Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest

The stable C isotope composition (δ¹³C) of CO₂ respired by trunks was examined in a mature temperate deciduous oak forest (Quercus petraea). Month-to-month, day-to-day and diurnal, measurements were made to determine the range of variations at different temporal scales. Trunk growth and respiration rates were assessed. Phloem tissue was sampled and was analysed for total organic matter and soluble sugar ¹³C composition. The CO₂ respired by trunk was always enriched in ¹³C relative to the total organic matter, sometimes by as much as 5‰. The δ¹³C of respired CO₂ exhibited a large seasonal variation (3.3‰), with a relative maximum at the beginning of the growth period. The lowest values occurred in summer when the respiration rates were maximal. After the cessation of radial trunk growth, the respired CO₂ δ¹³C values showed a progressive increase, which was linked to a parallel increase in soluble sugar content in the phloem tissue (R = 0.95; P < 0.01). At the same time, the respiration rates declined. This limited use of the substrate pool might allow the discrimination during respiration to be more strongly expressed. The late-season increase in CO₂ δ¹³C might also be linked to a shift from recently assimilated C to reserves. At the seasonal scale, CO₂ δ¹³C was negatively correlated with air temperature (R = −0.80; P < 0.01). The diurnal variation sometimes reached 3‰, but the range and the pattern depended on the period within the growing season. Contrary to expectations, diurnal variations were maximal in winter and spring when the leaves were missing or not totally functional. By contrast to the seasonal scale, these diurnal variations were not related to air temperature or sugar content. Our study shows that seasonal and diurnal variations of respired ¹³C exhibited a similar large range but were probably explained by different mechanisms.     

A recombinant multivalent combination vaccine protects against Chlamydia and genital herpes

Chlamydia trachomatis and Herpes simplex virus type 2 (HSV-2) genital infections pose a considerable public health challenge worldwide. Considering the high incidence of coinfections by the two pathogens, a combination vaccine that can be administered as a single regimen would be highly desirable. Recombinant Vibrio cholerae ghosts (rVCG) offer an attractive approach for the induction of humoral and cellular immune responses against human and animal pathogens. In this study, we evaluated a bivalent combination vaccine formulation comprising rVCG expressing chlamydial MOMP and HSV-2 glycoprotein D in mice for immunogenicity and protective efficacy against genital challenge with either pathogen. Mice immunized with the combination vaccine elicited secretory IgA and IgG2a antibodies to both chlamydial and HSV-2 antigens in serum and vaginal secretions. Robust antigen-specific mucosal and systemic T helper type 1 responses were induced in mice as measured by increased interferon-gamma levels produced by immune T cells in response to restimulation with target antigen in vitro. In addition, mice immunized with the combination vaccine were prophylactically protected from genital challenge with high doses of live Chlamydia and HSV-2. Thus, the combination vaccine regimen delivered by rVCG elicited adequate immune effectors that simultaneously protected against the individual pathogens.     

Lanthanide ion catalysis of the trans-cis isomerization of trans-bis(oxalato)-diaquochromate(III) and trans-bis(malonato)diaquochromate(III) [1]

The lanthanide ion catalyzed trans-cis isomerizations of trans-bis(oxalato)diaquochromate(II) and trans-bis(malonato)diaquochromate(III) have been studied. A linear free energy relationship was found correlating the catalytic rate constants for the oxalate reaction with the corresponding formation constants of complexes formed between simple monocarboxylic acids and the light (LaGd) members of the lanthanide series. The results indicates that for this portion of the series, the reaction mechanism is related to the formation of monocarboxylate complex intermediates. When the ionic radius of the lanthanide ion decreases below a particular value (as in the latter half of the series), the metal ion remains coordinated to both carboxylates of the oxalate ion rather than simply binding to only one carboxylate. In either situation, isomerization to the cis product eventually occurs, and the lanthanide ion is released.The reaction rates associated with the trans-bis(malonato)diaquochromate(III) reaction were found to be significantly slower than those of the corresponding oxalate system. However, in the malonate system, no linear free energy relationship was found relating the catalytic rate constants with the corresponding formation constants of monocarboxylic acids. One does find a linear relationship between the catalytic rate constants for the malonate reaction and the log K₁ values for the corresponding lanthanide/malonate complexes. During the course of the trans-cis isomerization, the lanthanide ion chelates the dissociated malonate group of a pentavalent Cr(III) intermediate. In the mechanism the lanthanide ion does not aid in ring opening, and neither does it singly bond to the intermediate     

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.     

Expression of the gap junction protein connexin43 in embryonic chick lens: Molecular cloning,ultrastructural localization,and post-translational phosphorylation

Summary Lens epithelial cells are physiologically coupled to each other and to the lens fibers by an extensive network of intercellular gap junctions. In the rat, the epithelial-epithelial junctions appear to contain connexin43, a member of the connexin family of gap junction proteins. Limitations on the use of rodent lenses for the study of gap junction formation and regulation led us to examine the expression of connexin43 in embryonic chick lenses. We report here that chick connexin43 is remarkably similar to its rat counterpart in primary amino acid sequence and in several key structural features as deduced by molecular cDNA cloning. The cross-reactivity of an anti-rat connexin43 serum with chick connexin43 permitted definitive immunocytochemical localization of chick connexin43 to lens epithelial gap junctional plaques and examination of the biosynthesis of connexin43 by metabolic radiolabeling and immunoprecipitation. We show that chick lens cells synthesize connexin43 as a single, 42-kD species that is efficiently posttranslationally converted to a 45-kD form. Metabolic labeling of connexin43 with³²P-orthophosphate combined with dephosphorylation experiments reveals that this shift in apparent molecular weight is due solely to phosphorylation. These results indicate that embryonic chick lens is an appropriate system for the study of connexin43 biosynthesis and demonstrate for the first time that connexin43 is a phosphoprotein.     

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA^Gln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNA^Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA^Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA^Gln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA^Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.