Nanopore analysis of tethered peptides

Peptides of 12 amino acids were tethered via a terminal cysteine to mono‐, di‐, tri‐, and tetrabromomethyl‐substituted benzene to produce bundles of one to four peptide strands (CY12‐T1 to CY12‐T4, respectively). The interaction of the bundles with the α‐hemolysin pore was assessed by measuring the blockade currents (I) and times (T) at an applied potential of ? 50, ? 100, and ? 150 mV. Three types of events could be distinguished: bumping events, with small I and short T where the molecule transiently interacts with the pore before diffusing away; translocation events, where the molecule threads through the pore with large I and the value of T decreases with increasing voltage; and intercalation events, where the molecule transiently enters the pore but does not translocate with large I and the value of T increases with increasing voltage. CY12‐T1 and CY12‐T2 gave only bumping and translocation events; CY12‐T3 and CY12‐T4 also gave intercalation events, some of which were of very long duration. The results suggest that three uncoiled peptide strands cannot simultaneously thread through the α‐hemolysin pore and that proteins must completely unfold in order to translocate. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Microcinematographic Studies of Mycoplasma hominis Cells

Cells of two strains of Mycoplasma hominis growing in liquid medium on a glass surface were observed continuously, and cinematographic pictures were taken. Most of the observed structures showed reversible changes of their shape, suggesting the presence of contractile material in membrane or cytoplasma. The frequency and speed of such variations were measured. The deformations seem to be related to multiplication. The mechanisms of these phenomena are unknown.     

Numerical analysis of Leptospira DNA-restriction endonuclease patterns

Abstract Fifteen test strains, representing 5 leptospire serogroups were studied by DNA-restriction endonuclease ( Eco RI) analysis. The resulting patterns for each strain were scanned by a densitometer and the absorbance values were recorded along the pattern at regular intervals. The absorbance profiles were compared using the correlation coefficient and the similarity matrix was clustered by the UPGMA algorithm. The results were displayed as a simplified dendrogram. The numerical analysis of DNA-restriction endonuclease patterns can be used successfully in the identification of Leptospira strains.     

Phospholipases of Leptospira. I. Presence of phospholipase A1 and lysophospholipase in Leptospira biflexa

The hydrolysis of phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and trioleoylglycerol by Leptospira biflexa strain Urawa was studied in vitro. Phospholipase A₁ was identified by the formation of ³²P- and ¹⁴C-labeled lyso-derivatives from ³²P-phosphatidylcholine, ³²P-phosphatidylethanolamine, or 1-acyl-2-[1-¹⁴C]oleoyl-sn-glycero-3-phosphorylcholine. Phospholipase A₁ activity was independent of lipase in the microorganism since ¹⁴C-labeled trioleoylglycerol was scarcely attacked under the same conditions in which the phospholipids were hydrolyzed. Lysophospholipase activity was also demonstrated using ³²P- and non-labeled lysophosphatidylcholine. The activity of phospholipase A₁ was found in a broad range of pH but no optimal pH was determined. The pH optimum of lysophospholipase was 8.0. Both enzymes were labile to heat. Phospholipase C activity, however, could not be detected because no radioactive di- and monoacylglycerol was found in the experiment with 1-acyl-2-[1-¹⁴C]-oleoyl-sn-glycero-3-phosphorylcholine as the substrate. It was inferred that phosphatidylethanolamine, which was the major component of phospholipids in leptospirae, was hydrolyzed serially by phospholipase A (A₁ and/or A₂?) and lysophospholipase to glycerophosphorylethanolamine via 2-acyl-type-lyso-derivative as one metabolic pathway of the substrate.     

Microcinematographic study of pancreatic islet cells from fetal cattle]

The growth characteristics of pancreatic islet cell cultures of bovine fetuses were investigated by the method of time-lapse cinemicrography. These monolayer cultures consisted of epithelial cells only. Under the influenc of high glucose concentration in the growth medium (up to 300 mg per 100 ml) there occurred an activation of mitochondrial apparatus of the islet cells, stimulation of cytogranulokinesis, and intensification of accumulation of the secretory granules with the subsequent degranulation of the islet cells. These data were compared with the results of cytological control of fixed and stained culture.     

Behavioral analysis of polarization vision in tethered flying locusts

For spatial navigation many insects rely on compass information derived from the polarization pattern of the sky. We demonstrate that tethered flying desert locusts (Schistocerca gregaria) show e-vector-dependent yaw-torque responses to polarized light presented from above. A slowly rotating polarizer (5.3° s^–1) induced periodic changes in yaw torque corresponding to the 180° periodicity of the stimulus. Control experiments with a rotating diffuser, a weak intensity pattern, and a stationary polarizer showed that the response is not induced by intensity gradients in the stimulus. Polarotaxis was abolished after painting the dorsal rim areas of the compound eyes black, but remained unchanged after painting the eyes except the dorsal rim areas. During rotation of the polarizer, two e-vectors (preferred and avoided e-vector) induced no turning responses: they were broadly distributed from 0 to 180° but, for a given animal, were perpendicular to each other. The data demonstrate polarization vision in the desert locust, as shown previously for bees, flies, crickets, and ants. Polarized light is perceived through the dorsal rim area of the compound eye, suggesting that polarization vision plays a role in compass navigation of the locust.     

