Impact of Diflubenzuron on Bombus impatiens (Hymenoptera: Apidae) Microcolony Development.

Reliance on the honey bee as a surrogate organism for risk assessment performed on other bees is widely challenged due to differences in phenology, life history, and sensitivity to pesticides between bee species. Consequently, there is a need to develop validated methods for assessing toxicity in non-Apis bees including bumble bees. The usefulness of small-scale, queenless colonies, termed microcolonies, has not been fully investigated for hazard assessment. Using the insect growth regulator diflubenzuron as a reference toxicant, we monitored microcolony development from egg laying to drone emergence using the Eastern bumble bee Bombus impatiens (C.), a non-Apis species native to North America. Microcolonies were monitored following dietary exposure to diflubenzuron (nominal concentrations: 0.1, 1, 10, 100, and 1,000 µg/liter). Microcolony syrup and pollen consumption was significantly reduced by diflubenzuron exposure. Pupal cell production was also significantly decreased at the highest diflubenzuron concentration assessed. Ultimately, diflubenzuron inhibited drone production in a concentration-dependent manner and a 42-d 50% inhibitory concentration (IC50) was determined. None of the dietary concentrations of diflubenzuron tested affected adult worker survival, or average drone weight. These data strengthen the foundation for use of this methodology, and provide valuable information for B. impatiens; however, more work is required to better understand the utility of the bumble bee microcolony model for pesticide hazard assessment.

A cost-effective colourimetric assay for quantifying hydrogen peroxide in honey.

Honey is a natural product with many beneficial properties including antimicrobial action. Production of hydrogen peroxide (H2O2) in diluted honey is central to this action. Here, we describe an optimized method for measuring levels of H2O2 in honey. This method is based on established methods, with the level of dilution, the time between dilution and reading the assay, and aeration of the samples during the assay identified as critical points for ensuring reliability and reproducibility. The method is cost-effective and easy to perform using common laboratory equipment. Using this method, we quantified the hydrogen peroxide content of five different, unprocessed polyfloral honeys collected in NC, USA. Our results show that H2O2 production by these honeys varies greatly, with some samples producing negligible levels of H2O2. We assessed the effect of colour on the assay by measuring the recovery of spiked H2O2 from light and dark honey and from serially diluted dark corn syrup, and found the amount of H2O2 that could be detected was lower in dark corn syrup and darker honey samples.

Effect of oxygen on the synthesis and assembly of mitochondrial encoded subunits of cytochrome oxidase and cytochrome b.c1 in mouse embryo fibroblasts.

Mouse embryo fibroblasts were grown in low and control O2 for 24 h (average medium oxygen tensions, 7 torr and 143 torr, respectively). Relative to controls, there was a reduction in radiolabeled subunits in immunoprecipitates of cytochrome oxidase and cytochrome b.c1 prepared from low O2 cells. Incorporation of radiolabeled amino acids into subunit I of cytochrome oxidase and the apocytochrome b protein of the b.c1 complex ranged from 51-100% of control, whereas the appearance of these pulse-labeled subunits into holoenzymes immunoprecipitated from low O2 cells was in the range of 6-39% of control. The synthesis of subunit II of cytochrome oxidase by low O2 cells ranged from 63-100% of control, and assembly of this protein into the low O2 immunoprecipitated enzyme ranged from 15-61% of control. Thus, the data suggest that O2 had an effect on the assembly of these mitochondrially translated proteins that was independent of any effect on their synthesis.

Stimulatory and persistent effect of acute hyperoxia on respiratory gas exchange of the chick embryo.

The hypothesis that oxygen availability limits growth of the normal chick embryo late in development predicts that an increase in oxygen availability would accelerate the rate of growth and, therefore, metabolism. We tested the prediction concerning metabolism by comparing the oxygen consumption (VO2) and carbon dioxide production (VCO2) of 14-18 day embryos acutely exposed to either 50% or 100% O2 with those of normoxic (21% O2) controls. Two hours of hyperoxia produced increases in both VO2 and VCO2; however, repeated measurements over time in normoxia also demonstrated a significant increase in gas exchange, presumably due to normal growth of the embryos. After correcting for the increase in VO2 due to growth, there was no effect of 60% O2 on day 14. Thereafter the stimulatory effect of 60% O2 increased gradually, reaching 6.1% on day 18. VCO2 was 4 to 6% higher in embryos acutely exposed to 60% O2 than in normoxic controls throughout the observation period, although the difference was significant only on day 18. The VO2 of embryos acutely exposed to 100% O2 was not significantly different from that observed in 60% O2, and was still significantly elevated 3 h after the eggs were returned to 21% O2. We conclude that acute hyperoxia late in incubation elicits an increase in embryonic VO2 and VCO2, with little or no effect on the respiratory exchange ratio, and that the stimulation of gas exchange by 100% O2 persists after the embryo is returned to normoxic conditions. These findings support the hypothesis that oxygen availability limits growth and metabolism of the normoxic chick embryo late in development.

