New insights into the surface Enhanced Raman Scattering (SERS) response of adenine using chemometrics.

The SERS response of adenine is one of the most studied, due to its outstanding exaltation. However, the spectra obtained strongly differ according to the experimental conditions and still remain not well understood. To be able to search for the presence of this molecule in complex environments, it is essential to better understand the SERS response of adenine alone. After a brief presentation of the literature on the subject, we present results suggesting that the experimental spectra would result from the overlap of different spectroscopic signatures, that may probably be due to different non-covalent interactions or different electromagnetic fields experienced by adenine molecules. An independent component analysis is reported. Our results underline the difficulty to precisely analyze the experimental data, the need to continue this research and to constitute data banks that would allow comparing the spectra obtained in different laboratories according to the experimental conditions.

Assessment LOPU-IVF in Japanese sika deer (Cervus nippon nippon) and application to Vietnamese sika deer (Cervus nippon pseudaxis) a related subspecies threatened with extinction.

In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 ± 1.0 vs. 8.2 ± 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep.

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.

A cis and trans adenine-dependent hairpin ribozyme against Tpl-2 target.

Ribozymes are catalytic RNAs that possess the property of cutting an RNA target via site-specific cleavage after sequence-specific recognition. Ribozymes can moreover cleave multiple substrate molecules. An increasing number of studies show that ribozymes are particularly well adapted tools against cancer, silencing or down-regulating gene expression at the RNA level. We have constructed an adenine-dependent hairpin ribozyme that cleaves the sequence at nucleotides A(225)(downward arrow)G(226) relative to the start codon of translation of the Tpl-2 kinase mRNA; this serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in breast cancer. An adenine-dependent hairpin ribozyme 1 (ADHR1) was previously isolated using the Systematic Evolution of Ligands by EXponential enrichment procedure. Switch on/switch off ribozymes are particularly useful since high amounts of stable ribozyme can be produced in the absence of adenine and the ribozyme specifically cleaves its target in the presence of adenine. The ADHR1 target sequence was replaced by a sequence derived from the Tpl-2 kinase mRNA. The resulting Tpl-2 ribozyme is active in cis cleavage: kinetic studies have been performed as a function of Mg2+ concentration, adenine concentration, as well as at different pH and with various cofactors. Finally, the Tpl-2 ribozyme was shown to cleave its target in trans successfully. These findings demonstrate that a potential therapeutic ribozyme can be produced by simple sequence modification.

Evaluation of a peptide ELISA for the detection of rituximab in serum.

Pharmacokinetic studies of therapeutic monoclonal antibodies necessitate the measurement of their biologically active fraction. The aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for rituximab, a chimeric anti-CD20 monoclonal antibody, based on its binding to a 20-mer peptide (P20) derived from the extracellular loop of human CD20 (residues 165-184). Derivatives of P20 were prepared by conjugation to bovine serum albumin (BSA-P20ACM) or biotin (Biot-P20ACM). Interactions of P20 and its derived peptides with rituximab were analyzed by surface plasmon resonance (SPR) and by ELISA. A monoclonal anti-idiotype antibody (MB2A4) was used as the reference in each case. SPR analysis showed that P20 (conjugated or unconjugated) had a lower affinity for rituximab than MB2A4. ELISA methods based on P20 or MB2A4 were both highly accurate and reproducible for rituximab measurement in spiked samples, but the MB2A4-based assay had a lower limit of quantification. Interestingly, discrepant results were obtained with the two ELISA methods when analyzing pharmacokinetic samples, with the rituximab concentrations obtained with the MB2A4-based method being systematically higher than those determined by the P20-based method. Possible interference of circulating CD20 with the P20-based method was supported by competition experiments. Rituximab aggregation in the bloodstream may also account for the bias observed in samples from healthy mice. The P20-based ELISA is far less sensitive than the MB2A-based ELISA, thus limiting its utility for pharmacokinetic studies. However, the discrepancy observed between two different approaches for rituximab measurement indicates that data from different studies should be interpreted with care.

Oligomerization of thioglutamic acid: encapsulated reactions and lipid catalysis.

For cellular life to begin on the early Earth, a permeation mechanism would be required to allow polar solutes to enter a membrane-bounded compartment. A second process--internal polymerization of peptides from amino acid--would also be an essential step toward the first compartmented metabolic pathways leading to biosynthesis. Here we report a study of amino acid permeation into lipid vesicles, in which thioglutamic acid penetrated lipid bilayer membranes at rates sufficient to support internal polymerization reactions. We also investigated spontaneous non-enzymatic polymerization reactions of thioglutamic acid to form oligopeptides. We found that oligomers up to 11mers are produced in the reaction mixture, and conclude that certain lipid surfaces can act as catalysts in promoting an oligomerization reaction. These observations are pertinent to understanding processes by which peptide bond synthesis could take place in primitive cellular life on the early Earth.

