Whole-embryo culture of Drosophila : development of embryonic tissues in vitro

Summary We have devised techniques to culture whole, dissected embryos of Drosophila melanogaster. We examine multiple aspects of the morphological and physiological development of the epidermis, musculature, nervous system, and internal organs in this cultured preparation, and show that in vitro development closely parallels normal embryogenesis. These techniques permit a wide range of experimental manipulations during embryogenesis and allow us to extend observations through late embryonic stages, after cuticle deposition. Applications of this technique are presented.     

Abscisic acid biosynthesis during corn embryo development

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro-and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.Abbreviations ABA abscisic acid - DAP days after pollination     

Nitrogen assimilation in field-grown winter wheat: Direct measurements of nitrate reduction in roots using15N

Summary Nitrate fertiliser labelled with¹⁵N was applied to a field grown crop of winter wheat. Uptake and assimilation of fertiliser nitrate was studied by monitoring the appearance of labelled nitrate and labelled amino acids in the xylem sap. Shortly after applying¹⁵N-nitrate to the soil about 30 per cent of recently absorbed¹⁵N was in the reduced form, indicating that roots of cereal crops can make a substantial contribution in reducing nitrate. Seasonal changes in crop growth andin vivo NRA are also described.     

Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

The reduction of methemerythrin by e-aq and CO2- from pulse radiolysis studies.

The rate constants for reduction of methemerythrin from Phascolopsis gouldii and Themiste pyroides by hydrated electrons are 2.0 and 3.9 x 10(9) M(-1)s(-1), respectively, at pH 8.2, I = 0.03 M, and 25 degrees C. There is only a small increase in rate when the pH is lowered to 6.3 and a very small decrease when the ionic strength is raised to 0.1 M. Adding azide ion (to form the met-azide adduct) has little effect on the reactivity towards e-aq. For the monomer form, metmyohemerythrin from T. pyroides, the reaction rate constant is 4.5 x 10(9) M(-1)s(-1). Methemerythrin from T. pyroides reacts with CO2- with a rate constant 6.8 x 10(7) M(-1)s(-1). The reactivity sequence e-aq greater than CO2- greater than SO2- (from dithionite reduction) towards methemerythrin is the same as that observed with reduction of heme proteins but the rate constants are some 10 to 100 times smaller for the former. Only 10 to 20% of the e-aq or CO2- radicals generated effect reduction of the iron centers in methemerythrin.     

Tracing lake trophic history with a chironomid–total phosphorus inference model

1. In the absence of historical water chemistry data, predictive biological indicator groups preserved in lake sediments can be employed to reconstruct the history of lake eutrophication. Diatoms are well established in this role, but to augment diatom‐based inferences of nutrient status we investigate the potential use of chironomid midges (Insecta: Chironomidae). 2. Canonical correspondence analysis (CCA) of modern chironomid assemblages in surface sediments from 44 lakes in the English Midlands and Wales, U.K., shows that five environmental variables (total phosphorus (TP), bottom dissolved oxygen, maximum lake depth, Secchi depth and surface water temperature) make a statistically significant (P < 0.05) contribution to explaining the variance in the chironomid data, of which TP makes the largest contribution (29%). 3. The relationship is used to develop a series of weighted averaging (WA) and partial least squares (PLS), (WA‐PLS) models to infer log₁₀TP. The models are evaluated by leave‐one‐out (jack‐knifing) cross‐validation. The simplest minimal adequate model is provided by WA with unweighted inverse deshrinking of root mean square error of prediction (RMSEP_jack=0.34 and r²_jack=0.60). 4. Using this model, the trophic history of Betton Pool, Shropshire, U.K., is reconstructed from the mid‐19th century to the present day and the results from the chironomid‐TP model are compared with inferences from a diatom‐TP model ( 13 ). Both reconstructions suggest that there was a gradual rise in TP since 1850 AD until about 1974, followed by a more pronounced and rapid increase that has continued until the present. Inferred TP values from the WA chironomid inference model agree with diatom‐inferred values. 5. The study demonstrates that fossil chironomid assemblages can be used to investigate quantitatively the trophic history of lakes.     

Diurnal variation in vascular function: role of sleep

Although vascular function is lower in the morning than afternoon, previous studies have not assessed the influence of prior sleep on this diurnal variation. The authors employed a semiconstant routine protocol to study the contribution of prior nocturnal sleep to the previously observed impairment in vascular function in the morning. Brachial artery vascular function was assessed using the flow-mediated dilation technique (FMD) in 9 healthy, physically active males (mean ± SD: 27 ± 9 yrs of age), at 08:00 and 16:00 h following, respectively, 3.29 ± .37 and 3.24 ± .57 h prior sleep estimated using actimetry. Heart rate and systolic and diastolic blood pressures were also measured. The data of the experimental sleep condition were compared with the data of the "normal" diurnal sleep condition, in which FMD measurements were obtained from 21 healthy individuals who slept only during the night, as usual, before the morning test session. The morning-afternoon difference in FMD was 1 ± 4% in the experimental sleep condition compared with 3 ± 4% in the normal sleep condition (p =?.04). This difference was explained by FMD being 3 ± 3% lower in afternoon following the prior experimental sleep (p =?.01). These data suggest that FMD is more dependent on the influence of supine sleep than the endogenous circadian timekeeper, in agreement with our previous finding that diurnal variation in FMD is influenced by exercise. These findings also raise the possibility of a lower homeostatic "set point" for vascular function following a period of sleep and in the absence of perturbing hemodynamic fluctuation.     

BGX: a fully Bayesian integrated approach to the analysis of Affymetrix GeneChip data

We present Bayesian hierarchical models for the analysis of Affymetrix GeneChip data. The approach we take differs from other available approaches in two fundamental aspects. Firstly, we aim to integrate all processing steps of the raw data in a common statistically coherent framework, allowing all components and thus associated errors to be considered simultaneously. Secondly, inference is based on the full posterior distribution of gene expression indices and derived quantities, such as fold changes or ranks, rather than on single point estimates. Measures of uncertainty on these quantities are thus available. The models presented represent the first building block for integrated Bayesian Analysis of Affymetrix GeneChip data: the models take into account additive as well as multiplicative error, gene expression levels are estimated using perfect match and a fraction of mismatch probes and are modeled on the log scale. Background correction is incorporated by modeling true signal and cross-hybridization explicitly, and a need for further normalization is considerably reduced by allowing for array-specific distributions of nonspecific hybridization. When replicate arrays are available for a condition, posterior distributions of condition-specific gene expression indices are estimated directly, by a simultaneous consideration of replicate probe sets, avoiding averaging over estimates obtained from individual replicate arrays. The performance of the Bayesian model is compared to that of standard available point estimate methods on subsets of the well known GeneLogic and Affymetrix spike-in data. The Bayesian model is found to perform well and the integrated procedure presented appears to hold considerable promise for further development.