2-Methoxy-3-isobutylpyrazine in grape berries and its dependence on genotype

2-Methoxy-3-isobutylpyrazine (MIBP) contributes a bell pepper aroma to many grape cultivars and has a reported aroma threshold of ～2 ng L(-1) in water. The purpose of this study was twofold: (1) develop a procedure using headspace solid phase micro-extraction combined with GC-MS in the selected ion monitoring mode (HS-SPME-GC-MS-SIM) for analysis of MIBP in grape berries, and (2) determine the location of MIBP biosynthesis in grapevines by approach grafting clusters of Vitis vinifera L. cvs Cabernet Sauvignon and Muscat blanc onto each other. The soluble solids and pH of the grape juice/homogenate matrix from different grape berry developmental stages influenced the method precision; therefore, quantification via the method of standard addition was used. Using our developed method, the limit of detection (LOD) and limit of quantitation (LOQ) of MIBP were 0.1 ng L(-1) and 2 ng L(-1), respectively, measured in a model juice and non-MIBP containing Chardonnay juice. Spiked recoveries averaged between 91% and 112% in Cabernet Sauvignon and Pinot noir homogenates and the overall relative standard deviation was less than 10%. The method was used to analyze MIBP in 29 grape cultivars and in fruit from clusters grafted to Cabernet Sauvignon or Muscat vines. Quantifiable levels were found only in Cabernet franc, Cabernet Sauvignon, Merlot, Sauvignon blanc and Semillon, providing information on the genetic connection for the occurrence of MIBP in grapes. No MIBP was detected in the berries of Muscat blanc clusters grafted onto Cabernet Sauvignon vines when sampled at fruit maturity. MIBP was detected in all berries of Cabernet Sauvignon regardless the graft configuration. The data indicate that MIBP or its precursors originate in the berry and its formation depends upon grape genotype.     

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O₂) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni²⁺-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.     

Facial bristle feather histology and morphology in New Zealand birds: Implications for function

Knowledge of structure in biology may help inform hypotheses about function. Little is known about the histological structure or the function of avian facial bristle feathers. Here we provide information on morphology and histology, with inferences for function, of bristles in five predominantly insectivorous birds from New Zealand. We chose species with differing ecologies, including: brown kiwi (Apteryx mantelli), morepork (Ninox novaezealandae), hihi (Notiomystis cincta), New Zealand robin (Petroica australis), and New Zealand fantail (Rhipidura fuliginosa). Average bristle length corrected for body size was similar across species. Bristles occurred in distinct groups on different parts of the head and upper rictal bristles were generally longest. The lower rictal bristles of the fantail were the longest possessed by that species and were long compared to bristles of other species. Kiwi were the only species with forehead bristles, similar in length to the upper rictal bristles of other species, and the lower rictal bristles of fantails. Herbst corpuscles (vibration and pressure sensitive mechanoreceptors) were found in association with bristle follicles in all species. Nocturnal and hole‐nesting birds had more heavily encapsulated corpuscles than diurnal open‐nesting species. Our results suggest that avian facial bristles generally have a tactile function in both nocturnal and diurnal species, perhaps playing a role in prey handling, gathering information during flight, navigating in nest cavities and on the ground at night and possibly in prey‐detection. These differing roles may help explain the observed differences in capsule thickness of the corpuscles. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

A fluorescence-based high-throughput assay for antimicrotubule drugs

With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.     

Measurement of Reactive Oxygen Species in the Culture Media Using Acridan Lumigen PS-3 Assay

Reactive oxygen species (ROS) are generated continuously during aerobic metabolism. ROS are highly reactive molecules and in excessive amounts, can lead to protein and DNA oxidation, protein cross-linking, and cell death. Cell-culture models provide a valuable tool in understanding the mechanisms that lead to cell death. Accumulation of ROS within cells and/or their release into the culture media are highly cell type-specific. The ability to estimate ROS levels in the culture media is an important step in understanding the mechanisms contributing to disease processes. In this paper, we describe the optimization of a simple method to estimate ROS levels in the culture media using the Acridan Lumigen PS-3 reagent provided in the Amersham ECL Plus kit (GE Healthcare, UK). We have shown that the Acridan Lumigen PS-3 assay generates ROS-specific chemiluminescence in fresh as well as media stored at −20°C, in as little as 10–20 μl of samples. The method was able to detect the dose (of stimulants)- and time (acute and chronic)-dependent changes in ROS levels in media collected from various cell types. Our results suggest that the kit reagents, PBS buffer, and various media did not contribute significantly to the overall chemiluminescence generated in the assay; however, we suggest that the unused medium specific for each cell type should be used as blanks and final readings of test samples normalized against these readings. As this method uses commonly available laboratory equipment and commercially available reagents, we believe this assay is convenient, economical, and specific in estimating ROS released extracellularly into the culture media.     

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.