducible nitrate reductase form present in soybeans, however, appears to be similar to most substrateinduced nitrate reductases found in higher plants.

     

Cytochrome b-245 from human neutrophils is a glycoprotein.

Cytochrome b-245 is a glycoprotein. It runs as a broad band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its apparent Mr varies with the concentration of acrylamide. It stained positively with Schiff reagent and with silver stains after oxidation with periodic acid. It preferentially bound the lectin of Phaseolus vulgaris (type III), and cleavage of carbohydrate with endoglycosidase F resulted in a sharp band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent Mr of 55000 G.l.c. analysis of carbohydrate showed this to account for about 15% of the Mr and N-acetylglucosamine and galactose to be the major sugars.