On the Process of Cellular Division in Escherichia coli VI. Use of a Methocel-Autoradiographic Method for the Study of Cellular Division in Escherichia coli

A technique allowing chain formation by Escherichia coli for the analysis of the segregation of certain cellular components by autoradiographic methods has been developed. Deoxyribonucleic acid labeled with (3)H-thymidine does not appear to segregate preferentially with one or another of the bacteria. Membrane lipids labeled with either (3)H-oleate or (3)H-glycerol are evenly distributed among all progeny cells. A similar pattern of distribution of cell wall material is observed when (3)H-diaminopimelate is used as the marker.     

In vivo evidence for translesion synthesis by the replicative DNA polymerase δ

The intolerance of DNA polymerase δ (Polδ) to incorrect base pairing contributes to its extremely high accuracy during replication, but is believed to inhibit translesion synthesis (TLS). However, chicken DT40 cells lacking the POLD3 subunit of Polδ are deficient in TLS. Previous genetic and biochemical analysis showed that POLD3 may promote lesion bypass by Polδ itself independently of the translesion polymerase Polζ of which POLD3 is also a subunit. To test this hypothesis, we have inactivated Polδ proofreading in pold3 cells. This significantly restored TLS in pold3 mutants, enhancing dA incorporation opposite abasic sites. Purified proofreading-deficient human Polδ holoenzyme performs TLS of abasic sites in vitro much more efficiently than the wild type enzyme, with over 90% of TLS events resulting in dA incorporation. Furthermore, proofreading deficiency enhances the capability of Polδ to continue DNA synthesis over UV lesions both in vivo and in vitro. These data support Polδ contributing to TLS in vivo and suggest that the mutagenesis resulting from loss of Polδ proofreading activity may in part be explained by enhanced lesion bypass.     

Structure and ligand binding properties of myoglobins reconstituted with monodepropionated heme: functional role of each heme propionate side chain

Two heme propionate side chains, which are attached at the 6 and 7 positions of the heme framework, are linked with Arg45 and Ser92, respectively, in sperm whale myoglobin. To evaluate the role of each propionate, two kinds of one-legged hemins, 6-depropionated and 7-depropionated protohemins, were prepared and inserted into the apomyoglobin to yield two reconstituted proteins. Structural data of the reconstituted myoglobins were obtained via an X-ray crystallographic analysis at a resolution of 1.1-1.4 A and resonance Raman spectroscopy. It was found that the lack of the 6-propionate reduces the number of hydrogen bonds in the distal site and clearly changes the position of the Arg45 residue with the disrupting Arg45-Asp60 interaction. In contrast, the removal of the 7-propionate does not cause a significant structural change in the residues of the distal and proximal sites. However, the resonance Raman studies suggested that the coordination bond strength of the His93-Fe bond for the protein with the 7-depropionated protoheme slightly increases compared to that for the protein with the native heme. The O2 and CO ligand binding studies for the reconstituted proteins with the one-legged hemes provide an important insight into the functional role of each propionate. The lack of the 6-propionate accelerates the O2 dissociation by ca. 3-fold compared to those of the other reconstituted and native proteins. The lack of the 7-propionate enhances the CO affinity by 2-fold compared to that of the protein with the native heme. These results indicate that the 6-propionate clearly contributes to the stabilization of the bound O2, whereas the 7-propionate plays an important role in the regulation of the Fe-His bond.     

A human homolog of Drosophila warts tumor suppressor, h-warts, localized to mitotic apparatus and specifically phosphorylated during mitosis.

We identified a human homolog of Drosophila warts tumor suppressor gene, termed h-warts, which was mapped at chromosome 6q24-25.1. The h-warts protein has a serine/threonine kinase domain and is localized to centrosomes in interphase cells. However, it becomes localized to the mitotic apparatus, including spindle pole bodies, mitotic spindle, and midbody, in a highly dynamic manner during mitosis. Furthermore, h-warts is specifically phosphorylated in cells at mitotic phase, most likely by Cdc2 kinase. These findings suggest that h-warts functions as a component of the mitotic apparatus and is involved in proper progression of mitosis.     