Transferrin overexpression alters testicular function in aged mice.

Many studies have shown a correlation between transferrin (Tf) concentration and sperm yield in several mammalian species. We have used transgenic mice expressing human Tf (hTf) to investigate if overexpression of Tf increases the efficiency of mouse spermatogenesis. We demonstrated that a 36% increase of Tf does not ameliorate the efficiency of mouse spermatogenesis but on the contrary resulted in a 36% decrease of testis sperm reserves. Tf overexpression had no effect on testicular determination and development, however testicular function of these transgenic mice was affected in an age-dependent manner. At 16 months of age, testicular and epididymal weights were significantly reduced. While spermatogenesis was qualitatively normal, testicular functions were perturbed. In fact, testosterone rate after human chorionic gonadotropin (hCG) stimulation was lower in Tf overexpressing mice. Intratesticular concentration of estradiol-17beta was increased and fluid accumulation after ligation of rete testis was more abundant in these transgenic mice. Surprisingly, we found that endogenous Tf levels were also increased in Tf overexpressing mice and we demonstrated for the first time that Tf may serve to upregulate its own expression in testis. Collectively, our data show that Tf overexpression has negative effects on testicular function and that Tf levels require strict regulation in the testis.

Fast, reversible interaction of prion protein with RNA aptamers containing specific sequence patterns.

One of the unsolved problems in prion diseases relates to the physiological function of cellular prion protein (PrP), of which a misfolded isoform is the major component of the transmissible spongiform encephalopathies agent. Knowledge of the PrP-binding molecules may help in elucidating its role and understanding the pathological events underlying prion diseases. Because nucleic acids are known to bind PrP, we attempted to identify the preferred RNA sequences that bind to the ovine recombinant PrP. An in vitro selection approach (SELEX) was applied to a pool of 80-nucleotide(nt)-long RNAs containing a randomised 40-nt central region. The most frequently isolated aptamer, RM312, was also the best ligand (20 nM KD value), according to both surface plasmon resonance and filter binding assays. The fast rates of association and dissociation of RM312 with immobilized PrP, which are reminiscent of biologically relevant interactions, could point to a physiological function of PrP towards cellular nucleic acids. The minimal sequence that we found necessary for binding of RM312 to PrP presents a striking similarity with one previously described PrP aptamer of comparable affinity. In addition, we here identify the two lysine clusters contained in the N-terminal part of PrP as its main nucleic-acid binding sites.

[Immune response to equine Chorionic Gonadotropin used for the induction of ovulation in goats and ewes]. / Réponse immunitaire à la eCG utilisée dans le traitement de l'induction d'ovulation chez la chèvre et la brebis.

In dairy goats and ewes the use of equine Chorionic Gonadotropin (eCG) as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). Treatment for induction and synchronization of ovulation consists of a progestagen delivered by vaginal sponge, followed by an eCG injection. In some females, the first injection of eCG induces a humoral response with high concentrations of anti-eCG antibodies in contrast to other females displaying a very low concentration of anti-eCG antibodies. Females eliciting a low response were also poor responders after the following treatments. Conversely, high responders at the first treatment systematically yielded high immune responses upon the following treatment. By a molecular genetic approach using microsatellites we showed that the anti-eCG immune response phenotypes were associated with MHC class II polymorphism. Females with high residual antibody concentrations at the time of eCG injection exhibited a much lower kidding rate than other females did. Lower fertility of these females, inseminated at a fixed time after eCG treatment (43H for goats and 55H for ewes), might be due to the delay in estrus occurrence and the pre ovulatory LH surge. Consequently, under field conditions old females selected for AI are only those with low residual anti-eCG antibody concentrations and old females with high residual antibody concentration are culled from AI breeding because of their low fertility during the previous year. So we have undertaken comparative studies to establish if the anti-eCG immune response is correlated with the global immunity in animals.

RNA-acting antibiotics: in-vitro selection of RNA aptamers for the design of new bioactive molecules less susceptible to bacterial resistance.

During the last few years, antibiotic multiresistance has been increasing, not only in hospitals, but also, more worryingly, in general medicine. Different ways are being explored to bypass this problem. RNA-acting antibiotics such as aminosides (aminoglycosides) bind to bacterial RNA causing premature termination of proteins and mistranslation in bacteria. It is now possible to study the interactions of such antibiotics with their target by in-vitro selection of RNA molecules that recognize these antibiotics (RNA aptamers, SELEX method). The knowledge of the antibiotic-RNA interactions represents a promising way for the rational design of new bioactive compounds less susceptible to bacterial resistance.

Why biologists should support the exploration of Mars.

