Transient glucose intolerance during attacks of thyrotoxic periodic paralysis

In a 19-year-old Japanese male (case 1) with thyrotoxic periodic paralysis (TPP), an increase of plasma glucose concentration together with abnormally high levels of serum immunoreactive insulin (IRI) was observed preceding a spontaneous attack of paralysis. Therefore, the plasma glucose, glucagon, epinephrine, norepinephrine, serum IRI, growth hormone and cortisol levels, and the erythrocyte insulin receptors were measured in case 1 and a 40-year-old Japanese male (case 2) with TPP during attacks of paralysis induced by prolonged glucose loading. In case 1, the serum IRI concentration was elevated to the extraordinarily high level of 655.0 microU/ml at the beginning of paralysis, and at that time, the plasma glucose concentration was 147 mg/dl. However, when paralysis was not induced by a similar glucose loading during methimazole treatment, the serum IRI and plasma glucose levels at the corresponding time after glucose loading were 20.9 microU/ml and 87 mg/dl, respectively. Furthermore, the affinity of the erythrocyte insulin receptors was decreased during the attack. In case 2, plasma glucose and serum IRI concentrations were increased in accordance with the initiation of paralysis although the blood levels of hormones counteracting insulin were not significantly changed. These findings suggest that there is something interacting with the normal action of the insulin in the early phase of paralysis.     

Genome type analysis of adenovirus type 4 isolates, recently obtained from clinically different syndromes in some areas in Japan

DNA cleavage analyses with EcoRI, HindIII and BamHI were carried out to investigate genome types of recent Ad 4 isolates obtained from acute respiratory disease (8 strains), and ocular disease (11 strains) in Japan. DNA cleavage patterns of all 19 isolates studied were identical regardless of whether they were recovered from respiratory tract or conjunctiva, but were distinct from that of the prototype strain.     

Affinity chromatography of urokinase on an agarose derivative coupled with pyroglutamyl-lysyl-leucyl-argininal

Pyroglutamyl-lysyl-leucyl-argininal (Pyr-Lys-Leu-Argal) immobilized on gel matrix through the epsilon-amino group of its lysine residue was shown to be an efficient biospecific affinity adsorbent for purification of urokinase. Pyr-Lys-Leu-Argal dibutylacetal, a precursor of this immobilized ligand, was synthesized by a fragment condensation procedure, in which one of the thermolysin-digestion products of leupeptin dibutylacetal, H-Leu-Argal dibutylacetal, was used as a key intermediate. The precursor was coupled to CH-Sepharose 4B with the aid of a water-soluble carbodiimide, and its acetal protecting group was then removed by mild acid treatment to free the essential aldehyde function. The Sepharose derivative thus prepared was shown to adsorb urokinase selectively and effectively from a crude human urine preparation at neutral pH and to release the bound enzyme under mild acidic conditions. The present technique afforded a highly purified urokinase preparation abundant in the high-molecular form with 90% recovery. The complex formed between urokinase and the immobilized ligand was found to have a dissociation constant of about 2 X 10(-4)M.     

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.     

Nucleosidediphosphate-kinase induction by human recombinant IL-2 in mouse NK cells

The ability of human recombinant IL-2 to induce NDP-kinase in mouse NK cells has been studied. A significant increase in the amount of NDP-kinase was observed when the cells were exposed to IL-2 (100 units/ml) for 3 h at 37 degrees C. The enzyme inducting ability of human recombinant IL-2 was similar to that of native mouse IL-2 in the cells. The enzymatic characteristics [chemical requirements for the phosphoenzyme formation and molecular size of two distinct subunits (18,000 and 20,000 daltons)] of NDP-kinase from IL-2 treated cells were similar to those of the enzymes from EAT cells. The enzyme's biological role in the initiation of cell proliferation by IL-2 has been discussed.     

Characterization of nucleosidediphosphate (NDP)-kinase-associated GTP binding proteins from human recombinant interleukin 2 (rIL-2)-treated mouse NK cells

Nucleosidediphosphate (NDP)-kinase-associated proteins from rIL-2-treated mouse NK cells have been biochemically characterized. The associated proteins could be separated from partially purified NDP-kinases by the 5-25% glycerol density gradient centrifugation method after treatment with 6 M urea in the presence of 1 mM EDTA. The associated proteins (approx. Mr 20,000) were defined as GTP binding proteins, since only [alpha-32P]GTP was bound to these proteins in the presence of 5 mM Mg2+ at 37 degrees C. We also found that these GTP binding proteins hydrolyzed only GTP in the presence of 5 mM Mg2+. The data presented here for: GTP specific binding activity; GTPase activity; and molecular size (approx. Mr 20,000) of the NDP-kinase-associated GTP binding proteins are similar to those reported for ras oncogene products (p21 proteins).     

