Seasonal variation in serum testosterone, testicular volume and semen characteristics in coatis (Nasua nasua)

The objective was to characterize seasonal changes in serum testosterone concentration, testicular volume and sperm quantity and quality in captive coatis (Nasua nasua) from Pantanal, MT, Brazil. Sampling was done once monthly for 1 y. Mean (± SEM) serum testosterone concentrations (767.37 ± 216.2 ng/ml) and total and progressive sperm motility (79.6 ± 3.9%; 3.8 ± 0.3, on a scale of 0 to 5) peaked in July. The highest combined testis volume (10.3 ± 0.4 cm³) and sperm concentration (403 million ± 102 sperm/ml) occurred in August, at the peak of the winter breeding season. No seasonal effects on percentages of morphologically normal sperm, acrosome integrity, or live sperm were detected; however, the percentage of secondary sperm defects was higher in the winter. In conclusion, intricate relationships between testosterone concentration, testis volume, semen concentration and total and progressive sperm motility with high levels of breeding activity were observed during the dry season in the winter (June, July, August), followed by a subsequent decline in these activities during the wet season (i.e., summer: December, January, February). There was no seasonal pattern for production of functionally intact and morphologically normal sperm.     

Proteolytic ectodomain shedding of membrane proteins in mammals—hardware,concepts, and recent developments

Proteolytic removal of membrane protein ectodomains (ectodomain shedding) is a post‐translational modification that controls levels and function of hundreds of membrane proteins. The contributing proteases, referred to as sheddases, act as important molecular switches in processes ranging from signaling to cell adhesion. When deregulated, ectodomain shedding is linked to pathologies such as inflammation and Alzheimer's disease. While proteases of the “a disintegrin and metalloprotease” (ADAM) and “beta‐site APP cleaving enzyme” (BACE) families are widely considered as sheddases, in recent years a much broader range of proteases, including intramembrane and soluble proteases, were shown to catalyze similar cleavage reactions. This review demonstrates that shedding is a fundamental process in cell biology and discusses the current understanding of sheddases and their substrates, molecular mechanisms and cellular localizations, as well as physiological functions of protein ectodomain shedding. Moreover, we provide an operational definition of shedding and highlight recent conceptual advances in the field. While new developments in proteomics facilitate substrate discovery, we expect that shedding is not a rare exception, but rather the rule for many membrane proteins, and that many more interesting shedding functions await discovery.     

Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism

Cholinesterases present homologies with some cell adhesion molecules; however, it is unclear whether and how they perform adhesive functions. Here, we provide the first direct evidence showing that neurite growth in vitro from various neuronal tissues of the chick embryo can be modified by some, but not all, anticholinesterase agents. By quantifying the neuritic G4 antigen in tectal cell cultures, the effect of anticholinesterases on neurite growth is directly compared with their cholinesterase inhibitory action. BW 284C51 and ethopropazine, inhibiting acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively, strongly decrease neurite growth in a dose-dependent manner. However, echothiophate which inhibits both cholinesterases, does not change neuritic growth. These quantitative data are supplemented by morphological observations in retinal explant cultures grown on striped laminin carpets, viz., defasciculation of neurite bundles by BW 284C51 and Bambuterol occurs, indicating that these drugs disturb adhesive mechanisms. These data strongly suggest that a) cholinesterases can participate in regulating axonal growth, b) both AChE and BChE can perform such a nonsynaptic function, and c) this function is not the result of the enzyme activity per se, since at least one drug was found that inhibits all cholinesterase activities but not neurite growth. Thus, a secondary site on cholinesterase molecules must be responsible for adhesive functions.     

Basolateral protein Scribble binds phosphatase PP1 to establish a signaling network maintaining apicobasal polarity

Scribble, a member of the LAP protein family, contributes to the apicobasal polarity (ABP) of epithelial cells. The LAP-unique region of these proteins, which is essential and sufficient for ABP, includes a conserved Leucine-Rich Repeat (LRR) domain. The major binding partners of this region that could regulate ABP remain unknown. Here, using proteomics, native gel electrophoresis, and site-directed mutagenesis, we show that the concave surface of LRR domain in Scribble participates in three types of mutually exclusive interactions—(i) homodimerization, serving as an auto-inhibitory mechanism; (ii) interactions with a diverse set of polarity proteins, such as Llgl1, Llgl2, EPB41L2, and EPB41L5, which produce distinct multiprotein complexes; and (iii) a direct interaction with the protein phosphatase, PP1. Analogy with the complex between PP1 and LRR domain of SDS22, a well-studied PP1 regulator, suggests that the Scibble-PP1 complex stores a latent form of PP1 in the basolateral cell cortex. Such organization may generate a dynamic signaling network wherein PP1 could be dispatched from the complex with Scribble to particular protein ligands, achieving fast dephosphorylation kinetics.     

