Dynamics of immunocyte activation during intravenous immunoglobulin treatment in Kawasaki disease.

Objectives: Kawasaki disease (KD) is a systemic vasculitis of early childhood. Intravenous immunoglobulin (IVIG) is the standard treatment for KD. However, IVIG is not effective in approximately 15% of children with KD, and the mechanisms for this are unclear. We investigated changes in monocyte and T-cell activation from pre- to post-IVIG in IVIG-effective and IVIG-resistant KD.Method: We analysed peripheral CD14+CD16+ cells and human leucocyte antigen-DR (HLA-DR) expression on CD4+ and CD8+ cells in 46 children with KD who were admitted to Yamaguchi University Hospital between January 2011 and May 2016. We compared the kinetics in the absolute numbers of CD14+CD16+ cells, CD4+HLA-DR+ cells, and CD8+HLA-DR+ cells before and after IVIG treatment between IVIG-effective and IVIG-resistant groups.Results: Among the 46 subjects, 30 had IVIG-effective KD and 16 had IVIG-resistant KD. The absolute number of CD14+CD16+ cells in the IVIG-effective group decreased significantly after IVIG, while that in the IVIG-resistant group showed no change after IVIG. The absolute number of CD4+HLA-DR+ cells increased significantly after IVIG in both groups. The absolute number of CD8+HLA-DR+ cells before IVIG was low and significantly increased after IVIG in the IVIG-resistant group, while that in the IVIG-effective group showed no change after IVIG.Conclusions: Our results suggest that insufficient control of monocyte suppression and T-cell activation, especially in terms of the CD8-related immune system, are associated with IVIG resistance. The restoration of T-cell suppression may be important for KD recovery. These findings provide insight into the mechanism of IVIG resistance.

Induction of apoptosis and cellular senescence in mice lacking transcription elongation factor, Elongin A.

Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To gain more insight into the physiological functions of Elongin A, we generated Elongin A-deficient mice. Elongin A homozygous mutant (Elongin A(-/-)) embryos demonstrated a severely retarded development and died at between days 10.5 and 12.5 of gestation, most likely due to extensive apoptosis. Moreover, mouse embryonic fibroblasts (MEFs) derived from Elongin A(-/-) embryos exhibited not only increased apoptosis but also senescence-like growth defects accompanied by the activation of p38 MAPK and p53. Knockdown of Elongin A in MEFs by RNA interference also dramatically induced the senescent phenotype. A study using inhibitors of p38 MAPK and p53 and the generation of Elongin A-deficient mice with p53-null background suggests that both the p38 MAPK and p53 pathways are responsible for the induction of senescence-like phenotypes, whereas additional signaling pathways appear to be involved in the mediation of apoptosis in Elongin A(-/-) cells. Taken together, our results suggest that Elongin A is required for the transcription of genes essential for early embryonic development and downregulation of its activity is tightly associated with cellular senescence.

Validation of a single-channel airflow monitor for screening of sleep-disordered breathing.

A simple screening method for sleep-disordered breathing (SDB) is desirable for primary care practices. In the present study, a simple monitor, which utilises a new type of flow sensor and a novel algorithm, was prospectively validated. Home recording for 2 nights with the monitor only, followed by in-laboratory recording with the monitor together with polysomnography, were carried out in consecutive patients (n = 100) suspected of SDB. A subjective sleep log was also recorded. The signal was analysed using power spectral analysis, which yielded the flow respiratory disturbance index (flow-RDI). There was no recording failure at home. The reproducibility of the flow-RDI between the 2 nights at home was high (intraclass correlation coefficient = 0.92). The sensitivity and specificity of the in-laboratory flow-RDI to diagnose SDB were 0.96 and 0.82, 0.91 and 0.82, and 0.89 and 0.96, for apnoea/hypopnoea index (AHI) > or =5, > or =15 and > or =30 events x h(-1), respectively. The diagnostic ability in low-severity subgroups (female, normal weight, AHI <15 events x h(-1)) was almost comparable to that in the entire group. Excluding subjective waking time on the sleep log from the recording time had no significant effect on the flow-RDI. The single-channel monitor is considered feasible for ambulatory sleep disordered breathing monitoring because of its easy applicability, high reproducibility and relatively high agreement with polysomnography results.