Physicists, chemists and geologists in the USA and Europe propose that the search for extraterrestrial life is an important justification for the exploration of Mars. Biologists, however, much more excited by the advent of the postgenome sequencing era, in general display little enthusiasm for planetary exploration. We argue that the search for traces of life on Mars represents a major thought-provoking challenge for the life sciences that should be taken up by the biological community.

Recent findings in the modern RNA world.

It is assumed that modern life forms arose from a molecular ancestor in which RNA molecules both stored genetic information and catalyzed biochemical reactions. In modern cells, these functions are carried out, respectively, by DNA and proteins, but diverse cellular RNAs are also involved in key cellular functions. In this paper, we review the cellular RNAs that are ubiquitous and/or that perform essential biological functions, and we discuss the evolutionary relationships of such RNAs with a prebiotic RNA world. This unexpected biological diversity of cellular RNAs and the crucial functions they perform in cellular metabolism demonstrate the complexity of an RNA-driven metabolism in an ancient RNA world and in modern life. Cellular RNAs are involved in translation (tRNA and rRNA) but also in ribosome maturation (snoRNA) and more generally in RNA processing (snRNA and snoRNA), replication (telomerase RNA), editing, protein translocation (SRP RNA), cellular transport (vRNA) and translation quality control (tmRNA). In addition, the function of many other cellular RNAs has not yet been determined. Future investigations of RNA function will allow us to better understand not only early evolutionary biological processes but also the central metabolism of modern cells.

Relationship between peri-oestrus progesterone levels and time of ovulation by echography in pigs and influence of the interval between ovulation and artificial insemination (AI) on litter size.

Two methods for the determination of ovulation were compared to one ultrasonography performed 5 times a day. Time of ovulation by echography was 40 +/- 5.8 h (mean +/- SD) after the onset of oestrus. Preovulatory LH rise (two blood samples per day) began near the onset of oestrus but, in our conditions, this parameter could not be used to predict ovulation. The basal level of progesterone (two blood samples per day) was determined with a non-linear model, the timing when progesterone rose more than one SD (0.3 ng x mL(-1)) coincided with the timing of ovulation determined by echography (R2 = 0.98). This method was efficient and was used in a field trial to measure the consequences of the variability of the interval between Al and ovulation on litter size. The interval between Al and ovulation had an effect on litter size; litter size decreased by one piglet when this interval increased by 10h.

Oligomerization of alpha-thioglutamic acid.

A decaglutamic acid primer is extended very slowly by alpha-thioglutamic acid. The addition of bicarbonate to the reaction mixture greatly accelerates the reaction. We believe the N-carboxyanhydride of glutamic acid is an intermediate in the accelerated reaction. When K3Fe(CN)6 and bicarbonate are both present in the reaction mixture, oligoglutamic acids up to at least the 15-mer are formed rapidly. The acylating agent is the oxidation product of thioglutamic acid, a diacyldisulfide.

Copper-adenine catalyst for O(2) production from H(2)O(2).

In solutions of CuCl2 and adenine copper can be bound to adenine. Two Cu(adenine)(2) complexes [Cu(C(5)H(5)N(5))(2)]2+/Cu(C(5)H(4)N(5))(2)] are in equilibrium with free adenine. Copper-adenine complexes present a catalytic activity (e.g., H(2)O(2) disproportionation into O(2) and water) but depending on complex concentration H(2)O(2) also strongly oxidizes the adenine within the complexes. Raman spectroscopy quantifies copper-adenine complex formation and H(2)O(2) consumption; polarography quantifies O(2) production. As for C(40) catalase, optimal catalytic capacities depend on physiological conditions, such as pH and temperature. The comparative analysis of kinetic parameters shows that the affinity for H(2)O(2) of Cu(adenine)(2) is 37-fold lower than that of C(40) catalase and that the molar activity for O(2) production is 200-fold weaker for Cu(adenine)(2) than for the enzyme. In the 10(-6)-10(-3) M range, the strong decrease of activity with raising complex concentration is explained by aggregation or stacking, which protects Cu(adenine)(2) entities from H(2)O(2) oxidation, but also decreases O(2) production.

Humoral immune response to equine chorionic gonadotropin in ewes: association with major histocompatibility complex and interference with subsequent fertility.

In dairy ewes, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination (AI). In this report we show the presence of anti-eCG antibodies in plasma of treated ewes. The major histocompatibility complex (MHC) was involved in the individual variability of the humoral immune responses to eCG. We found significant associations between the anti-eCG response phenotype and some MHC class II alleles. The low immune response phenotype was associated with one MHC class II allele only in Lacaune ewes, and the high immune response phenotype was associated with one MHC class II allele both in Manech and in Lacaune ewes. In herds, the impact of residual anti-eCG antibodies on subsequent fertility after AI seems minimal because of an indirect elimination of high-responder ewes from AI breeding. Therefore, the true magnitude of the association between residual anti-eCG antibody concentration and fertility has been underestimated. An additional experiment without any high-responder female elimination showed a significant correlation between high residual antibody concentrations and lower lambing rate after AI at a fixed time, possibly because of a delayed preovulatory LH surge. The results suggest that anti-eCG antibody concentration is one risk factor for infertility after AI.

