Occurrence and induction of spermidine-N1-acetyltransferase in Escherichia coli

Spermidine acetylase activity was detected in extracts prepared from $\text{Escherichia coli}$ and there was a marked increase in activity over the early period of growth. This increase reached a maximum 3 h after inoculation and was followed by an increase in ornithine decarboxylase activity. The acetylase was also able to use spermine as a substrate, but not putrescine. With spermidine and acetyl-CoA as substrate, the product formed was exclusively N¹-acetyl-spermidine. This is the first evidence for the occurrence in bacteria of spermidine-N¹-acetyltransferase, an enzyme which has previously been described in mammalian cells. These results suggest that acetylation of spermidine may be involved in the growth of $\text{Escherichia coli}$ and in the regulation of its polyamine content.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Characterization of O-trimethylsilyl derivatives of d-glucose,d-galactose,and d-mannose by gas-liquid chromatography-chemical-ionization mass spectrometry with ammonia as reagent gas

The per(trimethylsilyl) ethers of d-glucose, d-galactose, and d-mannose were analyzed by g.l.c.-c.i.m.s. with ammonia as the reagent gas. C.i.m.s. gave simple fragmentation and fragment ions of high intensity in the high-mass range where the QM⁺ ion is also detected. The β-d anomers gave ions at m/e 558 showing intensities 3–12 times those of the α-d anomers. The epimers could be distinguished by differences in the intensities of the ions and by the observation that d-glucose gave a base peak at m/e 198, d-galactose at m/e 468, and d-mannose at m/e 204. The pyranose and furanose structures could be distinguished by comparing the ion intensities at m/e 198, m/e 271, m/e 361, m/e 396, and m/e 451. A similar analysis was also performed with 2-methylpropane as the reagent gas.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.     

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

Characteristics of hepatitis B virus isolates of genotype G and their phylogenetic differences from the other six genotypes (A through F)

Eight hepatitis B virus (HBV) isolates of genotype G were recovered from patients and sequenced over the entire genome. Six of them had a genomic length of 3,248 bp and two had genomic lengths of 3,239 bp (USG15) and 3,113 bp (USG18) due to deletions. The 10 HBV/G isolates, including the 8 sequenced isolates as well as the original isolate (AF160501) and another isolate (B1-89), had a close sequence homology of 99.3 to 99.8% among themselves (excluding USG18 with a long deletion) but of <88.7% to any of the 68 HBV isolates of the other six genotypes with the full-length sequence known. The eight HBV/G isolates possessed an insertion of 36 bp in the core gene and two stop codons in the precore region, as did the AF160501 and B1-89 isolates. The 10 HBV/G isolates clustered on a branch separate from those bearing the other six genotypes (A through F [A-F]) in the phylogenetic tree constructed from full-length sequences of 78 HBV isolates as well as in those constructed from the core, polymerase, X, and envelope genes. Despite two stop codons in the precore region that prohibited the translation of the HBV e antigen (HBeAg), all of the eight patients with HBV/G infection possessed the HBeAg in serum. By restriction fragment length polymorphism of the surface gene, all of the eight patients were found to be coinfected with HBV of genotype A (HBV/A), which would be responsible for the expression of HBeAg in them. It is worthy of examination to determine how coinfection occurs and whether HBV/G needs HBV/A for replication.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.