Selective enhancement in striatal [H]nitrendipine binding following chronic treatment with morphine

Two parameters of Ca²⁺ dynamics in brain preparations (⁴⁵Ca-uptake to slices and [³H]nitrendipine binding to membrane fractions) were measured in naive and chronic morphine-administered rats. While morphine did not have any effect on ⁴⁵Ca-uptake to striatal slices in normal Krebs-Ringer solution, it inhibited K⁺-stimulated ⁴⁵Ca-uptake to slices. Furthermore, the effect of morphine was antagonized by naloxone. Inhibition of K⁺-stimulated ⁴⁵Ca-uptake to striatal slices by morphine was not observed in preparations obtained from chronic morphine-administered rats (6 mg/kg/b.i.d./7 days). In membrane fractions, [³H]nitrendipine binding increased by 34% in striatum following chronic morphine treatment, whereas no change was observed in the cortex and hippocampus. The results will be discussed in relation to the phenomena underlying chronic morphine administration.     

Effects of brain and mesenchyme upon the cytogenesis of rat adenohypophysis in vitro

Summary The objective of this in-vitro study was to examine whether the diencephalic floor or the mesenchyme is involved in differentiation of LH cells in the developing rat adenohypophysis. Overall growth of the adenohypophysial tissue was retarded when the adenohypophysial primordium was cultivated after enzymatic removal of the diencephalic floor on days 11.5 and 12.5 of gestation. This malgrowth was more marked when the brain was separated on day 11.5; most expiants retained a simple cystiform structure that consisted of a few layers of undifferentiated cells. Removal of the brain also caused a highly significant decrease (P < 0.001) in the number of immunoreactive LH cells, if it was performed on day 11.5 but not day 12.5. Mesenchyme had little effect on the adenohypophysial growth or the number of immunopositive cells. Cultivation of the adenohypophysial primordium with the diencephalic floor resulted in the appearance of many immunoreactive LH cells. The number of LH cells significantly decreased, however, when the co-cultivated brain completely surrounded the adenohypophysial tissue.These results indicate that in 11.5-day-old fetal rats the diencephalic floor is indispensable for the initial proliferation of adenohypophysial primordial cells and for the early determinating process of LH cells. Once determined, the development of LH cells may proceed without the surrounding tissues. The cytodifferentiation seems to be rather inhibited when in contact with the brain. The significance of the intimate spatial relationship between developing LH cells and the surrounding mesenchyme is also discussed.     

Scanning electron-microscopic studies on the three-dimensional structure of sarcoplasmic reticulum in the mammalian red,white and intermediate muscle fibers

Summary The three-dimensional structure of the sarcoplasmic reticulum (SR) in the red, white and intermediate striated muscle fibers of the extensor digitorum longus muscle of the rat was examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by the osmium-DMSO-osmium procedure.In all three types of fibers, the terminal cisternae and transverse tubules form triads at the level of the A-I junction. Numerous slender sarcotubules, originating from the A-band side terminal cisternae, extend obliquely or longitudinally and form oval or irregular shaped networks of various sizes in front of the A-band, then become continuous with the tiny mesh (fenestrated collar) in front of the H-band. The A-and H-band SR appears as a single sheet of anastomotic tubules. Numerous sarcotubules, originating from the I-band side terminal cisternae, extend in threedimensional directions and form a multilayered network over the I-band and Z-line regions. At the I-band level, paired transversely oriented mitochondria partly embrace the myofibril. The I-band SR network is poorly developed in the narrow space between the paired mitochondria, but is well developed in places devoid of these mitochondria.The three-dimensional structure of the SR is basically the same in all three muscle fiber-types. However, the SR is sparse on the surface of mitochondria, so the mitochondria-rich red fiber has a much smaller total volume of SR than the mitochondria-poor white fiber. Moreover, the volume of SR of the intermediate fiber is intermediate between the two.     

Structure and localization of genes encoding aberrant and normal epidermal growth factor receptor RNAs from A431 human carcinoma cells.

A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.