Equine sperm membrane phase behavior: the effects of lipid-based cryoprotectants

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.     

Marine reptiles (Thalattosuchia) from the Early Jurassic of Lombardy (northern Italy)

The fossil remains of two small reptiles recently discovered in the Sogno Formation (Lower Toarcian) near Cesana Brianza (Lecco Province), represent the first mesoeucrocodylians reported for Lombardy and some of the few Jurassic reptiles from Italy. Due to the absence of diagnostic skeletal elements (the skulls are lacking), it is not possible to refer the new specimens at genus level with confidence. Although the well developed dermal armour would characterise Toarcian thalattosuchians of the genera Steneosaurus (Teleosauridae) and Pelagosaurus (Metriorhynchidae), the peculiar morphology of the osteoderms allow to tentatively refer the remains to the latter taxon (cf. Pelagosaurus sp.). The small size, along with the opening of the neurocentral vertebral sutures and, possibly, the non sutured caudal pleurapophyses, indicate that the specimens were morphologically immature at death. These “marine crocodiles” confirm the affinities between the fauna of the Calcare di Sogno Formation and coeval outcrops of central Europe that also share the presence of similar fishes and crustaceans.     

Cathepsin B-mediated yolk protein degradation during killifish oocyte maturation is blocked by an H+-ATPase inhibitor: effects on the hydration mechanism

In teleost oocytes, yolk proteins (YPs) derived from the yolk precursors vitellogenins are partially cleaved into free amino acids and small peptides during meiotic maturation before ovulation. This process increases the osmotic pressure of the oocyte that drives its hydration, which is essential for the production of buoyant eggs by marine teleosts (pelagophil species). However, this mechanism also occurs in marine species that produce benthic eggs (benthophil), such as the killifish (Fundulus heteroclitus), in which oocyte hydration is driven by K+. Both in pelagophil and benthophil teleosts, the enzymatic machinery underlying the maturation-associated proteolysis of YPs is poorly understood. In this study, lysosomal cysteine proteinases potentially involved in YP processing, cathepsins L, B, and F (CatL, CatB, and CatF, respectively), were immunolocalized in acidic yolk globules of vitellogenic oocytes from the killifish. During oocyte maturation in vitro induced with the maturation-inducing steroid (MIS), CatF disappeared from yolk organelles and CatL became inactivated, whereas CatB proenzyme was processed into active enzyme. Consequently, CatB enzyme activity and hydrolysis of major YPs were enhanced. Follicle-enclosed oocytes incubated with the MIS in the presence of bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, underwent maturation in vitro, but acidification of yolk globules, activation of CatB, and proteolysis of YPs were prevented. In addition, MIS plus bafilomycin A1-treated oocytes accumulated less K+ than those stimulated with MIS alone; hence, oocyte hydration was reduced. These results suggest that CatB is the major protease involved in yolk processing during the maturation of killifish oocytes, whose activation requires acidic conditions maintained by a vacuolar-type H+-ATPase. Also, the data indicate a link between ion translocation and YP proteolysis, suggesting that both events may be equally important physiological mechanisms for oocyte hydration in benthophil teleosts.     

Networks of high mutual information define the structural proximity of catalytic sites: implications for catalytic residue identification

Identification of catalytic residues (CR) is essential for the characterization of enzyme function. CR are, in general, conserved and located in the functional site of a protein in order to attain their function. However, many non-catalytic residues are highly conserved and not all CR are conserved throughout a given protein family making identification of CR a challenging task. Here, we put forward the hypothesis that CR carry a particular signature defined by networks of close proximity residues with high mutual information (MI), and that this signature can be applied to distinguish functional from other non-functional conserved residues. Using a data set of 434 Pfam families included in the catalytic site atlas (CSA) database, we tested this hypothesis and demonstrated that MI can complement amino acid conservation scores to detect CR. The Kullback-Leibler (KL) conservation measurement was shown to significantly outperform both the Shannon entropy and maximal frequency measurements. Residues in the proximity of catalytic sites were shown to be rich in shared MI. A structural proximity MI average score (termed pMI) was demonstrated to be a strong predictor for CR, thus confirming the proposed hypothesis. A structural proximity conservation average score (termed pC) was also calculated and demonstrated to carry distinct information from pMI. A catalytic likeliness score (Cls), combining the KL, pC and pMI measures, was shown to lead to significantly improved prediction accuracy. At a specificity of 0.90, the Cls method was found to have a sensitivity of 0.816. In summary, we demonstrate that networks of residues with high MI provide a distinct signature on CR and propose that such a signature should be present in other classes of functional residues where the requirement to maintain a particular function places limitations on the diversification of the structural environment along the course of evolution.     