Apoptosis observed in BALB/3T3 cells having ingested Staphylococcus aureus.

Staphylococcus aureus was previously shown to be internalized by murine fibroblast. We examined the intracellular events of S. aureus ingested by BALB/3T3 cells. After uptake of strains A191 and A151, isolates from atopic lesion, and a laboratory strain, Cowan I, for 1 hr, BALB/3T3 cells were incubated with 1.25 microg/ml lysostaphin. Laddering of the DNA in multiples of approximately 180 bp occurred within 4 hr following bacterial addition in BALB/3T3 cells infected with A191 and within 18 hr in BALB/3T3 cells infected with A151: histochemical staining by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method revealed that the rate of the fragmentation of nucleic DNA in Cowan I-infected BALB/3T3 cells at 21 hr following bacterial addition was 0.52 +/- 0.25%, significantly higher than that in the control cells. Transmission electron micrographs of BALB/3T3 cells at 4 hr following A191 addition showed that the apoptotic features, including electron-dense nucleus and plasma membrane blebbing, occurred in some cells in which many staphylococci escaped the endosome and went on to cell division. At the same time, A151 organisms enclosed with endosome membrane were static in the intact BALB/3T3 cells. The significant increase of A191 was confirmed by counting intracellular live bacteria during 2- to 6-hr incubation. These results suggest that internalized S. aureus escapes the endosome, multiplies and induces apoptosis in the fibroblast cell.     

Inhibitory effect of iron‐oxidizing bacteria on ferrous‐promoted chalcopyrite leaching

It is generally accepted that iron‐oxidizing bacteria, Thiobacillus ferrooxidans, enhance chalcopyrite leaching. However, this article details a case of the bacteria suppressing chalcopyrite leaching. Bacterial leaching experiments were performed with sulfuric acid solutions containing 0 or 0.04 mol/dm³ ferrous sulfate. Without ferrous sulfate, the bacteria enhance copper extraction and oxidation of ferrous ions released from chalcopyrite. However, the bacteria suppressed chalcopyrite leaching when ferrous sulfate was added. This is mainly due to the bacterial consumption of ferrous ions which act as a promoter for chalcopyrite oxidation with dissolved oxygen. Coprecipitation of copper ions with jarosite formed by the bacterial ferrous oxidation also causes the bacterial suppression of copper extraction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 478–483, 1999.     

Histone-like DNA binding protein of Streptococcus intermedius induces the expression of pro-inflammatory cytokines in human monocytes via activation of ERK1/2 and JNK pathways

Streptococcus intermedius is a commensal associated with serious, deep-seated purulent infections in major organs, such as the brain and liver. Histone-like DNA binding protein (HLP) is an accessory architectural protein in a variety of bacterial cellular processes. In this study, we investigated the mechanisms of pro-inflammatory cytokine inductions in THP-1 cells by stimulation with recombinant HLP of S. intermedius (r Si -HLP). r Si -HLP stimulation-induced production of pro-inflammatory cytokines (IL-8, IL-1β and TNF-α) occurred in a time- and dose-dependent manner. In contrast with the heat-stable activity of DNA binding, the induction activity of r Si -HLP was heat-unstable. In subsequent studies, r Si -HLP acted cooperatively with lipoteichoic acid, the synthetic Toll-like receptor 2 agonist, Pam3CSK4, and the cytosolic nucleotide binding oligomerization domain 2 receptor agonist, muramyldipeptide. Furthermore, Western blot and blocking assays with specific inhibitors showed that r Si -HLP stimulation induced the activation of cell signal transduction pathways, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). In addition to its physiological role in bacterial growth through DNA binding, these results indicate that Si -HLP can trigger a cascade of events that induce pro-inflammatory responses via ERK1/2 and JNK signal pathways, and suggest that bacterial HLP may contribute to the activation of host innate immunity during bacterial infection.     

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.