Invariant NKT cells produce IL-17 through IL-23-dependent and -independent pathways with potential modulation of Th17 response in collagen-induced arthritis.

Invariant natural killer T (iNKT) cells play a protective role in the development of certain autoimmune diseases. However, their precise role in the pathogenesis of autoimmune arthritis remains unclear. In this study, we examined the possible contribution of iNKT cells in collagen-induced arthritis (CIA) by using iNKT cell-deficient mice (Jalpha281-/- mice). CIA in these mice was markedly suppressed and interleukin (IL)-17 production was reduced in a native type II collagen (CII)-specific T cell response. Draining lymph nodes of CII-immunized Jalpha281-/- mice contained a significantly low number of IL-17-producing T helper cells. To determine whether iNKT cells produce IL-17, we measured IL-17 by enzyme-linked immunosorbent assay in iNKT cells stimulated with the ligand, alpha-galactosylceramide (alpha-GalCer). Notably, splenocytes from Jalpha281-/- mice stimulated in this way were negative for IL-17, whereas those from C57BL/6 mice produced IL-17. Immunostaining for IL-17 in iNKT cells confirmed intracellular staining of the protein. RT-PCR analysis showed that iNKT cells expressed retinoid-related orphan receptor gammaT and IL-23 receptor. Moreover, cell sorting demonstrated that NK1.1- iNKT cells were the main producers of IL-17 compared with NK1.1+ iNKT cells. IL-17 production by iNKT cells was induced by IL-23-dependent and -independent pathways, since iNKT produced IL-17 when stimulated with either IL-23 or alpha-GalCer alone. Our findings indicate that iNKT cells are producers and activators of IL-17 via IL-23- dependent and -independent pathways, suggesting that they are key cells in the pathogenesis of CIA through IL-17.

Fulminant type 1 diabetes mellitus observed in insulin receptor substrate 2 deficient mice.

The objective of this study was to characterise the fulminant type 1 diabetes mellitus (DM) accompanying abrupt hyperglycaemia and ketonuria observed in insulin receptor substrate 2 (IRS2)-deficient mice. IRS2-deficient mice backcrossed onto the original C57BL/6J:Jc1 background (B6J-IRS2(-/-) mice) for more than 10 generations were used. Eight male IRS2-deficient mice with ketonuria and abrupt increase in plasma glucose concentrations over 25 mmol/l were used as the fulminant type 1 diabetic mice (diabetic mice) and 8 male IRS2-deficient mice (8 weeks old) without glycosuria were used as the control mice. Plasma metabolite, immunoreactive insulin (IRI) and C-peptide concentrations, hepatic energy metabolism related enzyme activities and histopathological change in pancreatic islets were investigated. The diabetic mice showed significantly higher plasma glucose and cholesterol concentrations and lower plasma IRI and C-peptide concentrations than the control mice. In livers of the diabetic mice, glycolytic and malate-aspartate shuttle enzyme activities decreased significantly and gluconeogenic, lipogenic and ketone body synthesis enzyme activities increased significantly compared to those in the control mice. The pancreatic islets of the diabetic mice decreased significantly in size and number of beta cells. The diabetic IRS2-deficient mice did not show the islet-related antibodies observed in the diabetic NOD mice in their sera. The characteristics of the diabetic IRS2-deficient mice resembled those of the human nonautoimmune fulminant type 1 DM. IRS2-deficient mice may be a useful animal model for studying the degradation mechanism of pancreatic beta cells in the process of development of fulminant type 1 DM.

p11 in Cholinergic Interneurons of the Nucleus Accumbens Is Essential for Dopamine Responses to Rewarding Stimuli.

