Retinal pigment epithelial cells undergoing mitotic catastrophe are vulnerable to autophagy inhibition.

The increased mitochondrial DNA damage leads to altered functional capacities of retinal pigment epithelial (RPE) cells. A previous study showed the increased autophagy in RPE cells caused by low concentrations of rotenone, a selective inhibitor of mitochondrial complex I. However, the mechanism by which autophagy regulates RPE cell death is still unclear. In the present study, we examined the mechanism underlying the regulation of RPE cell death through the inhibition of mitochondrial complex I. We report herein that rotenone induced mitotic catastrophe (MC) in RPE cells. We further observed an increased level of autophagy in the RPE cells undergoing MC (RPE-MC cells). Importantly, autophagy inhibition induced nonapoptotic cell death in RPE-MC cells. These findings indicate that autophagy has a pivotal role in the survival of RPE-MC cells. We next observed PINK1 accumulation in the mitochondrial membrane and parkin translocation into the mitochondria from the cytosol in the rotenone-treated RPE-MC cells, which indicates that increased mitophagy accompanies MC in ARPE-19 cells. Noticeably, the mitophagy also contributed to the cytoprotection of RPE-MC cells. Although there might be a significant gap in the roles of autophagy and mitophagy in the RPE cells in vivo, our in vitro study suggests that autophagy and mitophagy presumably prevent the RPE-MC cells from plunging into cell death, resulting in the prevention of RPE cell loss.

Structural deformation and intertube conductance of crossed carbon nanotube junctions.

We present a first-principles study of the structure and quantum electronic conductance of junctions consisting of two crossed (5,5) single-walled carbon nanotubes. The structures are determined by constrained minimization of total energy at a given force between the two tubes, simulating the effects of substrate-tube attraction or an applied force. We find that the intertube contact distance is very sensitive to the applied force in the range of 0--10 nN. The intertube conductance is sizable for realistic deformation expected from substrate interaction. The results explain the recent transport data on crossed nanotubes and show that these systems may be potentially useful as electromechanical devices.

US identification of the anal sphincter complex and levator ani muscle in neonates: infracoccygeal approach.

PURPOSE: To identify the anal sphincter complex and levator ani muscle at transperineal ultrasonography (US) with the infracoccygeal approach. MATERIALS AND METHODS: Infracoccygeal US was performed with a 7-MHz linear-array transducer in 40 healthy neonates. The babies were placed in the supine position, and both legs were drawn up to the chest. The buttocks were accordingly lifted up. The approach site was just inferior to the coccyx and posterior to the anus. Scanning was performed to obtain transverse images of the anorectal area. The thickness of the anal sphincter complex and the puborectalis muscle were measured. RESULTS: Infracoccygeal US revealed the internal anal sphincter (IAS), the external anal sphincter (EAS), and the puborectalis muscle in all babies. The IAS and EAS were depicted as inner and outer hypoechoic ringlike structures, respectively. A round, hyperechoic space (intersphincteral space) was present between the hypoechoic IAS and EAS. The puborectalis muscle was identified as a U-shaped hypoechoic structure. The bulbocavernosus and ischiocavernous muscles were also identified. Mean thicknesses were as follows: IAS, 1.3 mm +/- 0.3 (SD) (range, 0.8-1.9 mm); EAS, 1.6 mm +/- 0.3 (range, 1.2-2.3 mm); and puborectalis muscle, 1.1 mm +/- 0.3 (range, 0.6-1.9 mm). CONCLUSION: Infracoccygeal transperineal US is an excellent diagnostic modality for demonstrating the anal sphincter complex and levator ani muscle in neonates.

Cre/loxP-mediated in vivo excision of large segments from yeast genome and their amplification based on the 2microm plasmid-derived system.

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2microm plasmid-derived ori and Flp/FRT machinery. To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100kb apart. Moreover, 2microm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites. The strain carrying all these inserted elements was perfectly stable. Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2microm ori, as aided by FRT/Flp, provided the amplification function. Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.

Lumbricin I, a novel proline-rich antimicrobial peptide from the earthworm: purification, cDNA cloning and molecular characterization.

A novel antimicrobial peptide was isolated and characterized from the earthworm, Lumbricus rubellus. The antimicrobial peptide was purified to homogeneity by a heparin-affinity column and C18 reverse-phase HPLC, and named lumbricin I. Lumbricin I was a proline-rich antimicrobial peptide of 62 amino acids (15% proline in molar ratio; molecular mass, 7231 Da), whose complete sequence was determined by a combination of peptide sequence and cDNA analysis. The peptide and cDNA sequence analysis revealed that lumbricin I was produced as a precursor form consisting of 76 amino acids, with 14 residues in a presegment and 62 residues in mature lumbricin I. Lumbricin I showed antimicrobial activity in vitro against a broad spectrum of microorganisms without hemolytic activity. In addition, a 29-amino acid peptide, named lumbricin I(6-34), which was derived from residues 6-34 of lumbricin I, showed marginally stronger antimicrobial activity than lumbricin I. Northern blot analysis on total RNA revealed that expression of lumbricin I gene was not induced by bacterial infection, but was constitutively expressed. Furthermore, the expression of lumbricin I gene was specific in adult L. rubellus: Lumbricin I mRNA was detected only in adult L. rubellus, but not in eggs and young L. rubellus.

Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome.

The isolation and amplification of large, predetermined segments of a genome from its host have been explored. The prototype of our approach was the excisional replication of some viruses such as the lambda-lysogen. Similar machinery was used to excise and amplify large genomic segments of Escherichia coli in its host. Two loxP sequences for a site-specific recombinase Cre, together with a conditional replication origin (pi-dependent gamma-ori), were inserted into the genome by homologous recombination at predetermined sites, 50-100 kb apart. Cre and pir200 which encodes the site-specific recombinase Cre and an ori-specific replication protein pi, respectively, were also introduced into the genome. The predetermined genomic segments flanked by the loxP sequences were excised and amplified upon induction of the cre and pir200 genes which were under the control of the tet promoter. This excised and amplified DNA could be easily purified as a large plasmid. This procedure can provide an alternative to conventional cloning methods by obtaining predetermined large genomic segments directly from the original organisms. In this study, using the Cre/loxP site-specific recombination and pi/gamma-ori replication system of plasmid R6K, a procedure was devised that could isolate a large segment of the E. coli genome and demonstrated the feasibility of the procedure by excising and amplifying the 50-kb trg-narZ and 100-kb trg-hipA regions of the E. coli W3110 genome.