Sperm plasma membrane breakdown during Drosophila fertilization requires sneaky, an acrosomal membrane protein

Fertilization typically involves membrane fusion between sperm and eggs. In Drosophila, however, sperm enter eggs with membranes intact. Consequently, sperm plasma membrane breakdown (PMBD) and subsequent events of sperm activation occur in the egg cytoplasm. We previously proposed that mutations in the sneaky (snky) gene result in male sterility due to failure in PMBD. Here we support this proposal by demonstrating persistence of a plasma membrane protein around the head of snky sperm after entry into the egg. We further show that snky is expressed in testes and encodes a predicted integral membrane protein with multiple transmembrane domains, a DC-STAMP-like domain, and a variant RING finger. Using a transgene that expresses an active Snky-Green fluorescent protein fusion (Snky-GFP), we show that the protein is localized to the acrosome, a membrane-bound vesicle located at the apical tip of sperm. Snky-GFP also allowed us to follow the fate of the protein and the acrosome during fertilization. In many animals, the acrosome is a secretory vesicle with exocytosis essential for sperm penetration through the egg coats. Surprisingly, we find that the Drosophila acrosome is a paternally inherited structure. We provide evidence that the acrosome induces changes in sperm plasma membrane, exclusive of exocytosis and through the action of the acrosomal membrane protein Snky. Existence of testis-expressed Snky-like genes in many animals, including humans, suggests conserved protein function. We relate the characteristics of Drosophila Snky, acrosome function and sperm PMBD to membrane fusion events that occur in other systems.     

Expression pattern of Filamin-240 in Drosophila blood cells

The expression pattern of Filamin-240 was studied in subsets of Drosophila blood cells by means of immunofluorescent staining and Western blot analysis with use of an antibody specific to a "filamin-folding domain", a consensus motif profile generated from the 20 existing filamin repeats. Expression of Filamin-240 is restricted to lamellocytes - a special blood cell type of the cellular immune response - and is involved in the regulation of lamellocyte development. In the cher1 homozygous larvae, which lack Filamin-240 protein, a vigorous lamellocyte differentiation occurs which is further enhanced upon in vivo immune challenge by a parasitic wasp, Leptopilina boulardi. By introducing a full-length transgene encoding the Drosophila Filamin-240 protein into the cher1 Filamin-deficient homozygous mutant, the mutant blood cell phenotype was rescued. These data demonstrate that the expression of Filamin-240 is strictly lamellocyte specific in Drosophila blood cells and that the protein is a suppressor of lamellocyte development.     

Angiopoietin-1-induced angiogenesis is modulated by endothelial NADPH oxidase

Reactive oxygen species (ROS) play a central role in the pathogenesis of many cardiovascular diseases, such as atherosclerosis and hypertension. Endothelial NADPH oxidase is the major source of intracellular ROS. The present study investigated the role of endothelial NADPH oxidase-derived ROS in angiopoietin-1 (Ang-1)-induced angiogenesis. Exposure of porcine coronary artery endothelial cells (PCAECs) to Ang-1 (250 ng/ml) for periods up to 30 min led to a transient and dose-dependent increase in intracellular ROS. Thirty minutes of pretreatment with the NADPH oxidase inhibitors diphenylene iodinium (DPI, 10 microM) and apocynin (200 microM) suppressed Ang-1-stimulated ROS. Pretreatment with either DPI or apocynin also significantly attenuated Ang-1-induced Akt and p44/42 MAPK phosphorylation. In addition, inhibition of NADPH oxidase significantly suppressed Ang-1-induced endothelial cell migration and sprouting from endothelial spheroids. Using mouse heart microvascular endothelial cells from wild-type (WT) mice and mice deficient in the p47(phox) component of NADPH oxidase (p47(phox-/-)), we found that although Ang-1 stimulated intracellular ROS, Akt and p42/44 MAPK phosphorylation, and cell migration in WT cells, the responses were strikingly suppressed in cells from the p47(phox-/-) mice. Furthermore, exposure of aortic rings from p47(phox-/-) mice to Ang-1 demonstrated fewer vessel sprouts than WT mice. Inhibition of the Tie-2 receptor inhibited Ang-1-induced endothelial migration and vessel sprouting. Together, our data strongly suggest that endothelial NADPH oxidase-derived ROS play a critical role in Ang-1-induced angiogenesis.     

Selective transport by SecA2: An expanding family of customized motor proteins

The SecA2 proteins are a special class of transport-associated ATPases that are related to the SecA component of the general Sec system, and are found in an increasingly large number of Gram-positive bacterial species. The SecA2 substrates are typically linked to the cell wall, but may be lipid-linked, peptidoglycan-linked, or non-covalently associated S-layer proteins. These substrates can have a significant impact on virulence of pathogenic organisms, but may also aid colonization by commensals. The SecA2 orthologues range from being highly similar to their SecA paralogues, to being distinctly different in apparent structure and function. Two broad classes of SecA2 are evident. One transports multiple substrates, and may interact with the general Sec system, or with an as yet unidentified transmembrane channel. The second type transports a single substrate, and is a component of the accessory Sec system, which includes the SecY paralogue SecY2 along with the accessory Sec proteins Asp1-3. Recent studies indicate that the latter three proteins may have a unique role in coordinating post-translational modification of the substrate with transport by SecA2. Comparative functional and phylogenetic analyses suggest that each SecA2 may be uniquely adapted for a specific type of substrate. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Phylogenetic utility of ycf1 in orchids: a plastid gene more variable than matK

