Quick replication fork stop by overproduction of Escherichia coli DinB produces non-proliferative cells with an aberrant chromosome.

Escherichia coli dinB encodes the translesion DNA polymerase DinB, which can inhibit progression of replication forks in a dose-dependent manner, independent of exogenous DNA damage. We reported previously that overproduction of DinB from a multicopy dinB plasmid immediately abolished ongoing replication fork progression, and the cells rapidly and drastically lost colony-forming ability, although the mechanisms underlying this lethality by severe replication fork stress remained unclear. Here, we show that the reduced colony-forming ability in the dinB-overexpressing cells is independent of the specific toxin genes that trigger programmed bacterial cell death when replication is blocked by depletion of the dNTP pool. After DinB abolished replication fork progression and colony-forming ability, most of the cells were still viable, as judged by fluorescent dye staining, but contained irregularly shaped nucleoids in which chromosomal DNA was preferentially lost in the replication terminus region relative to the replication origin region. Flow cytometric analysis of the cells revealed chromosomal damage and the eventual appearance of cell populations with less than single-chromosome DNA content, reminiscent of sub-G1 cells with lethal DNA content produced during eukaryotic apoptosis. This reduced DNA content was not observed after replication fork progression was quickly stopped in temperature-sensitive dnaB helicase mutant cells at a non-permissive temperature. Thus, the quick replication stop provoked by excess DinB uniquely generates temporarily viable but non-reproductive cells possessing a fatally depleted chromosomal content, which may represent one of the possible fates of an E. coli cell whose replication is overwhelmingly compromised.

Overproduction of Escherichia coli DNA polymerase DinB (Pol IV) inhibits replication fork progression and is lethal.

Escherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.

Degradation of nuclear DNA by DNase II-like acid DNase in cortical fiber cells of mouse eye lens.

The eye lens is composed of fiber cells that differentiate from epithelial cells on its anterior surface. In concert with this differentiation, a set of proteins essential for lens function is synthesized, and the cellular organelles are degraded. DNase II-like acid DNase, also called DNase IIbeta, is specifically expressed in the lens, and degrades the DNA in the lens fiber cells. Here we report that DNase II-like acid DNase is synthesized as a precursor with a signal sequence, and is localized to lysosomes. DNase II-like acid DNase mRNA was found in cortical fiber cells but not epithelial cells, indicating that its expression is induced during the differentiation of epithelial cells into fiber cells. Immunohistochemical and immunocytochemical analyses indicated that DNase II-like acid DNase was colocalized with Lamp-1 in the lysosomes of fiber cells in a relatively narrow region bordering the organelle-free zone, and was often found in degenerating nuclei. A comparison by microarray analysis of the gene expression profiles between epithelial and cortical fiber cells of young mouse lens indicated that some genes for lysosomal enzymes (cathepsins and lipases) were strongly expressed in the fiber cells. These results suggest that the lysosomal system plays a role in the degradation of cellular organelles during lens cell differentiation.

[Spinal anesthesia in a parturient complicated with idiopathic thrombocytopenic purpura].

A 27-yr-old parturient with idiopathic thrombocytopenic purpura was scheduled to undergo resection of a left ovarian cyst at 15 weeks gestation. Platelet counts were between 46,000 and 64,000.microliter-1, bleeding time was 2 min, and she denied having unusual bleeding diathesis. As the patient was reluctant to receive general anesthesia for fear of latent adverse effects of the drugs on the fetus, we selected spinal anesthesia and the perioperative course was uneventful. However, it is questionable to perform regional anesthesia in patients with coagulation disorders, for spinal hematomas leading to paraplegia can be a rare but devastating complication of regional anesthesia. According to our extensive literature review, it was revealed that platelet insufficiency, both in terms of function and count, did not represent a major risk factor for spinal hematomas associated with regional anesthesia, especially for spinal anesthesia. We suggest that spinal anesthesia may be safely performed in patients if their platelet counts exceed around 50,000.microliter-1.

[Successful intravenous administration of low dose ketamine for pain caused by erythromelalgia: report of a case].

A 15-year-old female complained of reddening, edema, and pain in her hands and feet. The symptoms were relieved upon cooling. From these findings, a diagnosis of erythromelalgia was made. Because none of the oral medication prescribed by dermatologist was effective, the patient was consulted to our department. A low dose of ketamine, a drug considered to be effective for intractable pain, was administered intravenously and the pain subsided significantly. Furthermore, the pain became completely controllable with a combination of intramuscular ketamine injection and other oral medication.