The central region of RepE initiator protein of mini-F plasmid plays a crucial role in dimerization required for negative replication control.

The RepE protein (251 residues, 29 kDa) of mini-F plasmid, mostly found as dimers, plays a key role in mini-F replication. Whereas monomers bind to the origin to initiate replication, dimers bind to the repE operator to repress its own transcription. Among the host factors required for mini-F replication, a set of molecular chaperones (DnaK, DnaJ and GrpE) is thought to facilitate monomerization of RepE dimers. To further understand the structural basis of functional differentiation between the two forms of RepE, we examined the region(s) critical for dimerization by isolation and characterization of RepE mutants that were defective in autogenous repressor function. Such mutations were isolated from two separate regions of RepE, the central region (residues 111 to 161) and the C-terminal region (residues 195 to 208). The central region overlapped the region where the chaperone-independent copy-up mutations were previously isolated (residues 93 to 135). Likewise the mini-F mutant plasmids, carrying the mutations in the central region, could replicate in a dnaK null mutant host. One of them, S111P (111th serine changed to proline), showed a very high origin-binding activity vis-à-vis a severely reduced operator-binding activity, much like the RepE54 (R118P) mutant previously shown to form only monomers. Gel filtration and chemical crosslinking studies with purified RepE revealed that S111P primarily formed monomers, whereas other mutant proteins formed mostly dimers. On the other hand, analysis of deletion mutants revealed that the N-terminal 42 and the C-terminal 57 residues were dispensable for dimerization. Thus, the region spanning residues 93 to 161 of RepE (including Ser111 and Arg118) appeared to be primarily involved in dimerization, contributing to the negative regulation of plasmid replication.

Gentian mosaic virus: A New Species in the Genus Fabavirus.

ABSTRACT A viral isolate, designated N-1 and obtained from a gentian (Gentiana scabra) plant that exhibited mosaic symptoms, was transmitted mechanically to nine plant species in six families. These plants are known as hosts of fabaviruses. The N-1 isolate was composed of isometric particles 30 nm in diameter and included two RNA molecules of approximately 6.0 and 3.6 kb in length, as estimated by agarose gel electrophoresis. The RNAs were encapsidated separately in two of the three types of particle. Each particle contained two distinct proteins with Mr values of 39.3 x 10(3) and 26.6 x 10(3), as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of complete nucleotide sequences of the RNAs suggested that each encoded a single large polyprotein, in which putative functional proteins were arranged in a manner similar to those in Broad bean wilt virus 1 (BBWV-1) and Broad bean wilt virus 2 (BBWV-2), which are members of the genus Fabavirus (family Comoviridae). Analysis of the deduced amino acid sequences of the proteins indicated that those of isolate N-1 shared 38 to 66% identity with those of BBWV-1 and BBWV-2 but only 16 to 42% identity with those of a comovirus, Cowpea mosaic virus. Phylogenetic analysis, based on the amino acid sequences of RNA polymerase, placed isolate N-1 in a separate lineage from BBWV-1 and BBWV-2. In indirect-enzyme-linked immunosorbent assay, isolate N-1 exhibited distant serological relationship to BBWV-1, BBWV-2, and Lamium mild mosaic virus, another fabavirus. Our results suggest that N-1 represents a new species of Fabavirus. We propose the name Gentian mosaic virus for this new species.

The iteron regions necessary for the RepE-iteron interaction in vivo in mini-F plasmids of Escherichia coli.

We have determined the nucleotide positions of an incC iteron essential for RepE binding by analyzing mutated incC iterons defective in exerting incompatibility towards mini-F plasmids. The mutations affecting this incompatibility occurred mostly at two positions within the incC iteron, i.e. an iteron conserved position and a mini-F specific position. Most of the iterons with a base-change at either of these two positions had lost the binding affinity for RepE. This agrees with the crystallographic structure of the RepE-iteron complex which showed that the N and C terminal domains of RepE interact with the two major grooves on one face of the iteron DNA. These grooves contain the iteron conserved and mini-F specific positions necessary for RepE binding. Thus the binding mode may be common to in the case of mini-F like plasmids.

[Anesthetic management of a patient with Coffin-Lowry syndrome].

