A single molecule assay for measuring site-specific DNA cleavage

Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.     

Release,establishment and realised geographic distribution of Diorhabda carinulata and D. elongata (Coleoptera: Chrysomelidae) in California,U.S.A.

This report summarises efforts to establish Diorhabda carinulata (Desbrochers) and D. elongata (Brullé) in California for the control of invasive saltcedars (Tamarix spp.), which degrade riparian ecosystems in the western United States. Over 14,000 D. carinulata individuals were released in California among four locations between 1999 and 2002 but beetles only established at the Tinemaha Reservoir site, the most eastern release location. More than 236,000 D. elongata individuals were released between 13 sites from 2003–2009 and establishment was limited to two sites, along the Cache and Pope creeks in northwestern California. The D. carinulata population did not disperse beyond the release area despite the presence of nearby (ca. 20?km) patches of the host plant. In contrast, D. elongata spread along Cache Creek and branches of related tributaries within the same watershed at ca. 14?km per year. A survey of 122 Tamarix stands across 15 California counties revealed that neither introduced beetle colonised other host patches, including those in neighbouring watersheds. Despite exclusive use of T. parviflora for ca. 36 generations, field collected D. elongata adults demonstrated strong preferences for T. ramosissima over T. parviflora when selecting both resting and ovipositional sites in caged choice tests. The proportion of D. elongata ovipositing on T. parviflora varied over time but with no clear trend of shifting host preference despite strong selection pressure. Explanations for the limited establishment and spread of Diorhabda spp. as well as impact to the target weeds are discussed.     

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.     

Characteristics of the muscle mechanoreflex during quadriceps contractions in humans

We examined muscle sympathetic nerve activity (MSNA) in thenonexercising lower limb during repetitive static quadricepscontraction paradigm at 25% maximal voluntary contraction in eightmen. Subjects performed 20-s contractions with 5-s rest periods for upto 12 contractions. Although the workload was constant, we found that MSNA amplitude rose as a function of contraction number [0.6 ln (amplitude/min)/contraction]; this suggests chemicalsensitization of the muscle reflex response. We employedsignal-averaging techniques and then integrated the data to examine theonset latency of the MSNA response as a function of the 25-scontraction-rest period. We observed an onset latency of ~4-6 s.Moreover, although the onset latency did not appear to vary as afunction of contraction number, the rate of MSNA increase tookapproximately four contractions to reach a steady-state rate of rise;this suggests contraction-induced sensitization. The onset latencyreported here is similar to findings in recent animal studies, but itis at odds with latencies determined in prior human handgripcontraction studies. We believe our data suggest that1) mechanically sensitive afferentscontribute importantly to the MSNA response to the paradigm employedand 2) these afferents may besensitized by the chemical products of muscle contraction.     

Type-A retrovirus particles in heterohybridomas: precaution for human monoclonal antibody purification

Fine structural features of the murine myeloma MHFP-1 and two heterohybridomas secreting human IgM monoclonal antibody were examined. Intracisternal type-A retrovirus particles were found in both MHFP-1 and the heterohydribomas constructed by fusing MHFP-1 and human peripheral blood lymphocytes. The implications of this finding for the purification of human monoclonal antibody for therapeutic applications is discussed.     

Coronary artery embolization in closed-chest canines using flexible radiopaque plugs

A new technique induces localized myocardial infarction in closed-chest dogs by placing discrete plugs in coronary arteries without using cumbersome coaxial catheters or guide wires. Flexible plugs, essential to this method, are formed by extruding a dental impression polymer, rendered radiopaque with sodium iodide, into spaghetti-like strands. Segments of these strands can be injected through a catheter into a selected coronary artery. Contact with blood or saline causes plugs to swell. The mean increase in plug diameter due to swelling was 27 +/- 20%. Eight anesthetized dogs were embolized via carotid approach [6 left anterior descending (LAD), 1 left circumflex (LCX), and 1 LAD and LCX]. Plug positions were monitored fluoroscopically. One animal died at 2 days postembolization. The remaining seven dogs were killed after 14-37 days. Autopsies showed complete vessel occlusion and localized infarction. Infarcts resulting from coronary artery occlusion with one, two, or three plugs involved 2-26% of the left ventricular mass.     

Ten years of vegetation dynamics in two rivulets in Lower Saxony (FRG)

The vegetation dynamics in six permanent plots in two lowland rivulets of the Federal Republic of Germany are analyzed. The year-to-year change in species cover is displayed by means of tables. In each site there are core species (both hydrophytes and helophytes) which have been able to successfully reproduce within the sampling plots over the total observation period. There are also transient hydrophytes which regularly become washed in from the upper course, and transient helophytes growing permanently into the river from the banks.A numerical analysis of the performance of the 12 most frequent and abundant hydrophytes in relation to various independent variables was carried out using canonical correspondence analysis. There is no directional temporal variation within the vegetation data set. The hydrochemical variables were almost constant within the observation period. Rainfall in summer has some influence via discharge and turbidity. Most of the variance in the data set is explained by the position of the sites along the rivers. Most of the residual variance can be explained by a binary disturbance variable. The processes observed can mostly be explained from life history characteristics of the dominant species, particularySparganium emersum, Ranunculus peltatus andPotamogeton natans. The spatial scale of the study site was relatively adequate. A smaller size would have produced noisy data (suggesting erratic change), while a greater size would have produced no change at all. The adequate temporal scale for observation is the comparison of the yearly maxima because of the seasonality of most of the species. An exact prediction of dominance and species composition of the following year is impossible.