2DE identification of proteins exhibiting turnover and phosphorylation dynamics during sea urchin egg activation

The animal egg is a unique quiescent cell, prepackaged with maternal mRNAs and proteins that have functions in early development. Rapid, transient signaling at fertilization alters egg physiology, resulting in Ca²⁺ release from the endoplasmic reticulum (ER) and cytoplasmic alkalinization. These events trigger the zygote developmental program through initiation of DNA synthesis and entry into mitosis. Post-translational modifications of maternal proteins are responsible for the spatio-temporal regulation that orchestrates egg activation. We used functional proteomics to identify the candidate maternal proteins involved in egg activation and early development. As the first step of this analysis, we present the data on the baseline maternal proteome, in particular, on proteins exhibiting changes in abundance and in phosphorylation state upon egg activation. We identify 94 proteins that were stable, reproducibly displayed a shift in isoelectric point, or changed in relative abundance at specific times after activation. The identities of these proteins were determined by quadrupole time-of-flight tandem mass spectrometry. The set of the most dynamic proteins appear to be enriched in intermediary metabolism proteins, cytoskeletal proteins, gamete associated proteins and proteins that have Ca²⁺ mediated activities.     

Adiponectin Receptor-1 C-Terminal Fragment (CTF) in Plasma: Putative Biomarker for Diabetes

Introduction

Polypeptide fragments from cell surface receptors when found in plasma may be indicators of receptor regulation in disease conditions. It is known that subjects with diabetes have significantly lower plasma concentrations of adiponectin, a hormone released by adipose tissue, compared with nondiabetic controls. This hormone interacts with cell surface receptors in muscle (AdipoR1) and liver (AdipoR2).

Methods

We analyzed the relative distribution of specific fragments of AdipoR1 in healthy and diabetic individuals using an immunoaffinity mass spectrometry approach. We used antibodies raised against AdipoR1 immobilized on pre-activated protein chip surfaces to determine the molecular weights of bound polypeptide fragments using immunomass spectrometry (immuno-MS).

Results

Initially, immuno-MS analyses using a polyclonal antibody revealed two peaks (m/z 3,902 and 7,812) in plasma from normal, healthy individuals (n?=?5) that were not present in the plasma of diabetics (n?=?5). To confirm the detection of these fragments, a monoclonal antibody was developed against the last 25 amino acids of the AdipoR1 C-terminal fragment (CTF). Using the immuno-MS method, the monoclonal antibody detected the AdipoR1 CTF (m/z 3475) in all healthy controls (n?=?10), but did not detect these fragments in the diabetic patients (n?=?10).

Discussion

These preliminary observations suggest that the plasma levels of this receptor fragment may serve as an indicator of diabetic condition.     

Calcium-mediated inactivation of the MAP kinase pathway in sea urchin eggs at fertilization.

We have evaluated the regulation of a 43-kDa MAP kinase in sea urchin eggs. Both MAP kinase and MEK (MAP kinase kinase) are phosphorylated and active in unfertilized eggs while both are dephosphorylated and inactivated after fertilization, although with distinct kinetics. Reactivation of MEK or the 43-kDa MAP kinase prior to or during the first cell division was not detected. Confocal immunolocalization microscopy revealed that phosphorylated (active) MAP kinase is present primarily in the nucleus of the unfertilized egg, with some of the phosphorylated form in the cytoplasm as well. Incubation of unfertilized eggs in the MEK inhibitor U0126 (0.5 microM) resulted in the inactivation of MEK and MAP kinase within 30 min. Incubation in low concentrations of U0126 (sufficient to inactivate MEK and MAP kinase) after fertilization had no effect on progression through the embryonic cell cycle. Microinjection of active mammalian MAP kinase phosphatase (MKP-3) resulted in inactivation of MAP kinase in unfertilized eggs, as did addition of MKP-3 to lysates of unfertilized eggs. Incubation of unfertilized eggs in the Ca(2+) ionophore A23187 led to inactivation of MEK and MAP kinase with the same kinetics as observed with sperm-induced egg activation. This suggests that calcium may be deactivating MEK and/or activating a MAP kinase-directed phosphatase. A cell-free system was used to evaluate the activation of phosphatase separately from MEK inactivation. Unfertilized egg lysates were treated with U0126 to inactivate MEK and then Ca(2+) was added. This resulted in increased MAP kinase phosphatase activity. Therefore, MAP kinase inactivation at fertilization in sea urchin eggs likely is the result of a combination of MEK inactivation and phosphatase activation that are directly or indirectly responsive to Ca(2+).     

Infertility in CFW/R1 mice associated with cystic dilatation of the bulbourethral gland

A third of male inbred CFW/R1 mice in a breeding colony developed subcutaneous, bilateral, perineal masses determined to be cystic bulbourethral glands. The masses developed in mice between 4 and 15 months of age. After development of these perineal masses, diseased males were unable to produce offspring. Gross examination revealed the masses impinging on the scrotum and displacing the testes into the inguinal canal. The perineal masses were paired, membranous, translucent cysts, 6 to 10 mm3, attached to the bulbocavernosus muscle and connected to the pelvic urethra by way of a non-patent duct. The cysts contained a clear to tan, minimally cellular, viscous fluid with high mucus content, as documented by examination of Wright Giemsa-stained cytologic preparations. Histologic examination of hematoxylin and eosin-stained sections revealed cystic tubuloalveolar glands surrounded by striated muscle and lined by a single layer of pyramidal cuboidal to columnar epithelial cells with pale, basophilic, lacy cytoplasm and round, basal, condensed nuclei. These gross and histopathologic findings were consistent with cystic dilatation of the bulbourethral gland.     

