Quantitative PCR: Procedures and precisions

We develop a multitype branching-process model for the Polymerase Chain Reaction (PCR). We apply the model to a comparison of three methods for estimating the initial number of molecules of target present in a PCR. These three methods are: one which uses a coamplified, internal control; one which uses an external control series; and one which uses simple extrapolation of log outputvs time (no control). We identify assumptions for each method which permit mathematical analysis of bias and precision. All three methods perform well if: (1) replication efficiencies are stable among reactions; (2) other method-specific conditions on efficiencies are met; and (3) product accumulates exponentially throughout the range where it is observed. When replication efficiencies vary among reactions but other optimal conditions for each method hold, the no-control and external-control methods lose precision relative to the internal control method, but they may still perform satisfactorily for many applications. The internal control method continues to perform well even if accumulation of product plateaus. This method depends, however, on a condition we call equivalence of replication efficiencies, the attainability of which in practice remains to be proven.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.     

Insect fungal symbionts: A promising source of detoxifying enzymes

Summary Many species of insects cultivate, inoculate, or contain symbiotic fungi. Insects feed on plant materials that contain plant-produced defensive toxins, or are exposed to insecticides or other pesticides when they become economically important pests. Therefore, it is likely that the symbiotic fungi are also exposed to these toxins and may actually contribute to detoxification of these compounds. Fungi associated with bark beetles, ambrosia beetles, termites, leaf-cutting ants, long-horned beetles, wood wasps, and drug store beetles can variously metabolize/detoxify tannins, lignins, terpenes, esters, chlorinated hydrocarbons, and other toxins. The fungi (Attamyces) cultivated by the ants and the yeast (Symbiotaphrina) contained in the cigarette beetle gut appear to have broad-spectrum detoxifying abilities. The present limiting factor for using many of these fungi for large scale detoxification of, for example, contaminated soils or agricultural commodities is their slow growth rate, but conventional strain selection techniques or biotechnological approaches should overcome this problem.Presented at the Symposium on Fungal Detoxification at the 48th Annual Meeting of the Society for Industrial Microbiology, Philadelphia, PA, August 4–9, 1991.     

High-performance liquid chromatographic and gas—liquid chromatographic determination of diazepam and nordiazepam in plasma

A one-step method for extraction of diazepam, nordiazepam, and internal standard into toluene is followed by chromatographic separation and detection with either dual-wavelength high-performance liquid chromatography or electron-capture gas—liquid chromatography. Agreement between the two methods was excellent for diazepam (r = 0.99, n = 38) and good for nordiazepam (r = 0.96, n = 79) over a concentration range that included subtherapeutic, therapeutic, and toxic plasma levels.     

Une nouvelle sous-famille,les Poulpinae, et quatre nouvelles espèces de Saitoum radiolaires mésozoiques téthysiens

Studies of skeleton elements of several jurassic species of Saitoum allow to compare them with Poulpus from Trias. The sub-family Poulpinae is introduced, caracterised by three cephalic arcs and the collar position of the cephalic structure. Among the jurassic forms, 4 species are newly described: S. corniculum, S. elegans, S. levium and S. trichylum.     

The effects of mine drainage and organic enrichment on benthos in the river nent system,Northern Pennines

A preliminary survey of the benthic fauna of sites on the Nent system affected by elevated zinc levels, acid water and organic enrichment is described. Data on faunal composition are presented for 17 sites. A clustering technique was applied to the taxa/site matrix to demonstrate the degree of association between sites varying in zinc concentration. Chandler biotic scores were found to be lowest at sites affected by high zinc levels and/or dense growths of the alga Stigeoclonium tenue. The role of elevated zinc levels and algal growth in determining the diversity and abundance of benthos is discussed.     

Exercise increases the release of NAMPT in extracellular vesicles and alters NAD + activity in recipient cells

Aging is associated with a loss of metabolic homeostasis, with cofactors such as nicotinamide adenine dinucleotide (NAD⁺) declining over time. The decrease in NAD⁺ production has been linked to the age‐related loss of circulating extracellular nicotinamide phosphoribosyltransferase (eNAMPT), the rate‐limiting enzyme in the NAD⁺ biosynthetic pathway. eNAMPT is found almost exclusively in extracellular vesicles (EVs), providing a mechanism for the distribution of the enzyme in different tissues. Currently, the physiological cause for the release of eNAMPT is unknown, and how it may be affected by age and physical exercise. Here, we show that release of small EVs into the bloodstream is stimulated following moderate intensity exercise in humans. Exercise also increased the eNAMPT content in EVs, most prominently in young individuals with higher aerobic fitness. Both mature fit and young unfit individuals exhibited a limited increase in EV‐eNAMPT release following exercise, indicating that this mechanism is related to both the age and physical fitness of a person. Notably, unfit mature individuals were unable to increase the release of eNAMPT in EVs after exercise, suggesting that lower fitness levels and aging attenuate this important signalling mechanism in the body. EVs isolated from exercising humans containing eNAMPT were able to alter the abundance of NAD⁺ and SIRT1 activity in recipient cells compared to pre‐exercise EVs, indicating a pathway for inter‐tissue signalling promoted through exercise. Our results suggest a mechanism to limit age‐related NAD⁺ decline, through the systemic delivery of eNAMPT via EVs released during exercise.     

Cell specific expression of three genes involved in plasma membrane sucrose transport in developingVicia faba seed

Summary In developing seeds ofVicia faba, transfer cells line the inner surface of the seed coat and the juxtaposed epidermal surface of the cotyledons. Circumstantial evidence, derived from anatomical and physiological studies, indicates that these cells are the likely sites of sucrose efflux to, and influx from, the seed apoplasm, respectively. In this study, expression of an H⁺/sucrose symporter-gene was found to be localised to the epidermal-transfer cell complexes of the cotyledons. The sucrose binding protein (SBP) gene was expressed in these cells as well as in the thin-walled parenchyma transfer cells of the seed coat. SBP was immunolocalised exclusively to the plasma membranes located in the wall ingrowth regions of the transfer cells. In addition, a plasma membrane H⁺-ATPase was most abundant in the wall ingrowth regions with decreasing levels of expression at increasing distance from the transfer cell layers. The observed co-localisation of high densities of a plasma membrane H⁺-ATPase and sucrose transport proteins to the wall ingrowths of the seed coat and cotyledon transfer cells provides strong evidence that these regions are the principal sites of facilitated membrane transport of sucrose to and from the seed apoplasm.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - DIG digoxigenin - H⁺-ATPase plasma membrane H⁺-translocating adenosine triphosphatase - Ig immunoglobulin - LeSUT1 tomato H⁺/sucrose symporter - SBP sucrose binding protein     

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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