Validation of bacterial replication termination models using simulation of genomic mutations

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point.     

Ultrastructural stability under high temperature or intensive light stress conferred by a small heat shock protein in cyanobacteria

The role and sub-cellular localization of the small heat shock protein HspA under stress conditions was investigated comparing the cyanobacterium Synechococcus strain ECT16-1, which constitutively expresses HspA, with the reference strain ECT. The ultrastructure of ECT cells under elevated temperature or intensive light stress exhibited severe damage including aggregation of cytosol and disordered thylakoid membranes, but in ECT16-1 cells these ultrastructural changes were much less conspicuous. Immunocytochemical studies showed that the main localization of HspA in the ECT16-1 cells shifted from the thylakoid area to the cytoplasm, then back to thylakoid area during the heat stress. Expression of HspA stabilized the morphology of nucleoids. The results are discussed, in particular with respect to the unique property of HspA to associate with thylakoid membranes.     

Inhibition of poly (ADP-ribose) polymerase and caspase-3, but not caspase-1, prevents apoptosis and improves spatial memory of rats with twice-repeated cerebral ischemia

The effect of inhibition of PARP [(poly (ADP-ribose) polymerase], caspase-3 and caspase-1 on twice-repeated ischemia-induced apoptosis and memory impairment were examined. The twice repeated ischemia was induced by four-vessel occlusion method in which a 10 min ischemic episode was repeated once after 60 min. The spatial memory was assessed using 8-arm radial maze. The results of this study showed that the repeated ischemia impaired memory and induced apoptosis in hippocampus CA1 field after 7 days. Moreover, 3-aminobezamide (10 mg/kg i.v.), a PARP inhibitor, and Ac-DEVD-CHO (8.4 microg/5 microL i.c.v., bilaterally), a caspase-3 inhibitor, decreased apoptosis by 45% and 58% respectively. Both drugs reduced the error choices, but 3-aminobezamide additionally increased the correct choices and improved the memory when either drug was injected immediately after the ischemic insult. The results also showed that inhibition of interleukin-1beta-converting enzyme, ICE (caspase-1) by Z-ASP-DCB-CH2 (100 microg/kg i.c.v., bilaterally) neither decreased apoptosis (13% reduction) nor improved memory of the ischemic rats. These results suggest that direct inhibition of PARP and caspase-3, but not of caspase-1, prevents apoptosis and improves spatial memory impaired by repeated ischemia.     

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.     

Immunohistochemical characterization of brush cells in the rat larynx

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.     

Asymmetric Reduction of 4-Oxoisophorone Using Immobilized Thermophilic Bacteria Under Conditions of Controlled Cell Growth

Asymmetric reduction of 2,6,6,-trimethyl-2-cyclohexene-l,4-dione (4-oxoisophrone) to (6R)-2,2,6-trimethyl-1,4-cyclohexane-dione((3R)-dihydro-4-oxoisophorone) was catalysed by immobilized thermophilic bacteria, Thermomonospora curvata JTS 321. Because of leakage of entrapped cells from gel beads during reactions using culture medium, we optimized the medium to allow the microbial conversion under conditions of controlled cell growth. Of the media screened, liver infusion medium was found to be the most suitable and microbial conversion was achieved without cell leakage from the immobilized gels. Immobilized T. curvata cells were repeatedly used for the asymmetric reduction of 4-oxoisophorone, more than 15 times, with an extent of conversion of 50%.     

Identification of IL-18 and Th17 cells in salivary glands of patients with Sjögren's syndrome, and amplification of IL-17-mediated secretion of inflammatory cytokines from salivary gland cells by IL-18

IL-18 is a proinflammatory cytokine and plays an important pathogenic role in inflammatory and autoimmune disorders. IL-17 is also a proinflammatory cytokine and IL-17-secreting Th17 cells are involved in autoimmunity. However, the pathological roles of IL-18 and Th17 cells in Sj?gren's syndrome (SS) remain to be elucidated. This study showed that the expression of IL-18 was detected in acinar cells, intraducts, and CD68(+) macrophages in salivary glands of SS patients, but not in those of healthy subjects or patients with chronic graft-vs-host disease, by immunohistochemistry, and immunoblot analysis revealed that 24-kDa precursor form of IL-18 (proIL-18) and 18-kDa mature IL-18 were detected in SS salivary glands. The majority of the infiltrating cells in the salivary glands of SS patients were CD4(+) T cells, and CD8(+) T cells were infiltrated to a lesser extent. The predominant expression of IL-17 was found in infiltrating CD4(+) T cells, whereas a small number of infiltrating CD8(+) T cells expressed IL-17. Human salivary gland HSY and acinar AZA3 cells constitutively expressed proIL-18 and caspase-1, and a calcium ionophore A23187 induced the secretion of IL-18 from the cells. HSY and AZA3 cells expressed IL-18R and IL-17R on the cell surface, and IL-18 amplified the secretion of IL-6 and IL-8 that were induced by low amounts of IL-17. Primary salivary gland cells from normal subjects partially confirmed these findings. These results suggest that IL-18 and Th17 cells detected in the salivary glands in SS patients are associated with the pathogenesis of SS in the salivary glands.     

Deuterium NMR of Raft Model Membranes Reveals Domain-Specific Order Profiles and Compositional Distribution

In this report, we applied site-specifically deuterated N-stearoylsphingomyelins (SSMs) to raft-exhibiting ternary mixtures containing SSM, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (Chol) and successfully acquired deuterium quadrupole coupling profiles of SSM from liquid-ordered (L_o) and liquid-disordered (L_d) domains. To our knowledge, this is the first report that shows detailed lipid chain dynamics separately and simultaneously obtained from coexisting L_o and L_d domains. We also found that the quadrupole profile of the L_o phase in the ternary system was almost identical to that in the SSM-Chol binary mixture, suggesting that the order profile of the binary system is essentially applicable to more complicated membrane systems in terms of the acyl chain order. We also demonstrated that ²H NMR spectroscopy, in combination with organic synthesis of deuterated components, could be used to reveal the accurate mole fractions of each component distributed in the L_o and L_d domains. As compared with the reported tie-line analysis of phase diagrams, the merit of our ²H NMR analysis is that the domain-specific compositional fractions are directly attainable without experimental complexity and ambiguity. The accurate compositional distributions as well as lipid order profiles in ternary mixtures are relevant to understanding the molecular mechanism of lipid raft formation.