Coffin-Lowry syndrome (CLS) is characterized by mental retardation, a peculiar face and deformities of the thorax and spine. A 33-year-old female with Coffin-Lowry syndrome (CLS), further complicated with atrial septal defect and ventricular tachycardia, underwent elective surgery for anterior cervical cyst. As difficult intubation had been anticipated, anesthesia was induced with continuous administration of propofol. After confirming that she could be ventilated by mask, vecuronium bromide, midazolam and fentanyl were given. The operation and anesthesia were conducted uneventfully. No complications occurred postoperatively. The use of propofol for slow induction of anesthesia was advantageous for hemodynamic stability in this case.

[Application of modified ultrafiltration to cardiac surgery in adults].

Modified Ultrafiltration (MUF) was developed for blood concentration and reduction of postoperative edema in cardiac surgery in children. Its beneficial effects on postoperative hemodynamics have been reported. We applied MUF to cardiac surgery in adults and evaluated its usefulness. Between August, 1995 and April, 1997, MUF was performed in 41 adult patients. MUF was carried out immediately after the cessation of cardiopulmonary bypass. The mean fluid volume removed was 1,135.9 +/- 274.1 ml. The patient's haematocrit significantly increased from 23.2 +/- 2.6% to 26.9 +/- 3.2% (p < 0.0001). The dose of inotropes administered was maintained constant during MUF, and no changes were observed in CVP and the heart rate. However, the systolic blood pressure increased from 99.5 +/- 14.7 to 113.2 +/- 16.2 mmHg (p < 0.0001) and cardiac index from 4.2 +/- 0.9 to 4.9 +/- 1.3 l/min/m2 (p = 0.0006). It was suggested that MUF was an useful technique of haemoconcentration and appeared to have beneficial effects on postoperative hemodynamics in adult cardiac surgery.

Nociceptin system does not affect MAC of volatile anaesthetics.

BACKGROUND: Nociceptin is the endogenous agonist of the opioid receptor-like (ORL) 1 receptor (NOP), and both nociceptin and NOP are widely expressed in the brain and spinal cord, which are target organs of general anaesthetics. As nociceptin has been reported to be involved in modulating pain mechanisms and stress responses, it is possible that the activity of the nociceptin system affects the anaesthetic potency of general anaesthetics. To address this possibility, we investigated the minimum alveolar concentrations (MACs) of various volatile anaesthetics in nociceptin receptor knockout mice (NOP-/-) and wild-type mice (NOP+/+). METHODS: We used male NOP-/- mice and NOP+/+ mice. MACs for halothane, isoflurane and sevoflurane were determined by the tail-clamp method. RESULTS: MACs for halothane, isoflurane and sevoflurane in NOP-/- mice were 1.60 (SD 0.06), 1.68 (0.08) and 3.36 (0.07)%, respectively. In NOP+/+ mice, MACs for halothane, isoflurane and sevoflurane were 1.59 (SD 0.07), 1.72 (0.07) and 3.38 (0.09)%, respectively. CONCLUSION: MACs in NOP-/- mice did not significantly differ from those in NOP+/+ mice for halothane, isoflurane and sevoflurane. This result suggests that the nociceptin system does not affect the anaesthetic potency of volatile anaesthetics.

Regulation of DNA replication by iterons: an interaction between the ori2 and incC regions mediated by RepE-bound iterons inhibits DNA replication of mini-F plasmid in Escherichia coli.

In bacteria, plasmids and some DNA viruses, DNA replication is initiated and regulated by binding of initiator proteins to repetitive sequences. To understand the control mechanism we used the plasmid mini-F, whose copy number is stringently maintained in Escherichia coli, mainly by its initiator protein RepE and the incC region. The monomers of RepE protein bound to incC iterons, which exert incompatibility in trans and control the copy number of mini-F plasmid in cis. Many incompatibility defective mutants carrying mutations in their incC iterons had lost the affinity to bind to RepE, while one mutant retained high level binding affinity. The mutated incC mini-F plasmids lost the function to control the copy number. The copy number of the wild-type mini-F plasmid did not increase in the presence of excess RepE. These results suggested that the control of replication by incC iterons does not rely on their capacity to titrate RepE protein. Using a ligation assay, we found that RepE proteins mediated a cross-link structure between ori2 and incC, for which the dimerization domain of RepE and the structure of incC seem to be important. The structure probably causes inhibition of extra rounds of DNA replication initiation on mini-F plasmids, thereby keeping mini-F plasmid at a low copy number.

Opioid potentiation of N-type Ca2+ channel currents via pertussis-toxin-sensitive G proteins in NG108-15 cells.

