Transport of hepatitis B virus precore protein into the nucleus after cleavage of its signal peptide.

The precore and core proteins of hepatitis B virus have identical deduced amino acid sequences other than a 29-residue amino-terminal extension (precore region) on the precore protein. The first 19 of these residues serve as a signal sequence to direct the precore protein to the endoplasmic reticulum, where they are cleaved off with formation of precore protein derivative P22 for secretion. In this report, we show that P22 can alternatively be transported into the nucleus following signal peptide cleavage. Experiments with deletion mutants indicated that this nuclear transport proceeds via the cytosol and is dependent on the amino-terminal portion of P22. Thus, the hepatitis B virus precore protein is a secreted, cytosolic, and nuclear protein.     

Purification and properties of deacetoxycephalosporin C synthase from recombinant Escherichia coli and its comparison with the native enzyme purified from Streptomyces clavuligerus

A putatively rate-limiting synthase (expandase) of Streptomyces clavuligerus was stabilized in vitro and purified 46-fold from cell-free extracts; a major enriched protein with a Mr of 35,000 was further purified by electrophoretic elution. Based on a 22-residue amino-terminal sequence of the protein, the synthase gene of S. clavuligerus was cloned and expressed in Escherichia coli (Kovacevic, S., Weigel, B.J., Tobin, M.B., Ingolia, T.D., and Miller, J. R. (1989) J. Bacteriol. 171, 754-760). The synthase protein was detected mainly from granules of recombinant E. coli. The recombinant synthase was solubilized from the granules by urea, and for the first time a highly active synthase was purified to near homogeneity. The synthase was a monomer with a Mr of 34,600 and exhibited two isoelectric points of 6.1 and 5.3. Its catalytic activity required alpha-ketoglutarate, Fe2+, and O2, was stimulated by dithiothreitol or ascorbate but not by ATP, and was optimal at pH 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and at 36 degrees C. The Fe2+ requirement was specific, and at least one sulfhydryl group in the purified enzyme was apparently essential for the ring expansion. The Km values of the enzyme for penicillin N and alpha-ketoglutarate were 29 and 18 microM, respectively, and the Ka for Fe2+ was 8 microM. The recombinant synthase was indistinguishable from the native synthase of S. clavuligerus by those biochemical properties. In addition to the enzymic ring expansion of penicillin N to deacetoxycephalosporin C, the recombinant synthase catalyzed a novel hydroxylation of 3-exomethylenecephalosporin C to deacetylcephalosporin C.     

Synaptic contacts between embryonic Xenopus neurons and myotubes formed from a rat skeletal muscle cell line

Acetylcholine receptors (AChRs) accumulate at the junctional region during early development. In an attempt to characterize this process of AChR accumulation, we combined embryonic Xenopus neurons with myotubes formed from a rat skeletal muscle cell line. Xenopus neurons in culture are known to induce AChR accumulation in Xenopus muscles [Anderson, M. J., Cohen, M. W., and Zorychta, E. (1977). J. (London), 268, 731–756]. Rat myotubes, however, do not exhibit AChR accumulation in culture even when they are functionally innervated by the fetal rat spinal cord explant [Kidokoro, Y. (1980) Develop. Biol., 78, 231–241]. Establishment of synaptic transmission was examined electrophysiologically by recording synaptic potentials, while the distribution of AChR clusters was visualized using fluorescent α-bungarotoxin. Our results indicate that embryonic Xenopus neurons formed functional synaptic contacts but did not cause AChR accumulation in L6-myotubes. It seems that the ability of a nerve to cause AChR accumulation is separate from that to form the functional synapse. We also found that the mean amplitude of synaptic potentials in L6-myotubes interacted with Xenopus neurons was about half of that in L6-myotubes innervated by fetal rat spinal cord explants. Possible explanations for this finding are discussed.     

Intensity of light diffraction from striated muscle as a function of incident angle.