O2 effect on composition of chick embryonic heart and brain.

Heart ventricles from chick embryos incubated in 60% O2 (hyperoxia) on the 16th through the 18th days of incubation were 21% heavier than those from control embryos maintained in 21% O2 (normoxia). Heart ventricles from embyros incubated in 15% O2 (hypoxia) were 8% lighter than controls. Changes in ventricular weight were accompanied by proportional changes in protein content (21% more in hyperoxic ventricles; 8% less in hypoxic ventricles). Ventricular tissue DNA content showed a significant increase in hyperoxia. Tissue protein/DNA ratios were significantly higher in hyperoxia and lower in hypoxia. These data suggest that increased O2 availability led to hypertrophy of chick embryo ventricular cells and an increase in the level of DNA synthesis. Cytochrome oxidase activity per mg DNA was 15-25% higher in hyperoxic ventricles than in hypoxic ventricles. This result is consistent with our previous findings that alterations in O2 availability affect the O2 consumption rate of the chick emryo in ovo, and it provides direct evidence that a phenomenon repeatedly observed in vitro is of importance in vivo. In contrast to the heart, O2 availability did not affect the wet weight, protein or DNA contents, or cytochrome oxidase activity of the chick embryo brain.

Vitamin A deficiency leads to increased cell proliferation in olfactory epithelium of mature rats.

We have shown previously that vitamin A deficiency (VAD) leads to the decreased expression of gene products that are specifically synthesized by mature neurons in the olfactory epithelium (OE) of adult rats. These results support the hypothesis that retinoic acid, a derivative of vitamin A, is required for neurogenesis and neuron replacement in vivo. VAD does not cause gross degeneration of the OE, raising the question: what types of cells continue to populate VAD OE? In this study, we compared the cell densities of VAD and VA-sufficient (VAS) OE and investigated whether cell proliferation is upregulated in VAD OE. The results show that (1) total cell number in VAD and VAS OE are comparable; (2) localized areas of hyperplasia are present in the basal regions of VAD, but not VAS, OE; (3) there is a substantial increase in the number of PCNA (proliferating cell nuclear antigen) positive cells in the basal region of VAD OE relative to VAS OE; and (4) there is a relative increase in the levels of mRNA encoding the transcription factor, MASH I, in VAD OE. We conclude that reduced availability of vitamin A derivatives, such as retinoic acid, leads to a loss of control over proliferation, hyperplasia, and increased numbers of pro-neural cells in vivo.

Evolutionary conservation of the AU-rich 3' untranslated region of messenger RNA.

AU-rich sequence motifs (specifically sequences containing reiterations of AUUUA) are found in the 3' untranslated region of mammalian mRNAs encoding cytokines, adhesion molecules, and protooncogenes. Because these AU-rich elements (3'AURE) have been observed to reduce the stability and translational efficiency of transcripts that contain them, and because many of these transcripts accumulate in cells exposed to inflammatory stimuli, we reasoned that mRNAs with 3'AURE may be highly conserved and that the AURE is a marker of mRNAs that are inducible by environmental stressors. To test this hypothesis, we developed a polymerase chain reaction (PCR) strategy to isolate specifically mRNAs with 3'AURE. We first validated the effectiveness of this approach by selectively amplifying two mRNAs containing 3'AURE from interleukin 1 (IL-1)-induced human endothelial cells, then used the same primers in reverse transcriptase-PCR of sea urchin RNA, and used the radiolabeled reaction products to screen a cDNA library prepared from endotoxin-exposed sea urchin coelomocytes. We identified 124 positive clones and isolated a 1608-base-pair fragment that contains an AU-rich consensus sequence upstream from a poly(A) tail. This sea urchin transcript hybridizes with immobilized poly(A)(+)-selected RNA prepared from living coelomocytes maintained in vitro for 8.5-13 h but not with RNA prepared from freshly harvested coelomocytes. Our results provide support for the growing body of evidence that 3' AURE are both conserved and functional and indicate further that isolation and short-term in vitro culture of sea urchin coelomocytes is sufficient to induce the expression of transcripts containing 3'AURE.

Purification and primary structure of Capl, an S-100-related calcium-binding protein isolated from bovine retina.

