ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.     

Robust circadian rhythmicity of Per1 and Per2 mutant mice in constant light,and dynamics of Per1 and Per2 gene expression under long and short photoperiods

The Per1 and Per2 genes are components of the mammalian circadian clock. Mutations in these genes alter phase resetting in response to a nocturnal light pulse, and Per2 mutant mice are known to become arrhythmic in constant darkness. We show that under constant light conditions, Per2 mutant mice exhibit robust activity rhythms as well as body temperature rhythms with a period length that is less than 24 h. In Per1 mutants, the period length of both activity and body temperature rhythms is longer than 24 h in constant light. Per1 mutants prolong their period length (tao) when illuminance is increased, whereas Per2 mutants shorten their endogenous period. Additionally, the authors show that the circadian pattern of Per1 and Per2 gene expression in mice is modified under different photoperiods and that there is a mutual influence of these genes on their timing of expression. We propose that, in mice, the phase relationship between Per1 and Per2 gene expression might be critical for transducing day length information to the organism. Per1 could be part of a morning oscillator tracking dawn, and Per2 could be part of an evening oscillator tracking dusk.     

Pattern recognition and call preferences in treefrogs (Anura: Hylidae): a quantitative analysis using a no-choice paradigm

Studies of mate attraction have traditionally employed one of two experimental methods: choice tests in which female preferences from among two or more signals are tabulated, or single-stimulus tests in which the female response to one signal is quantified. Choice tests have long been preferred for examining mate attraction in anurans. Inspired by Wagner's (1998, Animal Behaviour,55, 1029-1042) discussion of the strengths and weaknesses of the two experimental approaches, we used a single-stimulus design to examine mate attraction in two sibling species of treefrogs (Hyla versicolor and H. chrysoscelis). We quantified female responses based upon the relative time required to approach signals varying in pulse rate, pulse rise time and pulse number. The data were used to generate response functions providing a quantitative measure of female attraction to the stimuli. Comparisons with data from choice experiments reveal broad similarities, as well as properties of female responses that had not been detected with choice tests. The results are discussed with regard to female selectivity for call parameters that are likely to mediate sexual selection and homospecific pairing.     

Brucella abortus strain RB51 vaccination in elk. I. Efficacy of reduced dosage

Bovine brucellosis is a serious zoonotic disease affecting some populations of Rocky Mountain elk (Cervus elaphus nelsoni) and bison (Bison bison) in the Greater Yellowstone Area, USA. The fear that elk and/or bison may spread Brucella abortus to livestock has prompted efforts to reduce or eliminate the disease in wildlife. Brucella abortus strain RB51 (RB51) vaccine has recently been approved for use in cattle. Unlike strain 19 vaccine, RB51 does not cause false positive reactions on standard brucellosis serologic tests. If effective, it may become the vaccine of choice for wildlife. In February 1995, 45 serologically negative female elk calves were trapped and taken to the Sybille Wildlife Research and Conservation Education Unit near Wheatland, Wyoming, USA. In May 1995, 16 of these elk calves were hand-vaccinated with 1 x 10(9) colony forming units (CFU) of RB51, 16 were vaccinated with 1 x 10(8) CFU RB51 by biobullet, and 13 were given a saline placebo. The elk were bred in fall of 1996 and they were challenged with 1 x 10(7) CFU of B. abortus strain 2308 by intraconjunctival inoculation in March 1997. Thirteen (100%) control elk aborted, 14 (88%) hand-vaccinated elk aborted, and 12 (75%) biobullet vaccinated elk aborted or produced nonviable calves. These results suggest that a single dose of 1 x 10(8) to 1 x 10(9) CFU RB51 does not provide significant protection against B. abortus induced abortion in elk. However, the vaccine appears to be safe at this dose and additional study may reveal a more effective RB51 vaccine regimen for elk.     

Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif

Agonist binding to the CC chemokine receptor 5 (CCR5) induces the phosphorylation of four distinct serine residues that are located in the CCR5 C terminus. We established a series of clonal RBL-2H3 cell lines expressing CCR5 with alanine mutations of Ser(336), Ser(337), Ser(342), and Ser(349) in various combinations and explored the significance of phosphorylation sites for the ability of the receptor to interact with beta-arrestins and to undergo desensitization and internalization upon ligand binding. Receptor mutants that lack any two phosphorylation sites retained their ability to recruit endogenous beta-arrestins to the cell membrane and were normally sequestered, whereas alanine mutation of any three C-terminal serine residues abolished both beta-arrestin binding and rapid agonist-induced internalization. In contrast, RANTES (regulated on activation normal T cell expressed and secreted) stimulation of a S336A/S349A mutant triggered a sustained calcium response and enhanced granular enzyme release. This mutational analysis implies that CCR5 internalization largely depends on a beta-arrestin-mediated mechanism that requires the presence of any two phosphorylation sites, whereas receptor desensitization is independently regulated by the phosphorylation of distinct serine residues. Surface plasmon resonance analysis further demonstrated that purified beta-arrestin 1 binds to phosphorylated and nonphosphorylated C-tail peptides with similar affinities, suggesting that beta-arrestins use additional receptor sites to discriminate between nonactivated and activated receptors. Surface plasmon resonance analysis revealed beta-arrestin 1 binding to the second intracellular loop of CCR5, which required an intact Asp-Arg-Tyr triplet. These results suggest that a conserved sequence motif within the second intracellular loop of CCR5 that is known to be involved in G protein activation plays a significant role in beta-arrestin binding to CCR5.     

The preprotein conducting channel at the inner envelope membrane of plastids

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of beta-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.     

Methods for homocysteine analysis and biological relevance of the results

It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B(2), B(6), B(9), B(12)) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

Familiarity and dominance relations among female sooty mangabeys in the Taï National Park

Dominance relationships of female sooty mangabeys have thus far been studied exclusively in captive groups. In captivity, adult females form a stable linear hierarchy as would be expected in species exhibiting strong contest competition. However, the same individuals do not exhibit other aspects of behavior that would be expected where contest competition occurs. For example, they show no kin‐based alliances leading to hierarchies in which the members of each matriline occupy adjacent ranks. The goal of this study was to provide the first data on dominance relationships of sooty mangabey females in their natural environment in the Taï National Park, Ivory Coast. In our study group, adult females formed a linear dominance hierarchy. Aggression over food increased in food patches, as would be expected for species that experience contest competition. Moreover, females formed highly differentiated social relationships, showing particular affinities with females of adjacent rank. Am. J. Primatol. 56:137–153, 2002. © 2002 Wiley‐Liss, Inc.     

Promoter-trapping in Saccharomyces cerevisiae by radiation-assisted fragment insertion

Non-homologous insertion (NHI) of DNA fragments into genomic DNA is a method widely used in insertional mutagenesis screens. In the yeast Saccharomyces cerevisiae, the efficiency of NHI is very low. Here we report that its efficiency can be increased by γ-irradiation of recipient cells at the time of transformation. Radiation-assisted NHI depends on YKU70, but its efficiency is not improved by inactivation of RAD5 or RAD52. In a pilot study, we generated 102 transformant clones expressing a lacZ reporter gene under standard conditions (30°C, rich medium). The site of insertion was determined in a subset of eight clones in which lacZ expression was altered by UV-irradiation. A comparison with published data revealed that three of the eight genes identified in our screen have not been targeted by large-scale transposon-based insertion screens. This suggests that radiation-assisted NHI offers a more homogeneous coverage of the genome than methods relying on transposons or retroviral elements.