Nucleotide sequence analysis of the Leptospira biflexa serovar patoc rpsL and rpsG genes.

The Leptospira biflexa rpsL and rpsG genes were sequenced. Although similar in many respects, proteins encoded by these L. biflexa genes had several unusual features when compared with homologous proteins of other organisms. Unlike the rpsL genes of other eubacteria, the L. biflexa rpsL gene is adjacent to a rpoC-like gene.     

Molecular and phenotypic characterization of Leptospira johnsonii sp. nov., Leptospira ellinghausenii sp. nov. and Leptospira ryugenii sp. nov. isolated from soil and water in Japan

In a previous study, 50 of 132 soil samples collected throughout Japan were found to be Leptospira‐positive. In the present study, three strains identified in the collected specimens, three, E8, E18 and YH101, were found to be divergent from previously described Leptospira species according to 16S ribosomal RNA gene sequence analysis. These three strains have a helical shape similar to that of typical Leptospira and were not re‐isolated from experimental mice inoculated with the cultured strains. Upon 16S ribosomal RNA gene sequence analysis, E8 was found to belong to the intermediate Leptospira species clade and E18 and YH101 to belong to the saprophytic Leptospira species clade. Based on analyses of genome‐to‐genome distances and average nucleotide identity in silico using whole genome sequences and DNA–DNA hybridization in vitro, these isolates were found to be distinct from previously described Leptospira species. Therefore, these three isolates represent novel species of the genus Leptospira for which the names Leptospira johnsonii sp. nov., (type strain E8 ^T, = JCM 32515 ^T = CIP111620 ^T), Leptospira ellinghausenii sp. nov., (type strain E18 ^T, = JCM 32516 ^T = CIP111618 ^T) and Leptospira ryugenii sp. nov., (type strain YH101 ^T, = JCM 32518 ^T = CIP111617 ^T) are proposed.     

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.     

Phylogenetic analysis of Leptospira strains of pathogenic serovars using 23S rDNA gene sequences.

The 23S ribosomal DNAs were amplified from 11 strains of Leptospira interrogans sensu lato by polymerase chain reaction (PCR) and sequenced. The PCR products of about 290-bp DNA fragments indicated more than 97% sequence similarity to each other. The phylogenetic tree based on the 23S ribosomal DNAs obtained in this study revealed that 11 strains of L. interrogans examined composed a cluster distinct to that of L. weilii and L. borgpetersenii, confirming that these strains were similar to strain Moulton of L. interrogans serovar canicola in 23S rDNA sequence.     

A comparative analysis of the attachment of Leptospira interrogans and L. borgpetersenii to mammalian cells

Leptospirosis, the world's most ubiquitous zoonosis, is caused by pathogenic Leptospira. As microbe-host interactions are specific in pathogenesis, it is likely that there are several molecules mediating the attachment of the Leptospira to mammalian cells. In this study, we analysed the attachment of Leptospira interrogans serovar Portlandvere and Leptospira borgpetersenii serovar Jules to untreated HEp-2 cells or HEp-2 cells treated with the various enzymes, lectins or sugars and to integrins αVβ3 and α5β1, relative to control wells. We found that both serovars bound equally well to HEp-2 cells; however, serovar Jules showed a higher level of attachment to integrins. Both serovars showed an increase in attachment to HEp-2 cells coated with lectins peanut agglutinin, Ulex europaeus agglutinin, soybean agglutinin and Erythrina cristagalli agglutinin (p < 0.05); in the case of Concanavalin A, Jules showed an increase, while Portlandvere showed a significant decrease in attachment. Trypsinizing monolayers resulted in a decrease in attachment for both serovars, while when chondroitinase, neuraminidase and heparinase were used an increase in attachment was recorded. Leptospires coated with sugars showed a decrease in attachment. These results show that serovar Jules' general greater affinity for the mediators examined may suggest a greater potential for virulence over serovar Portlandvere.     

Unique organization of Leptospira interrogans rRNA genes.

We cloned Sau3AI fragments containing the rRNA genes for Leptospira interrogans serovar canicola strain Moulton in the BamHI site of lambda EMBL3 bacteriophage DNA. Physical maps of the fragments were constructed, and the locations of the rRNA genes were determined by Southern blot hybridization and S1 protection. Each fragment of the 23S or the 16S rRNA gene contained at least one copy of the 23S or the 16S sequence. Genomic hybridization showed that there were two genes for the 23S rRNA and the 16S rRNA but only one gene for the 5S rRNA on the chromosome of L. interrogans. The results revealed the important fact that each rRNA gene is located far from the other rRNA genes. Our findings, accordingly, also suggest that these rRNA genes are expressed independently in this organism.     

Genome conservation in isolates of Leptospira interrogans.

Reference strains for each of the 23 serogroups of Leptospira interrogans yielded different pulsed-field gel electrophoresis patterns of NotI digestion products. This was also the case for the 14 serovars belonging to serogroup Icterohaemorrhagiae (with one exception). The NotI restriction patterns of 45 clinical leptospiral isolates belonging to serovar icterohaemorrhagiae were analyzed and compared with those of type strains. No differences were observed between isolates from countries of different continents, namely, France, French Guiana, New Caledonia, and Tahiti. The pattern was indistinguishable from that of the reference strain of serovar icterohaemorrhagiae.