Calcium protein placental homolog (Capl) is an S-100-related calcium-binding protein selectively expressed in cell lines that have been induced to grow or differentiate. In addition, the expression of Capl correlates with the induction of the metastatic phenotype in tumor cell lines and the transformation of normal cells by activated oncogenes or chemical carcinogens. Although not previously associated with the nervous system, in this study, Capl was purified from bovine neural retina by a combination of phenyl-Sepharose and organomercurial chromatography. The complete amino acid sequence of bovine Capl was established primarily by Edman degradation of peptides generated by cleavage of methionyl, lysyl, glutamyl, and aspartyl bonds. NH2-terminal methionyl and aspartyl peptides were analyzed by tandem mass spectrometry, which provided the sequence of the first 8 residues and identified the NH2-terminal blocking group as an acetyl moiety. The molecular mass of the intact protein determined by electrospray mass spectrometry (M(r) = 11,716.75 +/- 0.42) and the calculated molecular mass deduced from the amino acid composition (M(r) = 11,718) were in agreement, thus supporting the accuracy of the sequence assignment. Capl isolated from the retina was shown to be indistinguishable by mass and immunochemical properties from its counterpart in the bovine aorta, which previously was the only source of purified Capl. Northern analysis using cloned Capl cDNA revealed that Capl mRNA is present not only in the retina but the choroid as well. Further support for choroidal localization came from immunohistochemical experiments using specific anti-Capl antibodies. The physiological significance of Capl in ocular tissues and the aorta is discussed.

Rod outer segment retinol dehydrogenase: substrate specificity and role in phototransduction.

The reaction catalyzed by all-trans-retinol dehydrogenase of rod outer segments completes the quenching of photoactivated rhodopsin and initiates the cycle of reactions leading to regeneration of visual pigment. The goal of this study was to determine the kinetic parameters of the dehydrogenase at physiological levels of bleaching, to investigate its specificity, and to determine its possible role in modulating phototransduction. Reduction of all-trans-retinal could be measured after bleaching < 0.15% rhodopsin. Kinetic parameters for the forward reaction determined with endogenous all-trans-retinal were Km = 1.1 microM; Vmax = 7 nmol/min/mg rhodopsin. The low enzymatic activity suggests that at high bleach rates, all-trans-retinal could accumulate, increasing the steady state level of bleaching intermediates or promoting formation of pseudophotoproducts. Active pseudophotoproducts, which stimulate Gt activation and opsin phosphorylation by rhodopsin kinase, are formed with opsin and all-trans-retinal as well as retinal analogues lacking the 13 methyl or the terminal two carbons of the polyene chain. Addition of all-trans-retinol, NADP, and [32P]ATP to rod outer segments increased rhodopsin phosphorylation. Kinetic parameters for the reverse reaction determined with exogenous all-trans-retinol were Km = 10 microM; Vmax = 11 nmol/min/mg rhodopsin. Our results support the hypothesis that all-trans-retinol dehydrogenase could influence the phototransduction cascade, including activities of Gt, rhodopsin kinase, and binding of arrestin, by impeding the recycling of rhodopsin at high bleach levels.

Characterization and localization of an aldehyde dehydrogenase to amacrine cells of bovine retina.

An enzyme of bovine retina that catalyzes oxidation of retinaldehyde to retinoic acid was purified to homogeneity and a monoclonal antibody (mAb H-4) was generated. MAb H-4 recognized a single component (Mr = 55,000) in extracts of bovine retina and other bovine tissues. The antibody showed no cross-reactivity with extracts of rat, monkey, or human retinas. A 2067 bp cDNA was selected from a retina cDNA expression library using mAb H-4. The cDNA hybridized with a similarly sized, moderately abundant mRNA prepared from bovine retina. Nucleotide sequence analysis indicated that the cDNA contained a single open reading frame encoding 501 amino acids that have 88% sequence identity with the amino-acid sequence of human hepatic Class 1 aldehyde dehydrogenase. Amino-acid sequence analysis of purified enzyme demonstrated that the cDNA encodes the isolated enzyme. MAb H-4 specifically labeled the somata and processes of a subset of amacrine cells in bovine retinal sections. Labeled amacrine somata were located on both sides of the inner plexiform layer, and their processes ramified into two laminae within the inner plexiform layer. The inner radial processes of Müller (glial) cells were weakly reactive with mAb H-4. Weak immunostaining of amacrine cells was found in monkey retina with mAb H-4, but no signal was detected in rat or human retina. The results provide further evidence for metabolism and function of retinoids within cells of the inner retina and define a novel class of retinal amacrine cells.