In a recently developed theory of light diffraction by single striated muscle fibers, we considered only the case of normal beam incidence. The present investigation represents both an experimental and theoretical extension of the previous work to arbitrary incident angle. Angle scan profiles over a 50 degrees range of incident angle (+25 degrees to -25 degrees) were obtained at different sarcomere lengths. Left and right first-order scan peak separations were found to be a function of sarcomere length (separation angle = 2 theta B), and good agreement was found between theory and experiment. Our theoretical analysis further showed that a myofibrillar population with a single common skew angle can yield an angle scan profile containing many peaks. Thus, it is not necessary to associate each peak with a different skew population. Finally, we have found that symmetry angle, theta s, also varies with sarcomere length, but not in a regular manner. Its value at a given sarcomere length is a function of a particular region of a given fiber and represents the average skew angle of all the myofibril populations illuminated. The intensity of a diffraction order line is considered to be principally the resultant of two interference phenomena. The first is a volume-grating phenomenon which results from the periodic A-I band structure of the fiber (with some contribution from Z bands and H zones). The second is Bragg reflection from skew planes, if the correct relation between incident angle and skew angle is met. This may result in intensity asymmetry between the left and right first order lines.     

Characterization of nuclear targeting signal of hepatitis delta antigen: nuclear transport as a protein complex.

Hepatitis delta antigen (HDAg) is the only protein encoded by hepatitis delta virus (HDV). HDAg has been demonstrated in the nuclei of HDV-infected hepatocytes, and its nuclear transport may be important for the replication of HDV RNA. In this report, we investigated the mechanism of nuclear transport of HDAg. By expressing fusion proteins consisting of the different portions of HDAg and alpha-globin, we have identified a nuclear localization signal (NLS) within the N-terminal one-third of HDAg. It consists of two stretches of basic amino acid domains separated by a short run of nonbasic amino acids. Both of the basic domains are necessary for the efficient nuclear transport of HDAg. The nonbasic spacer amino acids could be removed without affecting the nuclear targeting of HDAg significantly. Thus, the HDAg NLS belongs to a newly identified class of NLS which consists of two discontiguous stretches of basic amino acids. This NLS is separated from a stretch of steroid receptor NLS-like sequence, which is also present but not functioning as an NLS, in HDAg. Furthermore, we have shown that subfragments of HDAg which do not contain the NLS can be passively transported into the nucleus by a trans-acting full-length HDAg, provided that these subfragments contain the region with a leucine zipper sequence. Thus, our results indicate that HDAg forms aggregates in the cytoplasm and that the HDAg oligomerization is probably mediated by the leucine zipper sequence. Therefore, HDAg is likely transported into the nucleus as a protein complex.     

Regulation of arginase production by glucocorticoid in three human gastric cancer cell lines.

Gastric cancer tissues have high levels of glucocorticoid receptors (GR) and arginase. To investigate the interrelation of glucocorticoid, GR and arginase, three human gastric cancer cell lines (AZ-521, NUGC-3, KATO-III) were treated with hydrocortisone in the presence or absence of a glucocorticoid antagonist RU38486. GR were found to be present in all three lines, and hydrocortisone significantly increased the production of total arginase in all 3 lines. The induction of arginase production by hydrocortisone was inhibited by RU38486. These findings suggest that the regulation of arginase production by hydrocortisone in gastric cancer cells is mediated through GR.     

Aplysia peptide neurotransmitters beta-bag cell peptide, Phe-Met-Arg-Phe-amide, and small cardioexcitatory peptide B are rapidly degraded by a leucine aminopeptidase-like activity in hemolymph.

