Xenopus tropicalis allurin: expression, purification, and characterization of a sperm chemoattractant that exhibits cross-species activity

Previously we reported the identification of the first vertebrate sperm chemoattractant, allurin, in the frog Xenopus laevis (Xl) and demonstrated that it was a member of the CRISP family of proteins. Here we report identification, purification, and characterization of Xenopus tropicalis (Xt) allurin, a homologous protein in X. tropicalis. “Egg water” as well as purified allurin from both species exhibit efficient cross-species sperm chemoattractant activity. Western blots show that Xt egg water contains a single anti-allurin cross-reactive protein whose molecular weight (20,497 Da by MALDI MS) agrees well with the molecular weight of the hypothetical gene product for a newly recognized “Crisp A” gene in the X. tropicalis genome. A recombinant form of the protein, expressed in 3T3 cells, exhibits chemoattraction for both Xt and Xl sperm and cross reacts with anti-allurin antibodies. Examination of Crisp protein expression in the Xt oviduct using RT-PCR showed that of five documented Xt Crisp genes (Crisps 2, 3, LD1, LD2 and A) only Crisp A was expressed. In contrast, Crisp 2, Crisp 3, Crisp LD1, and Crisp LD2, but not Crisp A, were all found to be expressed in the Xt testes while subsets of Crisp proteins where expressed in the Xt ovary. These data suggest that Crisp proteins in amphibians may play multiple roles in sperm production, maturation and guidance just as they are thought to in mammals indicating that Crisp protein involvement in reproduction may not be limited to mammals.     

The early adaptive evolution of calmodulin

Interaction between gene duplication and natural selection in molecular evolution was investigated utilizing a phylogenetic tree constructed by the parsimony procedure from amino acid sequences of 50 calmodulin- family protein members. The 50 sequences, belonging to seven protein lineages related by gene duplication (calmodulin itself, troponin-C, alkali and regulatory light chains of myosin, parvalbumin, intestinal calcium-binding protein, and glial S-100 phenylalanine-rich protein), came from a wide range of eukaryotic taxa and yielded a denser tree (more branch points within each lineage) than in earlier studies. Evidence obtained from the reconstructed pattern of base substitutions and deletions in these ancestral loci suggests that, during the early history of the family, selection acted as a transforming force on expressed genes among the duplicates to encode molecular sites with new or modified functions. In later stages of descent, however, selection was a conserving force that preserved the structures of many coadapted functional sites. Each branch of the family was found to have a unique average tempo of evolutionary change, apparently regulated through functional constraints. Proteins whose functions dictate multiple interaction with several other macromolecules evolved more slowly than those which display fewer protein-protein and protein-ion interactions, e.g., calmodulin and next troponin-C evolved at the slowest average rates, whereas parvalbumin evolved at the fastest. The history of all lineages, however, appears to be characterized by rapid rates of evolutionary change in earlier periods, followed by slower rates in more recent periods. A particularly sharp contrast between such fast and slow rates is found in the evolution of calmodulin, whose rate of change in earlier eukaryotes was manyfold faster than the average rate over the past 1 billion years. In fact, the amino acid replacements in the nascent calmodulin lineage occurred at residue positions that in extant metazoans are largely invariable, lending further support to the Darwinian hypothesis that natural selection is both a creative and a conserving force in molecular evolution.      

Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix

This study examined the role of extracellular matrix in regulating matrix phenotype of hepatic lipocytes, the major source of matrix in liver. Lipocytes (Ito, stellate, or fat-storing cells) were purified from normal rat liver and established in primary culture on either uncoated plastic, plastic coated with individual matrix proteins, or a "complete" gel matrix, a basement membrane-like matrix derived from the Engelbreth-Holm-Swarm (EHS) murine tumor. The ultrastructure of lipocytes cultured on the gel matrix resembled that of cells in normal liver, whereas lipocytes on plastic had dispersed nuclear chromatin and expanded rough endoplasmic reticulum, consistent with active proliferation and secretion. Lipocytes on the gel matrix exhibited no proliferative activity; cells maintained on plastic proliferated and produced type I collagen predominantly. Total collagen secretion by lipocytes on the gel matrix was 29% of that of cells on plastic, and consisted of type III collagen only. This difference extended to proteoglycan production, which was less than 5% of the amount produced by cells in conventional culture on plastic. The effects of the EHS gel were not reproduced by the individual components of the gel (laminin, type IV collagen, and heparan sulfate proteoglycan) or by a type I collagen gel. They were also reversible upon transfer of the cells to conventional culture. In contrast to lipocytes, collagen synthesis by hepatocytes was similar whether cultured on EHS gel or on plastic. These results show that the extracellular matrix can modulate matrix protein production by lipocytes and imply that, in early hepatic inflammation, changes in the hepatic subendothelial matrix may underlie stimulation of lipocyte matrix production and progression of the fibrotic process.     

Release of growth hormone from ox pituitary slices after pronase treatment

Proteolytic enzymes have been used both to modify properties of the cell membrane and to dissociate cells from many tissues including pituitary (4, 5, 12). Exposure of secretory tissues to pronase can alter their secretory response. Thus incubation of pancreatic islets of Langerhans in the presence of low concentrations of pronase increased the subsequent release of insulin in the presence of stimulatory and nonstimulatory glucose concentrations (7). The purpose of the present investigation was to determine whether low concentrations of pronase have the same stimulatory effect on the release of a pituitary hormone, growth hormone. Such an effect on hormone release could be of some importance in view of the development of dissociated cell systems as models for the study of the control of hormone release (4, 5).     

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.     

Emerging threat of the 21st century lightscape to global biodiversity

Over the last century the temporal and spatial distribution of light on Earth has been drastically altered by human activity. Despite mounting evidence of detrimental effects of light pollution on organisms and their trophic interactions, the extent to which light pollution threatens biodiversity on a global scale remains unclear. We assessed the spatial extent and magnitude of light encroachment by measuring change in the extent of light using satellite imagery from 1992 to 2012 relative to species richness for terrestrial and freshwater mammals, birds, reptiles, and amphibians. The encroachment of light into previously dark areas was consistently high, often doubling, in areas of high species richness for all four groups. This pattern persisted for nocturnal groups (e.g., bats, owls, and geckos) and species considered vulnerable to extinction. Areas with high species richness and large increases in light extent were clustered within newly industrialized regions where expansion of light is likely to continue unabated unless we act to conserve remaining darkness. Implementing change at a global scale requires global public, and therefore scientific, support. Here, we offer substantial evidence that light extent is increasing where biodiversity is high, representing an emerging threat to global biodiversity requiring immediate attention.     

Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody

Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor‐alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB‐EGF (heparin‐binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C‐terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala⁴¹, Glu⁴⁴, and His⁴⁵. These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859.