A recent study showed that p11 expressed in cholinergic interneurons (CINs) of the nucleus accumbens (NAc) is a key regulator of depression-like behaviors. Dopaminergic neurons projecting to the NAc are responsible for reward-related behaviors, and their function is impaired in depression. The present study investigated the role of p11 in NAc CINs in dopamine responses to rewarding stimuli. The extracellular dopamine and acetylcholine (ACh) levels in the NAc were determined in freely moving male mice using in vivo microdialysis. Rewarding stimuli (cocaine, palatable food, and female mouse encounter) induced an increase in dopamine efflux in the NAc of wild-type (WT) mice. The dopamine responses were attenuated (cocaine) or abolished (food and female mouse encounter) in constitutive p11 knock-out (KO) mice. The dopamine response to cocaine was accompanied by an increase in ACh NAc efflux, whereas the attenuated dopamine response to cocaine in p11 KO mice was restored by activation of nicotinic or muscarinic ACh receptors in the NAc. Dopamine responses to rewarding stimuli and ACh release in the NAc were attenuated in mice with deletion of p11 from cholinergic neurons (ChAT-p11 cKO mice), whereas gene delivery of p11 to CINs restored the dopamine responses. Furthermore, chemogenetic studies revealed that p11 is required for activation of CINs in response to rewarding stimuli. Thus, p11 in NAc CINs plays a critical role in activating these neurons to mediate dopamine responses to rewarding stimuli. The dysregulation of mesolimbic dopamine system by dysfunction of p11 in NAc CINs may be involved in pathogenesis of depressive states.

RNA polymerase beta-subunit gene (rpoB) sequence analysis for the identification of Bacteroides spp.

Partial rpoB sequences (317 bp) of 11 species of Bacteroides, two Porphyromonas spp. and two Prevotella spp. were compared to delineate the genetic relationships among Bacteroides and closely related anaerobic species. The high level of inter-species sequence dissimilarities (7.6-20.8%) allowed the various Bacteroides spp. to be distinguished. The position of the Bacteroides distasonis and Bacteriodes merdae cluster in the rpoB tree was different from the position in the 16S rRNA gene tree. Based on rpoB sequence similarity and clustering in the rpoB tree, it was possible to correctly re-identify 80 clinical isolates of Bacteroides. In addition to two subgroups, cfiA-negative (division I) and cfiA-positive (division II), of Bacteroides fragilis isolates, two distinct subgroups were also found among Bacteroides ovatus and Bacteroides thetaiotaomicron isolates. Bacteroides genus-specific rpoB PCR and B. fragilis species-specific rpoB PCR allowed Bacteroides spp. to be differentiated from Porphyromonas and Prevotella spp., and also allowed B. fragilis to be differentiated from other non-fragilisBacteroides spp. included in the present study.

A novel in vivo model for predicting myelotoxicity of chemotherapeutic agents using IL-3/GM-CSF transgenic humanized mice.

Evaluating myelotoxicity is essential for ensuring the safety of novel drugs before they are approved for clinical applications. Although in vivo prediction of the maximum tolerated doses (MTDs) of anticancer drugs is usually performed in rodents, the results are not always applicable to clinical treatment because drugs may have different effects in human and rodent cells. Previously, we generated a human IL-3 and GM-CSF transgenic humanized mouse (hu-IL-3/GM Tg), in which human granulocytes effectively differentiated after hematopoietic stem cell transplantation. In this study, we established a novel in vivo preclinical evaluation model for predicting human myelotoxicity of anticancer drugs using these hu-IL-3/GM Tg mice. The myelotoxicity was investigated by kinetic flow cytometry of human or murine granulocytes and by colony-forming unit granulocyte/macrophage (CFU-GM) assays. In both in vivo and in vitro analyses, topotecan was more myelotoxic to human than murine granulocytes. In contrast, oxaliplatin was more myelotoxic to murine granulocytes. The level of myelotoxicity of paclitaxel treatment was comparable between human and mouse cells. These results demonstrate that our humanized mouse model can simultaneously evaluate myelotoxicity against human and mouse cells in vivo, and provides an effective preclinical tool for predicting appropriate doses of anticancer agents for clinical treatment.

Induction of squamous cell carcinoma in the rat lung by 1,6-dinitropyrene.