Plastid DNA sequences have been widely used by systematists for reconstructing plant phylogenies. The utility of any DNA region for phylogenetic analysis is determined by ease of amplification and sequencing, confidence of assessment in phylogenetic character alignment, and by variability across broad taxon sampling. Often, a compromise must be made between using relatively highly conserved coding regions or highly variable introns and intergenic spacers. Analyses of a combination of these types of DNA regions yield phylogenetic structure at various levels of a tree (i.e., along the spine and at the tips of the branches). Here, we demonstrate the phylogenetic utility of a heretofore unused portion of a plastid protein-coding gene, hypothetical chloroplast open reading frame 1 (ycf1), in orchids. All portions of ycf1 examined are highly variable, yet alignable across Orchidaceae, and are phylogenetically informative at the level of species. In Orchidaceae, ycf1 is more variable than matK both in total number of parsimony informative characters and in percent variability. The nrITS region is more variable than ycf1, but is more difficult to align. Although we only demonstrate the phylogenetic utility of ycf1 in orchids, it is likely to be similarly useful among other plant taxa.     

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Objectives

to assess the cardioprotective properties of a blueberry enriched diet (BD).

Background

Reactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.

Methods and Results

Following 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (t_MPT) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed t_MPT. The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.

Conclusion

A blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.     

Colony size and not nest density drives reproductive output in the Common Tern Sterna hirundo

Density is known to be an important factor in population size regulation. Several mechanisms of density limitation have been identified in colonial birds. We studied competition in Common Terns Sterna hirundo to assess whether the factor limiting reproductive output was competition for nest‐sites, which is dependent on local nest density, or density‐dependent competition for food resources, which is dependent on overall colony size using the same foraging area. We found strong associations of both colony size and nest density with reproductive output in five colonies of Common Terns in three different habitats (one marine, two freshwater). Based on detailed long‐term datasets of six separate sub‐colonies of the Banter See colony that differed in nest density, we found that reproductive success was not related to nest density but to overall colony size, possibly a result of resource depletion and food competition. We also found carry‐over effects of colony size during rearing on post‐fledging return rate. These results have important implications for the conservation management plans aimed at recovering declining populations of Common Terns.     

Conformational investigation of alpha,beta-dehydropeptides. XV: N-acetyl-alpha,beta-dehydroamino acid N 'N '-dimethylamides: conformational properties from infrared and theoretical studies.

The FTIR spectra were analysed in the region of the nu(s)(N-H), AI(C=O) and nu(s)(Calpha=Cbeta) bands for a series of Ac-DeltaXaa-NMe2, where DeltaXaa = DeltaAla, (Z)-DeltaAbu, (Z)-DeltaLeu, (Z)-DeltaPhe and DeltaVal, to determine a predominant solution conformation of these alpha,beta-dehydropeptide-related molecules. Measurements were taken in CCl4, DCM and MeCN solutions. In the same way, spectra of saturated analogues Ac-Xaa-NMe2, where Xaa = Ala, Abu, Leu, Phe and Val, were investigated. To help interpret the spectroscopic results, conformational maps were calculated by the B3LYP/6-31+G** method. Also, the relative energies of all conformers of the dehydro compounds in vacuo as well as in the studied solvents in addition to the theoretical IR frequencies of these conformers were calculated. For comparison, molecules of two saturated analogues, Ac-L-Ala-NMe2 and Ac-L-Phe-NMe2, were calculated in a similar way. Both unsaturated and saturated compounds, which have an aliphatic side chain, occur in CCl4 and DCM mainly as a mixture of extended conformers with the C5 H-bond and open conformers. As solvent polarity increases, participation of the open conformers also increases, and in MeCN, the model amides are almost exclusively in the open form, except Ac-DeltaAla-NMe2, which shows a small amount of the H-bonded conformer. Ac-DeltaAla-NMe2 and Ac-DeltaAbu-NMe2 have stronger C5 hydrogen bonds than those of their saturated counterparts. As the calculations indicate, the open conformation of the unsaturated amides is conformer H/F with phi, psi -44 +/- 5 degrees, 127 +/- 4 degrees. This is the second lowest in energy conformer in vacuo and in CCl4 and the lowest one in more polar solvents. The open conformation of Ac-L-Ala-NMe2 constitutes conformer C with phi, psi -101.5 degrees, 112.7 degrees. For Ac-DeltaAla-NMe2 and Ac-DeltaAbu-NMe2, FTIR also reveals the presence of a third conformer. Calculations indicate that is the semiextended conformer D with the N1-H1...N2 hydrogen bond/contact. In all solvents, Ac-L-Phe-NMe2 and Ac-(Z)-DeltaPhe-NMe2 show only the extended E and the open H/F, respectively. In both there is an amide/pi(Ph) interaction.