Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano-powders

The potential of an organic monolith with incorporated titanium dioxide (TiO(2)) and zirconium dioxide (ZrO(2)) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.     

Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using “off-the-shelf” microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with these methods.     

Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Down syndrome (DS) resulting from free trisomy 21 (FT21) has been largely associated with advanced maternal age. However, approximately 60% of FT21 cases are born to young couples. Thus, the etiological factors responsible for these FT21 children must differ from those proposed for maternal age-related FT21. These factors have not been defined. In this study, we analyzed the chromosomes of peripheral blood lymphocytes from three groups of couples aged ≤35 years, to identify chromosomal trisomies: Group I included 5 couples with normal offspring; Group II included 22 couples with one FT21 child; and Group III consisted of 3 couples with recurrent FT21. A total of 13,809 metaphases were analyzed with G-banding and 60,205 metaphases were analyzed with FISH using a 13/21 centromeric probe. Aneuploidy was significantly more frequent in Groups II and III. The frequencies of hyperdiploid cells were 0.19, 0.49 and 0.96% in Groups I–III, respectively. FISH analysis showed that trisomy 21 cell percentages were 0.08, 0.21 and 0.76 for Groups I–III, respectively, and were very similar to those obtained with G-banding. Trisomy 21 mosaicism was found in 2/22 couples with one FT21 offspring, and in 2/3 couples with recurrent FT21. Our data suggest that mosaicism is an important cause of FT21 offspring in young couples, and that aneuploidy is more frequent among couples with FT21 offspring. This may be related with age and other undetermined intrinsic and extrinsic factors.     

The gene for juvenile hyaline fibromatosis maps to chromosome 4q21

Juvenile hyaline fibromatosis (JHF) is an autosomal recessive condition characterized by multiple subcutaneous nodular tumors, gingival fibromatosis, flexion contractures of the joints, and an accumulation of hyaline in the dermis. We performed a genomewide linkage search in two families with JHF from the same region of the Indian state of Gujarat and identified a region of homozygosity on chromosome 4q21. Dense microsatellite analyses within this interval in five families with JHF who were from diverse origins demonstrate that all are compatible with linkage to chromosome 4q21 (multipoint LOD score 5.5). Meiotic recombinants place the gene for JHF within a 7-cM interval bounded by D4S2393 and D4S395.     

Microbial reduction and precipitation of vanadium by Shewanella oneidensis

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V(V)). The bacterium reduces V(V) (vanadate ion) to V(IV) (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V(IV)-containing solid.     

Specificity of binding of the plectin actin-binding domain to beta4 integrin

Plectin is a major component of the cytoskeleton and links the intermediate filament system to hemidesmosomes by binding to the integrin beta4 subunit. Previously, a binding site for beta4 was mapped on the actin-binding domain (ABD) of plectin and binding of beta4 and F-actin to plectin was shown to be mutually exclusive. Here we show that only the ABDs of plectin and dystonin bind to beta4, whereas those of other actin-binding proteins do not. Mutations of the ABD of plectin-1C show that Q131, R138, and N149 are critical for tight binding of the ABD to beta4. These residues form a small cavity, occupied by a well-ordered water molecule in the crystal structure. The beta4 binding pocket partly overlaps with the actin-binding sequence 2 (ABS2), previously shown to be essential for actin binding. Therefore, steric interference may render binding of beta4 and F-actin to plectin mutually exclusive. Finally, we provide evidence indicating that the residues preceding the ABD in plectin-1A and -1C, although unable to mediate binding to beta4 themselves, modulate the binding activity of the ABD for beta4. These studies demonstrate the unique property of the plectin-ABD to bind to both F-actin and beta4, and explain why several other ABD-containing proteins that are expressed in basal keratinocytes are not recruited into hemidesmosomes.     

Are Seafood PCB Data Sufficient to Assess Health Risk for High Seafood Consumption Groups?

Some populations in Washington's Puget Sound area consume much more seafood than the general population. Mean consumption rates are 61?g/person/day for the Tulalip and Squaxin Island Tribes and 117.2?g/person/day for Asian and Pacific Islanders (API). There is concern about possible health risks from seafood PCB exposure for these groups, but exposure evaluation is difficult due to inadequacies of environmental data. Available seafood PCB data were matched to results from recent seafood consumption surveys. To rate quality of matches and identify data gaps, a ranking system based on species specificity, data quality, and location compatibility was developed. Sensitivity of total PCB and congenerspecific PCB testing (for use in TCDD toxic equivalency approaches) necessary for cancer risk assessment was explored and included in the ranking scheme. For the Squaxin Tribe, appropriate total PCB data for risk assessment were available for 58% of seafood consumed, which is dominated by local salmon. For API, appropriate total PCB data were identified for only 4% of seafood consumed, which is dominated by commercial shellfish. Insufficient sensitivity of commercial seafood PCB analysis and overall lack of sufficiently sensitive PCB congener analysis are major gaps in ability to characterize PCB exposure and risk for these groups.     

Predictive systems ecology

Human societies, and their well-being, depend to a significant extent on the state of the ecosystems that surround them. These ecosystems are changing rapidly usually in response to anthropogenic changes in the environment. To determine the likely impact of environmental change on ecosystems and the best ways to manage them, it would be desirable to be able to predict their future states. We present a proposal to develop the paradigm of predictive systems ecology, explicitly to understand and predict the properties and behaviour of ecological systems. We discuss the necessary and desirable features of predictive systems ecology models. There are places where predictive systems ecology is already being practised and we summarize a range of terrestrial and marine examples. Significant challenges remain but we suggest that ecology would benefit both as a scientific discipline and increase its impact in society if it were to embrace the need to become more predictive.     

B-Type Natriuretic Peptide-Induced Delayed Modulation of TRPV1 and P2X3 Receptors of Mouse Trigeminal Sensory Neurons

Important pain transducers of noxious stimuli are small- and medium-diameter sensory neurons that express transient receptor vanilloid-1 (TRPV1) channels and/or adenosine triphosphate (ATP)-gated P2X3 receptors whose activity is upregulated by endogenous neuropeptides in acute and chronic pain models. Little is known about the role of endogenous modulators in restraining the expression and function of TRPV1 and P2X3 receptors. In dorsal root ganglia, evidence supports the involvement of the natriuretic peptide system in the modulation of nociceptive transmission especially via the B-type natriuretic peptide (BNP) that activates the natriuretic peptide receptor-A (NPR-A) to downregulate sensory neuron excitability. Since the role of BNP in trigeminal ganglia (TG) is unclear, we investigated the expression of BNP in mouse TG in situ or in primary cultures and its effect on P2X3 and TRPV1 receptors of patch-clamped cultured neurons. Against scant expression of BNP, almost all neurons expressed NPR-A at membrane level. While BNP rapidly increased cGMP production and Akt kinase phosphorylation, there was no early change in passive neuronal properties or responses to capsaicin, α,β-meATP or GABA. Nonetheless, 24 h application of BNP depressed TRPV1 mediated currents (an effect blocked by the NPR-A antagonist anantin) without changing responses to α,β-meATP or GABA. Anantin alone decreased basal cGMP production and enhanced control α,β-meATP-evoked responses, implying constitutive regulation of P2X3 receptors by ambient BNP. These data suggest a slow modulatory action by BNP on TRPV1 and P2X3 receptors outlining the role of this peptide as a negative regulator of trigeminal sensory neuron excitability to nociceptive stimuli.