Linear DNA must have free ends to transform rat cells efficiently

Summary We have observed that failure to remove certain restriction enzymes after digestion reduced the transforming ability of DNA from 10- to 50-fold. The DNA found integrated in the transformed cells isolated under these conditions had lost little or no sequences. We interpret these results as indicating that certain restriction enzymes remain bound to the DNA ends after digestion, thus generating a substrate unfavorable both for integration and exonucleolytic degradation. As expected from this interpretation, removal of the restriction enzymes before transfection restored the full transforming ability of linear DNA, but also resulted in the integrated sequences being significantly shorter than the transfected DNA. These findings strongly argue for the hypothesis that integration of linear DNA by illegitimate recombination requires free ends and further suggest that exonucleolytic degradation of such ends may generate a preferred substrate for integration. Finally, a comparison of the sequences found integrated after transfection with circular or linear molecules, led us to conclude that circular molecules need not be linearized to become integrated.     

In situ adsorption for enhanced alkaloid production byCatharanthus roseus

Summary A significant fraction (10–40%) of the indole alkaloids produced byCatharanthus roseus was observed to be secreted into the medium. When a neutral polymeric resin, known to adsorb these alkaloids, was added to the cultivation medium, the accumulation of total indole alkaloids and the specific alkaloids, ajmalicine and serpentine were stimulated. Sorbent addition was also observed to result in increased ratios of ajmalicine to serpentine, which suggests the potential of using in situ adsorption to direct metabolism toward a specific product or intermediate within a given pathway.     

The Association between Regional Environmental Factors and Road Trauma Rates: A Geospatial Analysis of 10 Years of Road Traffic Crashes in British Columbia,Canada

BackgroundBritish Columbia, Canada is a geographically large jurisdiction with varied environmental and socio-cultural contexts. This cross-sectional study examined variation in motor vehicle crash rates across 100 police patrols to investigate the association of crashes with key explanatory factors.MethodsEleven crash outcomes (total crashes, injury crashes, fatal crashes, speed related fatal crashes, total fatalities, single-vehicle night-time crashes, rear-end collisions, and collisions involving heavy vehicles, pedestrians, cyclists, or motorcyclists) were identified from police collision reports and insurance claims and mapped to police patrols. Six potential explanatory factors (intensity of traffic law enforcement, speed limits, climate, remoteness, socio-economic factors, and alcohol consumption) were also mapped to police patrols. We then studied the association between crashes and explanatory factors using negative binomial models with crash count per patrol as the response variable and explanatory factors as covariates.ResultsBetween 2003 and 2012 there were 1,434,239 insurance claim collisions, 386,326 police reported crashes, and 3,404 fatal crashes. Across police patrols, there was marked variation in per capita crash rate and in potential explanatory factors. Several factors were associated with crash rates. Percent roads with speed limits ≤ 60 km/hr was positively associated with total crashes, injury crashes, rear end collisions, and collisions involving pedestrians, cyclists, and heavy vehicles; and negatively associated with single vehicle night-time crashes, fatal crashes, fatal speeding crashes, and total fatalities. Higher winter temperature was associated with lower rates of overall collisions, single vehicle night-time collisions, collisions involving heavy vehicles, and total fatalities. Lower socio-economic status was associated with higher rates of injury collisions, pedestrian collisions, fatal speeding collisions, and fatal collisions. Regions with dedicated traffic officers had fewer fatal crashes and fewer fatal speed related crashes but more rear end crashes and more crashes involving cyclists or pedestrians. The number of traffic citations per 1000 drivers was positively associated with total crashes, fatal crashes, total fatalities, fatal speeding crashes, injury crashes, single vehicle night-time crashes, and heavy vehicle crashes. Possible explanations for these associations are discussed.ConclusionsThere is wide variation in per capita rates of motor vehicle crashes across BC police patrols. Some variation is explained by factors such as climate, road type, remoteness, socioeconomic variables, and enforcement intensity. The ability of explanatory factors to predict crash rates would be improved if considered with local traffic volume by all travel modes.     

Long-term study (1974-1998) of seasonal changes in the phytoplankton in Lake Geneva: a multi-table approach

The monitoring of Lake Geneva began after one decade of eutrophicationand has provided a uniform set of phytoplankton data. This studyaimed to define the mean annual pattern of the seasonal successionsof phytoplankton species, and to determine whether inter-annualdistortions in this seasonal structure occur. We analysed the25 annual patterns using the STATIS multi-table method. Thephytoplankton successions in the first part of the year fittedwell with the pattern predicted by the Plankton Ecology Group(PEG) model. But the temporal evolution in the summer phytoplanktoncommunity differed from the PEG model, and was subject to between-yearsdifferences. We identified three homogeneous periods for theannual patterns: 1974–1985, 1986–1991 (except 1988)and 1992–1996 (including 1988). During the first period,phytoplankton succession followed the reference annual pattern,the typical autumnal community was missing during the secondperiod, and this autumn community developed earlier during thethird period. This study reflects the good ability of the phytoplanktoncommunity of Lake Geneva to resist both inter-annual fluctuationsand brutal shifts in the annual functioning of the system. Webelieve these shifts resulted from a gradual change in environmentalparameters, including the deepening of the phosphorus-depletedlayer and a change in its timing as a result of the long-termmeteorological changes.     

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.     

Interaction of casein kinase 1 delta (CK1 delta) with the light chain LC2 of microtubule associated protein 1A (MAP1A)

CK1delta, a member of the casein kinase 1 family of serine/threonine specific kinases, has been shown to be involved in the regulation of microtubule dynamics. We have now identified a 176 aa fragment of the light chain LC2 of MAP1A (termed LC2-P16) specifically interacting with CK1delta. Two CK1delta interacting domains of LC2 were identified, located between aa 2629 and 2753 close to aa 2683 and between aa 2712 and 2805 of LC2. The two regions necessary for the interaction of LC2 with CK1delta have been mapped between aa 76-103 and aa 351-375 of CK1delta. Furthermore, LC2 has been identified as a new substrate of CK1delta. We therefore propose a model in which CK1delta could modulate microtubule dynamics by changing the phosphorylation status of the light chain LC2 of MAP1A.     

Antiviral effects of pan-caspase inhibitors on the replication of coxsackievirus B3

The induction of apoptosis during coxsackievirus B3 (CVB3) infection is well documented. In order to study whether the inhibition of apoptosis has an impact on CVB3 replication, the pan-caspase inhibitor Z-VAD-FMK was used. The decreased CVB3 replication is based on reduced accumulation of both viral RNA and viral proteins. These effects are due to an inhibitory influence of Z-VAD-FMK on the proteolytic activity of the CVB3 proteases 2A and 3C, which was demonstrated by using the target protein poly(A)-binding protein (PABP). The antiviral effect of the structurally different pan-caspase inhibitor Q-VD-OPH was independently of the viral protease inhibition and resulted in suppression of virus progeny production and impaired release of newly produced CVB3 from infected cells. A delayed release of cytochrome c into the cytoplasm was detected in Q-VD-OPH-treated CVB3-infected cells pointing to an involvement of caspases in the initial steps of mitochondrial membrane-permeabilization.