The carcinogenicity of 1-nitropyrene [(1-NP) CAS: 5522-43-0] and 1,6-dinitropyrene [(1,6-DNP) CAS: 42397-64-8] was examined by their direct injection in a beeswax-tricaprylin vehicle into the lung of male F344/DuCrj rats. Of 28 rats given 0.15 mg of 1,6-DNP, 21 (75%) developed squamous cell carcinomas, 2 (7%) developed undifferentiated carcinomas, and 2 (7%) had squamous metaplasias in the lung by 72 weeks. In 32 rats that received 1.5 mg of 1-NP, neither carcinoma nor squamous metaplasia was induced. In all 19 rats (100%) given 0.5 mg of 3-methylcholanthrene [(MCA) CAS: 56-49-5], squamous cell carcinomas were induced earlier than in rats treated with 1,6-DNP. In 1 of 31 rats (3%) given the beeswax-tricaprylin vehicle only, squamous metaplasia was induced. Distant metastases of induced tumors were observed in 4 rats treated with 1,6-DNP and in 1 rat receiving MCA. Two lung tumors induced by 1,6-DNP were successively transplanted into the same strain of rats for 3 generations.

1,6-Dinitropyrene: mutagenicity in Salmonella and carcinogenicity in BALB/c mice.

In tests on the carcinogenicity of 1,6-dinitropyrene [(1,6-DNP) CAS: 42397-64-8] and 1-nitropyrene [(1-NP) CAS: 5522-43-0], 0.1 mg of each compound was inoculated sc into BALB/c mice once a week for 20 weeks. In the group given injections of 1,6-DNP the first tumor appeared on day 112, and 10 of the 20 mice developed tumors at the injection site by 45 weeks after the first injection. However, no tumors were induced in any of the mice that received injections of 1-NP. All of the induced tumors were transplantable for more than five generations in male BALB/c mice. Most of the tumors showed the characteristic histologic features of malignant fibrous histiocytoma.

Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expression.

BACKGROUND: Amplification and rearrangement of the epidermal growth factor receptor (EGFR) gene is frequently associated with malignant gliomas. One type of EGFR mutation in primary gliomas results in overexpression of an aberrant EGFR messenger RNA (mRNA) that lacks sequences of exons II through VI of the human EGFR gene. We observed that the aberrantly spliced EGFR mRNA contains a ribozyme cleavable sequence (5'-AAG GUA AUU-3') created by the joining of EGFR exon I to exon VII. We hypothesized that an appropriately designed ribozyme RNA could mediate site-specific cleavage of the aberrant EGFR mRNA and reduce the growth of aberrant EGFR-producing tumor cells. METHODS: We synthesized aberrant EGFR mRNA substrates and a sequence-specific hammerhead ribozyme (abEGFR-rib) to examine the ribozyme's activity in vitro. We also constructed an abEGFR-rib plasmid and introduced it into ERM5-1 cells, which are murine NIH3T3 cells transfected to express an aberrant EGFR complementary DNA. We measured the growth potential of the cotransfected cells in culture and in nude mice. RESULTS: The synthesized abEGFR-rib efficiently and specifically cleaved aberrant EGFR mRNA substrates in vitro. Expression of the transfected abEGFR-rib suppressed expression of aberrant EGFR mRNA in ERM5-1 cells and reduced the growth of tumors formed by the cotransfected cells in nude mice. Finally, the incorporation of bromodeoxyuridine, a measure of mitotic activity, was also decreased in abEGFR-rib-producing ERM5-1 cells in vivo. CONCLUSION: Ribozymes targeted to aberrant EGFR mRNA can inhibit the growth of tumors formed by cells that express this mRNA.

Species differences in metabolic activation and inactivation of 1-nitropyrene in the liver.