The negative effect of repeated equine chorionic gonadotropin treatment on subsequent fertility in Alpine goats is due to a humoral immune response involving the major histocompatibility complex.

In dairy goats, the use of eCG as a convenient hormone for the induction of ovulation is necessary for out-of-season breeding and artificial insemination. However, repeated eCG treatments are followed by decreased fertility in goats inseminated at a fixed time after treatment. In this report, we show the presence of anti-eCG antibodies in plasma of treated goats. A 500 IU eCG injection induces a humoral response, with variable concentrations of anti-eCG antibody being produced in individual goats. The analysis of successive anti-eCG immune responses over several years has demonstrated the existence of different populations of goats, defined as low, medium, and high responders. By the use of two caprine microsatellites located inside (OLADRB) and outside (BM1258) the major histocompatibility complex (MHC), a significant association (p < 0.05) between the anti-eCG antibody response and some MHC-DRB alleles was found. Goats with high antibody concentrations at the time of eCG injection (> 2.5 microg/ml) exhibited a much lower kidding rate than did other females (41.3% vs. 66.7%). Lower fertility of these goats, inseminated at a fixed time after eCG treatment, might be due to the observed delay in estrus occurrence and the preovulatory LH surge.

Implications of recent advances in reproductive physiology for reproductive management of goats.

The control of reproduction in goats is interesting for technical reasons (synchronization of kiddings, adjustment to forage availability or to economy), and for genetic reasons (identification and dissemination of improved genotypes). The use of short-light rhythms leads to markedly increased production of semen per buck and prevents occurrence of a 'resting' season. Recent identification of a bulbourethral lipase in goat spermatozoa opens new perspectives in sperm preservation. Light plus 'short day' treatments also allow induction of out-of-season oestrous cycles and ovulations leading to enhanced fertility. Repeated use of eCG provokes the production of antibodies, delays the timing of ovulation and causes a reduction in fertility after fixed-time artificial insemination. All steps of embryo production, freezing and transfer are now controlled and allow the attainment of satisfactory numbers of kids born per donor female, which are compatible with the development of the technique for exchanging genotypes between countries. In vitro production of embryos allows high development rates to be achieved after in vitro maturation and fertilization of oocytes, and will ensure the production of synchronous populations of one-cell zygotes at the stage required by new biotechnologies.

From Panspermia to Bioastronomy, the evolution of the hypothesis of universal life.

During the 19th and early 20th centuries, ideas related to the possible origin in space of bioorganic molecules, or seeds, or even germs and organisms (and how they reached the Earth) included the Panspermia theory. Based on the idea of the eternity of life proposed by eminent physicists - such as Arrhenius and Kelvin - 'Panspermia' is mainly divided into two branches: lithopanspermia (transport of germs inside stones traveling in space) and radiopanspermia (transport of spores by radiative pressure of stellar light). We point out some arguments to help to understand whether 'Panspermia' could exist nowadays as the same theory defined one century ago. And we wonder about the kind of evolution 'Panspermia' could have undergone during only a few decades. This possible evolution of the 'Panspermia' concept takes place in the framework of the emergence of a new field, Bioastronomy. We present how this discipline has emerged during a few decades and how it has evolved. We consider its relationship with the progression of other scientific fields, and finally we examine how it is now included in different projects of space agencies. Bioastronomy researches having become more and more robust during the last few years, we emphasize several questions about new ideas and their consequences for the current hypothesis of 'Panspermia' and of universal life.

Use of an immunoenzymatic assay to detect the luteinizing hormone peak in bitches.

The success of artificial insemination with fresh chilled or frozen semen depends on the time of insemination. Determination of oestrous behaviour, use of vaginal smears or measurement of the vaginal resistivity are unreliable techniques and interpretation of the dosages of progesterone may be critical as it may vary from one laboratory to another. The plasma concentration of LH displays a peak of secretion that can be a good reference to date the events of the ovarian cycle. The plasma concentration of LH was measured in ten bitches by a new sandwich immunoenzymatic assay (Reprokit, Sanofi) and results were compared with the vaginal smears and plasma concentrations of progesterone. The LH peak was easily detected and lasted 3.3 days with maximum values ranging from 10 to 22 ng ml-1. At the time of the peak, the vaginal smears displayed features of pro-oestrus and the plasma concentration of progesterone was 2.9 ng ml-1. This immunoenzymatic assay, already used for many species, is an effective tool for determining the time of the LH peak. Added to the study of the progesterone concentration, it may help to determine the optimal time for artificial inseminations, particularly when frozen semen is used.