We have been investigating the role of proteolytic enzymes in the inactivation of peptide neurotransmitters in the marine snail Aplysia. Previous studies (Squire, C. R., Talebian, M., Menon, J. G., Dekruyff, S. D., Lee, T. D., Shively, J. E., and Rothman, B. S. (1991) J. Biol. Chem. 266, 22355-22363) showed that neuroactive fragments of the neurotransmitter alpha-bag cell peptide (alpha-BCP) were rapidly degraded (t1/2 = 0.5-2.7 min) in plasma, hemolymph that had been cleared by centrifugation. Degradation was caused by one or more enzymes resembling mammalian leucine amino-peptidase (LAP, EC 3.4.11.1). In this report we show that three other Aplysia peptide neurotransmitters, beta-BCP(1-5) (Arg-Leu-Arg-Phe-His), FMRFa (Phe-Met-Arg-Phe-amide), and SCPB(1-9) (Met-Asn-Tyr-Leu-Ala-Phe-Pro-Arg-Met-amide) are rapidly degraded (t1/2 = 0.3-2.4 min) in plasma by apparently the same LAP-like enzyme(s). Our findings strongly suggest that the LAP-like enzyme(s), by means of its broad substrate specificity and access to the extracellular spaces of the nervous system in vivo, plays a significant role in the inactivation of many Aplysia peptide neurotransmitters, and they raise the possibility that proteolytic enzymes in the extracellular fluid contribute significantly to the inactivation of peptide neurotransmitters in other animal species.     

Soluble complement receptor type 1 ameliorates the local and remote organ injury after intestinal ischemia-reperfusion in the rat.

We examined the role of C activation in ischemia reperfusion injury by inhibiting C activation in a rat model of mesenteric arterial occlusion. In anesthetized rats, 60 min of mesenteric arterial occlusion was followed by 3 h of reperfusion. PBS alone or containing soluble C receptor 1 (3 or 6 mg) was administered i.v. Controls underwent laparotomy without ischemia. Relative serum C activities were assessed by hemolytic assay, neutrophil (polymorphonuclear leukocyte) sequestration by tissue content of myeloperoxidase (MPO) activity, intestinal mucosal injury by histologic grading, lung vascular permeability by the ratio of bronchoalveolar lavage to blood concentration of radiolabeled BSA, and endothelial cell injury was quantified by measurement of plasma factor VIII-related Ag. After reperfusion, PBS-treated animals had increased intestinal MPO (0.048 +/- 0.007 U/g) compared to sham (0.022 +/- 0.005 U/g (p less than 0.05)) and intestinal mucosal injury score (2.490 +/- 0.221) compared to sham (0.331 +/- 0.045 (p less than 0.05)). Treatment with 6 mg soluble C receptor 1 15 min before reperfusion reduced intestinal MPO (0.017 +/- 0.003 U/g (p less than 0.05)) and mucosal injury (1.733 +/- 0.168 (p less than 0.05)) compared to PBS control. PBS-treated animals also demonstrated increased lung MPO (0.314 +/- 0.025 U/g vs 0.085 +/- 0.018 in sham (p less than 0.05)) and increased lung permeability (bronchoalveolar lavage/blood cpm 11.32 +/- 1.35 x 10(-3) vs sham 2.22 +/- 0.19 x 10(-3) (p less than 0.05)). Treatment with 6 mg soluble C receptor 1 15 min before reperfusion or at reperfusion reduced the lung permeability (bronchoalveolar lavage/blood cpm 3.90 +/- 0.79 x 10(-3) and 5.08 +/- 0.75, respectively (both p less than 0.05)) compared to PBS control, but did not reduce lung MPO (0.342 +/- 0.031 U/g and 0.246 +/- 0.025), respectively. Treatment with sCR1 also reduced the release of factor VIII-related Ag, 5-day mortality, and C hemolytic activity. In this model, C is a major mediator of intestinal injury and extraintestinal injury.     

Evidence that α-dihydrograyanotoxin II does not bind to the sodium gate

Summary The basis for the ability of -dihydrograyanotoxin II (-2HG-II) to promote Na⁺ conductance in axons was sought. The apparent binding of tritiated -2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes,Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the binding was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of binding in all tissues and its relative insensitivity to neuropharmacological agents. -2HG-II did not affect the Na⁺ conductance of phospholipid bilayers, nor did it permit transport of²²Na into a bulk organic phase. It was concluded that -2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per ² of cell membrane.