To extrapolate from animal studies to humans the risk of 1-nitropyrene (1-NP), we determined the differences between human and experimental animals in oxidative activation of 1-NP to 1-NP oxides and inactivation of 1-NP oxides by epoxide hydration and glutathione conjugation in hepatic subcellular fractions from 6 species including humans. Species differences were found in both activation of 1-NP and inactivation of 1-NP oxides. 1-Nitro-4,5-dihydro-4,5-epoxypyrene-producing activity was highest in guinea pig and dog, followed by hamster, rat, human, and mouse. 1-Nitro-9,10-dihydro-9,10-epoxypyrene-producing activity was highest in hamster, followed in order by guinea pig, rat, dog, mouse, and human. The ratio of 1-nitro-4,5-dihydro-4,5-epoxypyrene to 1-nitro-9,10-dihydro-9,10-epoxypyrene also varied with the animal species. Hydration of 1-nitro-4,5-dihydro-4,5-epoxypyrene was highest in human, followed by dog, guinea pig, hamster, rat, and mouse. 1-nitro-9,10-dihydro-9,10-epoxypyrene was a poor substrate for epoxide hydrolase in all species. Glutathione conjugation of 1-NP oxides in rodents was higher than that in human and dog. In humans, hepatic microsomes produced the lowest level of 1-NP oxides but hydrolyzed them most efficiently, and glutathione conjugation activity of the cytosol was as low as in dogs, and there was a wide degree of interindividual variations in these activities. No single species studied was a good model for humans, and the balance of activation/inactivation tends toward detoxification in these adult animals.

Mucosa-preferential DNA adduct formation by 2-amino-3-methylimidazo-[4,5-f]quinoline in the rat colonic wall.

The mechanism of mucosa-specific formation of DNA adducts, which was found recently in human intestines, was studied in male F344 rats treated with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). There are three conceivable pathways for p.o. administered IQ to reach the target colonic mucosal cells: pathway 1, through the digestive canal which exposes from the lumenal direction; pathway 2, following enterohepatic circulation re-expose from the lumenal direction; and pathway 3, exposure via blood circulation. To investigate these possible pathways, the following surgical procedures were performed: (a) portal catheterization for IQ administration to eliminate pathway 1 and (b) choledochal catheterization for bile drainage to eliminate pathway 2. When both procedures are combined, only pathway 3 is active. Four types of IQ-DNA adducts were commonly observed in the colons of all experimental groups, with no qualitative difference between the mucosal and muscular layers. When IQ-HCl was administered by p.o. gavage at a dose of 100 mumol/kg body weight, approximately 70% of the IQ-DNA adducts in the colonic mucosa (13.1 +/- 4.3 adducts/10(7) nucleotides) was induced through pathway 1. Pathway 3 induced the remaining 30% of mucosal adducts, producing equal adduct levels in both layers. Pathway 2 did not work for adduct formation. The DNA adduct formation was unaffected in the presence of intestinal flora, indicating that detoxified IQ does not reactivate by floral enzymes. In conclusion, mucosa-specific DNA adduct formation in the colon is caused most likely by the absorption of carcinogens through the lumen.

Intervention of T-cells in transportation of mouse mammary tumor virus (milk factor) to mammary gland cells in vivo.

Using BALB/c nu/nu, BALB/c nu/nufC3H (BALB/c nu/nu mice raised by C3H/HeN foster mother), BALB/c thymus-engrafted BALB/c nu/nufC3H, BALB/c nu/+, and BALB/c nu/+fC3H mice, we examined what kinds of cells are carriers of blood-borne mouse mammary tumor virus (B-MMTV). A radioimmunoassay and an immunoperoxidase assay revealed the presence of MMTV-gp52 antigen in the mammary glands of all BALB/c nu/+fC3H and BALB/c thymus-engrafted BALB/c nu/nufC3H mice (more than 60 days old) but only of some BALB/c nu/nufC3H mice (more than 120 days old): those that possessed a significant number of functional T-cells. BALB/c nu/+ mice did not show the antigen expression at any age. Transfer experiments of cells or plasma from young (less than 12 weeks) BALB/c nu/nufC3H to BALB/c +/+ virgins revealed that cells besides T-cells can also become carriers of B-MMTV. This was confirmed by Southern blotting analyses; exogenous provirus DNA sequences were found in B-cells as well as T-cells of BALB/c nu/+fC3H mice. However, when young BALB/c nu/nu mice were inoculated with BALB/c nu/nufC3H blood, they did not show the MMTV-gp52 antigen expression. Transfer experiments of purified T-cells, B-cells, natural killer cells, and macrophages from BALB/c fC3H mice to BALB/c nu/nu mice revealed that only T-cells have the ability to transfer viral activity to the mammary glands. These results suggest that B-MMTV is carried from the gastrointestinal tract to the mammary glands by lymphoid cells such as T-cells and B-cells, then transferred to the mammary gland cells by the T-cells.