Opioids have both inhibitory and stimulatory effects on neurotransmitter release. While the inhibitory effect has been ascribed to presynaptic inhibition of Ca2+ channels, the cellular mechanism underlying the stimulatory effect is not clear. In order to address this issue, we analyzed the effects of [d-Ala2, d-Leu5]-enkephalin (DADLE) on whole-cell Ba2+ currents (IBa) through voltage-gated Ca2+ channels in NG108-15 neuroblastoma x glioma hybrid cells. Application of DADLE inhibited and washout of DADLE transiently potentiated IBa. Furthermore, potentiation of IBa was elicited even in the presence of DADLE, when inhibition was relieved by a large depolarizing prepulse. DADLE-induced potentiation, as well as inhibition, had both voltage-sensitive and -insensitive components and was abolished by treatment with ICI174864, a delta-opioid antagonist, pertussis toxin (PTX) and omega-conotoxin GVIA. Potentiation developed over @3 min and took 5-20 min to recover, whereas inhibition was complete within 30 s and recovered within 1 min. Although this potentiation should contribute to DADLE-induced desensitization of Ca2+ channel inhibition, it was not the sole mechanism for desensitization. We conclude that the delta-opioid receptor exerts a dual action on N-type Ca2+ channels via PTX-sensitive G proteins, i.e., rapid inhibition followed by slowly developing potentiation.

Activation of mu-opioid receptor induces expression of c-fos and junB via mitogen-activated protein kinase cascade.

BACKGROUND: Opioid-induced long-term functional alterations of the nervous system, such as tolerance, addiction, and dependence, conceivably involve changes in gene expression. The authors have previously reported that opioid receptors are functionally coupled to extracellular signal-regulated kinase, a class of the mitogen-activated protein kinase. To address whether activation of the opioid receptor induces changes in gene expression through the activation of extracellular signal-regulated kinase, the authors examined mu-opioid receptor (MOR)-induced immediate early gene expression. METHODS: Chinese hamster ovary cells stably expressing MOR were used. Cells were stimulated by MOR agonists after 24-h serum starvation. Expression of c-fos and junB genes was analyzed by RNA blot hybridization. To explore the mechanism of MOR-mediated c-fos and junB expression, activity of a transcription factor, Elk-1, was assessed by reporter assay. Furthermore, to investigate the functional consequences of c-fos and junB induction, MOR-mediated formation of the functional transcription factor complex AP-1 was examined by reporter assay and electrophoretic mobility shift assay. RESULTS: Mu-opioid receptor activation induced c-fos and junB messenger RNAs, which were inhibited by pretreatment of the cells with pertussis toxin and PD98059, an inhibitor of extracellular signal-regulated kinase cascade. MOR stimulation elevated Elk-1-mediated transcriptional activity by about 10-fold. AP-1-mediated transcriptional activity was stimulated by MOR agonists by about twofold. Electrophoretic mobility shift assay revealed that AP-1 binding activity in the nuclear extract was elevated by MOR activation and further showed that products of c-fos and junB genes are involved in formation of AP-1 complex. CONCLUSIONS: Mu-opioid receptor activation induces c-fos and junB expression and elevates AP-1-mediated transcriptional activities via the mitogen-activated protein kinase cascade.

DNA phase transition promoted by replication initiator.

DNA is flexible and easily subjected to bending and wrapping via DNA/protein interaction. DNA supercoiling is known to play an important role in a variety of cellular events, such as transcription, replication, and recombination. It is, however, not well understood how the superhelical strain is efficiently redistributed during these reactions. Here we demonstrate a novel property of an initiator protein in DNA relaxation by utilizing a one-molecule-imaging technique, atomic force microscopy, combined with biochemical procedures. A replication initiator protein, RepE54 of bacterial mini-F plasmid (2.5 kb), binds to the specific sequences (iterons) within the replication region (ori2). When RepE54 binds to the iterons of the negatively supercoiled mini-F plasmid, it induces a dynamic structural transition of the plasmid to a relaxed state. This initiator-induced relaxation is mediated neither by the introduction of a DNA strand break nor by a local melting of the DNA double strand. Furthermore, RepE54 is not wrapped by DNA repeatedly. These data indicate that a local strain imposed by initiator binding can induce a drastic shift of the DNA conformation from a supercoiled to a relaxed state.