The coronary vasculature and the myocardium

The effect of bradykinin (BK) on myocardial inotropic state was tested on 5 isolated rat heart preparations, in which a proper ballon was placed to record left ventricular pressure, whose developed systolic value was taken as an index of contractility. A reduction of developed left ventricular pressure was observed when BK was added to the perfusion oxygenated Tyrode solution. However, when BK was given after 1-amino-benzotriazole, an inhibitor of Cytochrome P-450 (Cyt P-450), developed pressure did not change. Since Cyt P-450 is known to act on arachidonic acid inducing the production of epoxiecocistrienoic acids (EETs) which hyperpolarizes myocardial fibres, it was argued that the reduction in contractility by bradykinin was the result of the hyperpolarizing effect of EETs. The fact that the concentration of Cyt P-450 is higher in the vascular endothelial cells than in the sarcolemma of the myocytes and the observation that the coronary resistance decreases together with the contractility suggest that the endothelium plays a pivotal role in mediating the negative inotropic effect of BK.     

Development of serous cutaneous glands in Scinax nasica (Anura,Hylidae): patterns of poison biosynthesis and maturation in comparison with larval glands in specimens of other families

We examined the development of serous (poison) cutaneous glands in larval and juvenile Scinax nasica (Hylidae) at the ultrastructural level. We describe the biosynthesis and maturation of the cutaneous poison in comparison with the corresponding processes in representatives of Discoglossidae, Leptodactylidae, Pelobatidae and Pipidae. Serous biosynthesis in S. nasica starts in discrete adenoblasts and continues in the syncytial secretory unit. Biosynthetic processes involve rough endoplasmic reticulum and the Golgi apparatus, that releases membrane-bounded material, varying from fine grained to flocculent. During the post-Golgian secretory phase, this material undergoes initial maturation, and two products are formed: dense granules and larger vesicles holding a thin substance that will later be structured into a three-dimensional, honeycomb-like net. Both the secretory granules and vesicles change into glomerular-like aggregates of bowed, rod-shaped subunits (modules). In adult frogs, formation of dense granules is bypassed. The modular granule substructure seems to be related to the merocrine release of small amounts of poison, involved in regulating skin homeostasis. Comparison with maturational changes in larval glands of species representing four anuran families discloses similar patterns in the Leptodactylidae, but production of opaque homogeneous granules occurs in the Discoglossidae, clear vesicles in the Pelobatidae and aggregates of dense bars in the Pipidae.     

Regulation of c-Fes tyrosine kinase activity by coiled-coil and SH2 domains: analysis with Saccharomyces cerevisiae

The c-Fes protein-tyrosine kinase regulates the growth and differentiation of diverse cell types, including myeloid hematopoietic cells, vascular endothelial cells, and neurons. Structurally, Fes is composed of a unique N-terminal region with coiled-coil oligomerization motifs, followed by SH2 and kinase domains. Although Fes kinase activity is tightly regulated in cells, the structural basis for its negative regulation is not clear. In this report, c-Fes was expressed in Saccharomyces cerevisiae to determine whether regulation is kinase-intrinsic or dependent upon protein factors found in mammalian cells. Wild-type Fes kinase activity was completely repressed in yeast and did not affect cell growth. Mutation or deletion of the more N-terminal c-Fes coiled-coil domain reversed negative regulation, leading to strong kinase activation and suppression of yeast cell growth. Similarly, replacement of the wild-type SH2 domain with that of v-Src induced strong kinase activation and the growth-inhibitory phenotype. Immunoblotting with phosphospecific antibodies shows that activation of Fes by either mechanism induced autophosphorylation of the activation loop tyrosine residue (Tyr 713). These data support the idea that Fes naturally adopts an inactive conformation in vivo, and that maintenance of the inactive structure requires the coiled-coil and SH2 domains.     

Serous cutaneous glands in new world hylid frogs: an ultrastructural study on skin poisons confirms phylogenetic relationships between Osteopilus septentrionalis and Phrynohyas venulosa

Transmission electron microscope investigations of the serous (poison) skin glands in the New World tree frogs Osteopilus septentrionalis and Phrynohyas venulosa revealed that they produce granules with closely similar substructures, namely, a dense cortex and pale medulla. In both species these features, that contrast the complex, sometimes repeating patterns described in other hylid frogs, derive from similar secretory and maturational processes starting from the Golgi phase of poison biosynthesis. Observations on secretory discharge showed that the two species share common release mechanisms, based on bulk discharge (holocrine) processes. Our data provide novel evidence of the extensive ultrastructural polymorphism of serous skin products in Hylidae and agree with phylogenies that regard this family as polyphyletic in origin. Assuming that ultrastructural features of cutaneous poison biosynthesis and maturation are adequate clues for tracking anuran phylogeny, the present findings also support a close relationship between Osteopilus and Phrynohyas taxa as previously suggested by osteological evidence.