Identification of two common regions of allelic loss in chromosome arm 12q in human pancreatic cancer.

Using the method of microsatellite analysis, we studied 40 tissues with pancreatic ductal adenocarcinoma and identified two commonly deleted regions on the long arm of chromosome 12. One (region A) was found between D12S81 and D12S1719 at 12q21 at a frequency of 67.5%, and the other (region B) was located between D12S360 and D12S78 at 12q22-q23.1 at a frequency of 60%; the latter was reported previously (M. Kimura, et al. Genes Chromosomes Cancer, 17: 88-93, 1996). The results of microsatellite analyses were verified by fluorescence in situ hybridization. We further analyzed 19 pancreatic cancer cell lines by fluorescence in situ hybridization and found that 10 of them showed allelic loss at D12S81 and 6 showed allelic loss at D12S360. Yeast artificial chromosome contigs were constructed to cover the deleted regions. Region B was completely covered by a 650-kb yeast artificial chromosome clone. The frequently deleted regions in chromosome 12q in pancreatic cancer that were identified here may provide new avenues for isolating novel tumor suppressor genes.

Herbimycin A inhibits the association of p60v-src with the cytoskeletal structure and with phosphatidylinositol 3' kinase.

To search for the biochemical prerequisites for morphological transformation by p60v-src, we examined the effect of herbimycin A, a potent inhibitor of cell transformation, on chicken embryonic fibroblasts transformed by Rous sarcoma virus. A small dose of herbimycin (0.1-0.3 micrograms ml-1) was enough to convert the cell morphology to a normal phenotype with a concomitant reassembly of microfilament bundles. In the cells treated with the drug, the majority of the substrates for p60v-src remained phosphorylated and p60v-src was myristylated, membrane associated and fairly active as a protein kinase. Under the same conditions, however, the association of p60v-src with the cytoskeletal structure and with phosphatidylinositol 3' kinase was strongly inhibited, suggesting that the interactions of p60v-src with the cellular structure and the enzyme were indispensable for morphological transformation.

The roles of RNA polymerase and RNAase I in stable RNA degradation in Escherichia coli carrying the srnB+ gene.

In Escherichia coli cells carrying the srnB+ gene of the F plasmid, rifampin, added at 42 degrees C, induces the extensive rapid degradation of the usually stable cellular RNA (Ohnishi, Y., (1975) Science 187, 257-258; Ohnishi, Y., Iguma, H., Ono, T., Nagaishi, H. and Clark, A.J. (1977) J. Bacteriol. 132, 784-789). We have studied further the necessity for rifampin and for high temperature in this degradation. Streptolydigin, another inhibitor of RNA polymerase, did not induce the RNA degradation. Moreover, the stable RNA of some strains in which RNA polymerase is temperature-sensitive did not degrade at the restrictive temperature in the absence of rifampin. These data suggest that rifampin has an essential role in the RNA degradation, possibly by the modification of RNA polymerase function. A protein (Mr 12 000) newly synthesized at 42 degrees C in the presence of rifampin appeared to be the product of the srnB+ gene that promoted the RNA degradation. In a mutant deficient in RNAase I, the extent of the RNA degradation induced by rifampin was greatly reduced. RNAase activity of cell-free crude extract from the RNA-degraded cells was temperature-dependent. The RNAase was purified as RNAase I in DEAE-cellulose column chromatography and Sephadex G-100 gel filtration. Both in vivo and with purified RNAase I, a shift of the incubation mixture from 42 to 30 degrees C, or the addition of Mg2+ ions, stopped the RNA degradation. Thus, an effect on RNA polymerase seems to initiate the expression of the srnB+ gene and the activation of RNAase I, which is then responsible for the RNA degradation of E. coli cells carrying the srnB+ gene.

