Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

Potentiation and suppression of mouse liver cytochrome P-450 isozymes during the acute-phase response induced by bacterial endotoxin

Infection and inflammation are known to affect the metabolism and disposition of drugs and carcinogens. We report a detailed study of the effects of bacterial endotoxin on the constitutive and inducible expression and activities of cytochrome P-450 isozymes from families P-450I, P-450IIB, P-450IIC and P-450III. In general high doses of high endotoxin caused very marked suppression of P-450 isozymes and associated activities. However, this effect was differential, the expression of certain isozymes being only slightly reduced whereas others were suppressed to almost undetectable levels. Low doses of endotoxin also gave differential effects on cytochrome P-450 expression. Of particular interest was the very marked potentiation of the inductive effect of both 3-methylcholanthrene and phenobarbital. In the case of 3-methylcholanthrene the 10-fold induction of activity was increased to 24-fold by concomitant endotoxin administration. In this regard it was interesting that 3-methylcholanthrene was an effective inducer of a wide variety of acute-phase proteins including metallothionein, serum amyloid A, fibrinogen and hemopexin. These data show that endotoxin, and therefore bacterial infection and inflammation, can have profound and differential effects on components of the cytochrome-P-450 monooxygenase system which could result in significant changes in susceptibility to the effects of drugs, chemical toxins and carcinogens.     

Maintenance of the cellobiose utilization genes of Escherichia coli in a cryptic state

The genes for cellobiose utilization are normally cryptic in Escherichia coli. The cellobiose system was used as a model to understand the process by which silent genes are maintained in microbial populations. Previously reported was (1) the isolation of a mutant strain that expresses the cellobiose-utilization (Cel) genes and (2) that expression of those genes allows utilization of three beta- glucoside sugars: cellobiose, arbutin, and salicin. The Cel gene cluster has now been cloned from that mutant strain. In the course of locating the Cel genes within the cloned DNA segment, it was discovered that inactivation of the Cel-encoded hydrolase rendered the host strain sensitive to all three beta-glucosides as potent inhibitors. This sensitivity arises from the accumulation of the phosphorylated beta- glucosides. Because even the fully active genes conferred some degree of beta-glucoside sensitivity, the effects of cellobiose on a series of five Cel+ mutants of independent origin were investigated. Although each of those strains utilizes cellobiose as a sole carbon and energy source, cellobiose also acts as a potent inhibitor that reduces the growth rate on glycerol 2.5-16.5-fold. On the other hand, wild-type strains that cannot utilize cellobiose are not inhibited. The observation that the same compound can serve either as a nutrient or as an inhibitor suggests that, under most conditions in which cellobiose will be present together with other resources, there is a strong selective advantage to having the cryptic (Cel0) allele. In those environments in which cellobiose is the sole, or the best, resource, mutants that express the genes (Cel+) will have a strong selective advantage. It is suggested that temporal alternation between these two conditions is a major factor in the maintenance of these genes in E. coli populations. This alternation of environments and fitnesses was predicted by the model for cryptic-gene maintenance that was previously published.      

The role of aquatic moss on community composition and drift of fish-food organisms

Macroinvertebrate density, biomass and drift were studied from moss-covered and moss-free channels in the South Fork Salmon River, Idaho. Insect densities were compared for 10 different substrate types and locations involving moss (Fontinalis neo-mexicana), sand, pebbles and cobbles. An ANOVA test demonstrated that insect densities varied significantly with substrate type (P < 0.05), and that total insect density in moss clumps differed significantly from densities in mineral substrates. Insect densities were 4–18 times greater in moss clumps than in mineral substrates under and adjacent to moss; sands under moss supported the lowest densities. During most tests, densities in pebble and cobble substrates adjacent to moss clumps were not significantly different from those found in similar substrates in the moss-free channel. The 20% moss-covered channel had 1.6 to 7.2 greater insect density and 1.4 to 6.1 greater biomass than did the moss-free channel for the tests conducted. Generally, midges (Chironomidae) made up over 50% of the insect community; annelids were the principal non-insect invertebrates.In spite of greater insect density and biomass in a moss-covered than in the moss-free channel, we did not demonstrate universally increased drift of the immature stages from the moss-covered channel, at least during daylight hours. As a consequence, we infer that salmonid fishes, feeding primarily on drifting insects during the daytime, may not derive increased caloric benefit from moss habitats until the insects emerge as adults.     

Butterfly community structure and landscape composition in agricultural landscapes of the central United States

Agricultural landscapes worldwide are under increased pressure to provide food, feed, fiber, and fuel for a growing human population. These demands are leading to changes in agricultural landscapes and subsequent declines in biodiversity. We used citizen science data from the North American Butterfly Association and remotely-sensed land cover data from the US Department of Agriculture to study relationships between agricultural landscape composition and butterfly community structure in the Midwestern US. Landscape-level butterfly species richness (based on rarefaction estimates) was highest in agricultural landscapes with relatively low amounts of cropland, relatively high amounts of woodland, and intermediate amounts of grassland and wetland. Rarefied richness generally declined with the dominance of any of these land cover types. Unlike other land cover types, urban development had a consistent negative effect on rarefied richness. Butterfly community structure (based on relative abundance) was also significantly related to the amount of cropland, woodland, grassland, and wetland in the landscape. The rarest butterfly species were associated with woodland-, grassland-, and wetland-dominated landscapes, likely due to their association with plant species occurring in savannahs, prairies, and marshes, respectively. Assuming that variation across space reflects changes over time, our results support conclusions from previous studies that removal of natural and seminatural habitats alters butterfly community structure and decreases species diversity in agricultural landscapes.