Aptamers for pharmaceuticals and their application in environmental analytics

Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications.     

Multilocus analyses of seven candidate genes suggest interacting pathways for obesity-related traits in Brazilian populations

We investigated whether variants in major candidate genes for food intake and body weight regulation contribute to obesity-related traits under a multilocus perspective. We studied 375 Brazilian subjects from partially isolated African-derived populations (quilombos). Seven variants displaying conflicting results in previous reports and supposedly implicated in the susceptibility of obesity-related phenotypes were investigated: β2-adrenergic receptor (ADRB2) (Arg16Gly), insulin induced gene 2 (INSIG2) (rs7566605), leptin (LEP) (A19G), LEP receptor (LEPR) (Gln223Arg), perilipin (PLIN) (6209T > C), peroxisome proliferator-activated receptor-γ (PPARG) (Pro12Ala), and resistin (RETN) (-420 C > G). Regression models as well as generalized multifactor dimensionality reduction (GMDR) were employed to test the contribution of individual effects and higher-order interactions to BMI and waist-hip ratio (WHR) variation and risk of overweight/obesity. The best multilocus association signal identified in the quilombos was further examined in an independent sample of 334 Brazilian subjects of European ancestry. In quilombos, only the PPARG polymorphism displayed significant individual effects (WHR variation, P = 0.028). No association was observed either with the risk of overweight/obesity (BMI ≥ 25 kg/m2), risk of obesity alone (BMI ≥ 30 kg/m2) or BMI variation. However, GMDR analyses revealed an interaction between the LEPR and ADRB2 polymorphisms (P = 0.009) as well as a third-order effect involving the latter two variants plus INSIG2 (P = 0.034) with overweight/obesity. Assessment of the LEPR-ADRB2 interaction in the second sample indicated a marginally significant association (P = 0.0724), which was further verified to be limited to men (P = 0.0118). Together, our findings suggest evidence for a two-locus interaction between the LEPR Gln223Arg and ADRB2 Arg16Gly variants in the risk of overweight/obesity, and highlight further the importance of multilocus effects in the genetic component of obesity.     

Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members

Unique among fibroblast growth factors (FGFs), FGF19, -21, and -23 act in an endocrine fashion to regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis. These FGFs require the presence of Klotho/betaKlotho in their target tissues. Here, we present the crystal structures of FGF19 alone and FGF23 in complex with sucrose octasulfate, a disaccharide chemically related to heparin. The conformation of the heparin-binding region between beta strands 10 and 12 in FGF19 and FGF23 diverges completely from the common conformation adopted by paracrine-acting FGFs. A cleft between this region and the beta1-beta2 loop, the other heparin-binding region, precludes direct interaction between heparin/heparan sulfate and backbone atoms of FGF19/23. This reduces the heparin-binding affinity of these ligands and confers endocrine function. Klotho/betaKlotho have evolved as a compensatory mechanism for the poor ability of heparin/heparan sulfate to promote binding of FGF19, -21, and -23 to their cognate receptors.     

Regeneration of a chimeric retina from single cells in vitro: cell-lineage-dependent formation of radial cell columns by segregated chick and quail cells

Summary We report here that similar to E6-chicken retinal cells, dissociated cells from 5.5-day-old (E5.5) quail retinae reaggregate in rotary culture, multiply about tenfold and reestablish histotypical areas. These cellular aggregates include all nuclear layers either with inversed or correct laminar polarity, depending on the local origin of the cells (called rosetted and laminar in-vitro-retinae (IVR), respectively; Layer and Willbold 1989). In combined cultures, chick and quail cells are evenly mixed only during the first two days of culture. Along with the assembly of single cells into rosettes and then into discrete laminae, sectors of chick and quail cells begin to segregate. They are delineated by borders running radially through all three nuclear layers. Thus, interspecies migration of cells at this advanced stage of differentiation is strongly inhibited. Concomitant with this segregation, coherent radial columns spanning all three layers but containing cells from either species only, can be traced histologically. We conclude that a weak segregation of chick and quail retinal cells takes place already at the single cell level, but that the permanent segregation of entire tissue parts must be due to clonal cellular proliferation within the IVR in conjunction with some developmental-structural mechanism retaining clonal progenies within a columnar order.Abbreviations ECM extracellular matrix - E5.5 days of embryonic age - GCL ganglion cell layer - GC's ganglion cells - i.c. in culture - INL inner nuclear layer - rosetted in-vitro-retina retinal cell organoid aggregated from single cells of the central retina - IPL inner plexiform layer - MRE marginal retinal epithelium - ONL outer nuclear layer - OPL outer plexiform layer - OS ora serrate - PR photoreceptor cell - laminated in-vitro-retina fully laminated retinal cellorganoid resembling an E15-retina aggregated from cells of the eye periphery including RPE - RPE retinal pigment epithelium     

Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice

Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.