The pnd gene in E. coli plasmid R16: nucleotide sequence and gene expression leading to cell Mg2+ release and stable RNA degradation.

The pnd gene promotes the degradation of stable RNA in the presence of rifampicin at 42 degrees C, but is repressed during normal growth (Ohnishi, Y. and Akimoto, S. (1980) J. Bacteriol. 144, 833-835). We have determined the sequence of a third srnB-pnd-type gene, and have analyzed the effects of its expression from an inducible promoter. The nucleotide sequence of the pnd gene of the R16 plasmid exhibits an open reading frame for a polypeptide with 50 amino-acid residues, with high sequence homology to the pnd gene of a plasmid (R483) of a different incompatibility group. A possible base-paired stem and loop structure, which may participate in the regulation of gene expression, was detected between the promoter and the initiation codon, analogous to that in two comparable genes, srnB in the F and pnd in the R483 plasmid. When bacterial cells containing a lac-pnd fusion plasmid were incubated with a lac inducer at 30 degrees C, magnesium was released from the cells in bulk, and spheroplasts of the cells lysed even in hypertonic solution. Furthermore, when Mg2+ efflux was inhibited in the medium containing 5 mM Mg2+ or in Tris-HCl buffer, the degradation of stable RNA at 42 degrees C was inhibited. These results suggest that expression of the pnd gene effects a release of cellular magnesium by a membrane alterations, resulting in the stable RNA degradation at a higher temperature.

Expression of srnB gene of F plasmid by altered RNA polymerase in Escherichia coli.

Degradation of otherwise stable rRNA and tRNA takes place in the presence of rifampin, dependent on the F plasmid srnB gene. We have reported that a protein newly synthesized in the presence of rifampin might be a product of the srnB gene required for stable RNA degradation (Ito, R. and Ohnishi, Y. (1983) Biochim. Biophys. Acta 739, 27-34). Here we have further studied the mechanism of srnB expression. Among eighteen mutants with altered RNA polymerase, two (TJ2470 (rpoC4) and TJ302 (rpoC56)) showed RNA degradation at high temperature (42 degrees C) when the srnB gene was present. Labeling proteins at 42 degrees C in strain TJ2470 indicated that a protein of molecular weight 12 000 was a product of the srnB gene, and that expression of the srnB gene provoked RNA degradation. Using plasmid pTK4, in which the srnB gene is inserted downstream of the promoter of lacZ, lac promoter-dependent expression of the srnB gene, with production of the putative protein product, also induced RNA degradation at 42 degrees C, with no requirement for added rifampin or altered RNA polymerase. RNA degradation in these conditions was quite similar to that in the case of the addition of rifampin; e.g., it showed some responses to Mg2+, temperature and RNAase I content of the cells. Expression of the srnB gene dependent on lac promoter was also observed in minicells. Thus, it is inferred that the srnB gene is probably repressed under normal conditions with its own promoter; its expression initiates RNA turnover.

Plasmid genes increase membrane permeability in Escherichia coli.

The membrane permeability to o-nitrophenyl beta-D-galactoside is increased in the presence of rifampicin in Escherichia coli cells carrying srnB+ or pnd+ plasmids, but not in the cells carrying srnB- or pnd- mutant plasmids. The same permeability alteration was also observed at 42 degrees C when a rpoC4- mutant strain was used as a host strain in the absence of rifampicin. These results and the blockage of the effects by action of chloramphenicol suggest that the increase of permeability to o-nitrophenyl galactoside was caused by the expression of srnB+ or pnd+ gene, respectively. srnB+ gene expression leads to massive RNA degradation, probably through the activation of the rna+ gene product. In an rna- strain carrying the srnB+ plasmid, the extent of RNA degradation was reduced, whereas the permeability to o-nitrophenyl galactoside was increased to the same level as in the rna+ strain. Also, the increase in permeability to o-nitrophenyl galactoside was observed at 30 degrees C, although high-temperature incubation (42 degrees C) was necessary for the induction of RNA degradation. These results suggest that the alteration in permeability is a more direct effect of the expression of srnB+ or pnd+ gene and that the RNA degradation is a secondary phenomenon caused by the alteration in the membrane.