Method development and validation for quantitative determination of methadone enantiomers in human plasma by liquid chromatography/tandem mass spectrometry

A high-throughput method for quantitative determination of methadone enantiomers in human plasma was developed and validated by liquid chromatography/tandem mass spectrometry. The effects of pH and of types and concentrations of mobile-phase modifiers on the enantioselectivity of (R)- and (S)-methadone were investigated on a Chiral-AGP column. A baseline separation of the enantiomers was achieved with a retention time of less than 5 min. Ionization suppression and other matrix effects were evaluated. Morphine, cocaine, 6-monoacetylmorphine, benzoylecgonine and ecgonine methyl ester did not interfere with the performance of the assay. The specificity, linearity, intra- and inter-assay precision and accuracy, and extraction recovery were fully evaluated. The method showed excellent reproducibility (overall coefficient of variance < 8%) and accuracy (overall bias < 2.7%) with a broad linear range. The enantiomers were stable in human plasma after five freeze-thaw cycles, under bench-top storage at room temperature (RT) for 6h, in the extract reconstitution solution at RT for 17 h, and in processed-extracts stored at RT for 142 h. This validated LC/MS/MS assay offers high-throughput and improved specificity, sensitivity, linear range and ruggedness over previously published methods and has been successfully applied to the analysis of clinical samples.     

Substrate recognition by the mammalian proton-dependent amino acid transporter PAT1

The PAT family of proton-dependent amino acid transporters has recently been identified at the molecular level. This paper describes the structural requirements in substrates for their interaction with the cloned murine intestinal proton/amino acid cotransporter (PAT1). By using the Xenopus laevis oocytes as an expression system and by combining the two-electron voltage clamp technique with radiotracer flux studies, it was demonstrated that the aliphatic side chain of L-alpha-amino acids substrates can consist maximally of only one CH2-unit for high affinity interaction with PAT1. With respect to the maximal separation between the amino and carboxyl groups, only two CH2-units, as in gamma-aminobutyric acid (GABA), are tolerated. PAT1 displays no or even a reversed stereoselectivity, tolerating serine and cystein only in the form of D-enantiomers. A methyl-substitution of the carboxyl group (e.g. O-methyl-glycine) markedly diminishes substrate affinity and transport rates, whereas methyl-substitutions at the amino group (e.g. sarcosine or betaine) have only minor effects on substrate interaction with the transporter binding site. Furthermore, it has been shown (by kinetic analyses of radiolabelled betaine influx and inhibition studies) that the endogenous PAT system of human Caco-2 cells has very similar transport characteristics to mouse PAT1. In summary, one has defined the structural requirements and limitations thet determine the substrate specificity of PAT1. A critical recognition criterion of PAT1 is the backbone charge separation distance and the side chain size, whereas substitutions on the amino group are well tolerated.     

An Ancient Repeat Sequence in the ATP Synthase β-Subunit Gene of Forcipulate Sea Stars

A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase β-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Considerations for determining optimal mouse caging density

At the 2006 National Meeting of the American Association of Laboratory Animal Science, a panel discussed the question of what constitutes optimal or acceptable housing density for mice. Though there is a consensus that present guidelines are somewhat arbitrarily defined, scientific research has not yet been able to provide clear recommendations for amending them. Speakers explored the many factors that influence decisions on mouse housing, including regulatory requirements, scientific data and their interpretation, financial considerations and ethical concerns. The panel largely agreed that animal well-being should be the measure of interest in evaluating housing density and that well-being includes not only physical health, but also animals' behavior, productivity and preference.     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

Restoration of macroinvertebrates,fish, and habitats in streams following mining subsidence: replicated analysis across 18 mitigation sites

Human activities have led to declines in stream functioning and stream restoration seeks to reverse this trend. Longwall coal mining, an underground full‐extraction method, can cause surface subsidence, affecting streams by creating a series of deep pools that trap sediment, reduce habitat diversity, and impair macroinvertebrate and fish communities. Mining effects on streams must be mitigated to maintain the functions, values, and foreseeable uses of streams. Gate cutting is a procedure that alleviates pooling by reestablishing the stream grade, accompanied by procedures that stabilize the channel, restore substrates, and enhance in‐stream and riparian habitats. We evaluated effectiveness of gate cuts at restoring streams affected by subsidence pooling at 18 independent restoration sites over two mines in southwestern Pennsylvania, U.S.A. At each site, sampling stations were established to monitor effects of mining subsidence and its restoration on macroinvertebrates, fish communities, and habitats. We tested for effects of sequential interventions (subsidence and restoration) on biological and habitat variables in a replicated before–after design, controlling for potentially confounding temporal effects (sample month and antecedent effective precipitation). All biological indices and substrate‐related habitat indices declined following subsidence but improved following restoration. Macroinvertebrate indicex and taxa richness, substrates, and riparian vegetation continued to improve with time following restoration. Whereas other studies have concluded that biological communities may take many years to respond to restoration, these results indicate that where macroinvertebrate and fish communities are altered by subsidence pooling, they can be effectively restored using gate cuts to pre‐mining levels within relatively short time periods.