Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.     

Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.     

Solubilization and partial characterization of fatty acyl-CoA:sphingosine acyltransferase (ceramide synthetase) from rat liver and brain

Lignoceroyl-CoA:sphingosine lignoceroyltransferase, which catalyzes synthesis of lignoceroylsphingosine, the ceramide that is a major component of sphingolipids in mammalian tissues, has been solubilized from microsomes of rat brain and liver and partially purified. The microsomes were treated with 1 M sodium thiocyanate in N,N-bis(2-hydroxyethyl)glycine (Bicine) buffer containing 20% glycerol. The supernatant fraction obtained after centrifugation was fractionated by Sepharose CL-4B gel filtration. The ceramide synthetase activity was recovered in a small fraction containing high molecular weight proteins. Analysis of proteins and lipids indicated that the fraction was not simply a fragment of microsomes. The activity for synthesis of lignoceroylsphingosine, which is abundant in nervous system, was compared with that for the synthesis of stearoylsphingosine, which is more enriched in extraneural sphingolipids, in brain and liver microsomes. Despite the difference in relative abundance of molecular species of ceramides in these tissues, the activity for lignoceroylsphingosine synthesis was not more enriched in brain than in liver.     

Albumin inhibition of transferrin low-affinity binding to K562 cells

Several reports have suggested that variations of albumin concentration in the incubation medium can modulate the magnitude of transferrin binding to the cells. We have investigated this problem further using K562 cells. In the absence of human serum albumin, transferrin binding demonstrated a non-saturable curve which, upon Scatchard analysis, showed two components with high and low affinities. In the presence of 0.5% human serum albumin, the low-affinity but not the high-affinity component was totally inhibited and, thus, the binding showed a saturation plateau at transferrin concentration of 6 micrograms/ml. Increasing concentrations of human serum albumin in the incubation medium led to progressive inhibition of transferrin binding, reaching a plateau at 0.2% human serum albumin. At this concentration transferrin binding was about 12 ng/10(6) cells, corresponding to the saturation plateau for high-affinity binding. Low-affinity transferrin binding in the absence of human serum albumin could readily be displaced by subsequent addition of albumin. Similar inhibition was obtained by another serum protein, ceruloplasmin, suggesting that this inhibition is not unique to albumin and may be a common property of all proteins. Incubation at 37 degrees C with 59Fe-labeled transferrin indicated that all iron uptake occurs through high-affinity binding. We conclude that the reported variations in magnitude of transferrin binding by the cell due to variations in albumin concentration are the result of inhibition of low-affinity binding of transferrin by albumin.     

Effects of taurine on the electrical and mechanical activities of embryonic chick heart

The effect of taurine (2-aminoethanesulphonic acid) on myocardial slow action potentials (APs) and accompanying contractions was examined in isolated perfused chick hearts and reaggregated cultured cells. Isoproterenol (ISO), histamine (HIS), or tetraethylammonium (TEA) induced slow APs and contractions in hearts whose fast Na+ channels had been inactivated by elevated K+. Taurine (10 mM) not only failed to induce slow APs, but actually decreased ISO (10(-8) M), HIS (10(-4) M), or TEA (10 mM) induced slow APs and contractions transiently (about 30s-2 min after the addition of taurine). The properties of the slow APs recovered to control levels by 7-13 min after the addition of the taurine; at this time, there was an increase in developed tension of the contraction accompanying the slow APs. These results suggest that the positive inotropic action of taurine is not mediated through an increase in the slow inward Ca2+ current. However, the transient depression of Ca2+-dependent slow APs by taurine probably explains the transient negative inotropic effect of taurine.     

Genetic analysis for insulitis in NOD mice

Non-obese diabetic (NOD) mice spontaneously develop diabetic signs akin to those of Type I diabetes in man. Insulitis, i.e., lymphocytic infiltration around and into the pancreatic islets is one of the characteristics of such mice. It is also the etiologic pathological lesion in the development of diabetes mellitus in NOD mice. Thus, we chose insulitis as a marker for genetic analysis of the development of diabetes mellitus in NOD mice and clarified the mode of its inheritance. In breeding studies between NOD and C57BL/6J mice, insulitis was not observed in the F1 and (F1 X C57BL/6J) backcross generations, but was found with incidences of 3.9% in females and 1.4% in males in the F2 generation and 23.7% in females and 12.1% in males in the (F1 X NOD) backcross generation. These incidences in the F2 and (F1 X NOD) backcross females corresponded approximately to 1/16 and 1/4 of the incidences of 89.7% in the NOD females, respectively. A similar relationship was observed between the F2 and (F1 X NOD) backcross males and the NOD males. When the gene expressivity of both sexes for a double recessive homozygote was assumed to be the incidences of insulitis in 9-week-old NOD females and males, respectively, the expected numbers of both sexes with and without insulitis in the F2 and backcross generations agreed well with the observed ones. These observations suggest that two recessive genes on independent autosomal chromosomes are necessary for the development of insulitis in NOD mice.     

Suppressive effect of elcatonin, an eel calcitonin analogue, on excessive urinary hydroxyproline excretion in polyostotic fibrous dysplasia (McCune-Albright's syndrome)

A 12-year-old Japanese girl with polyostotic fibrous dysplasia and endocrine concomitants, was treated with elcatonin, a synthetic eel calcitonin analogue, 10 MRC unit/twice a week given by intramuscular injection. Significant decreases in 24 hr urinary content of hydroxyproline and other amino acids from bone collagen were observed during the course of treatment over 5 months. This biochemical result suggests that the synthetic eel calcitonin analogue exhibits the therapeutic effect in patients with polyostotic fibrous dysplasia by inhibiting bone resorption.