Influence of female age, sperm senescence and multiple mating on sperm viability in female Drosophila melanogaster

Sperm viability has been associated with the degree of promiscuity across species, as well as the degree of reproductive success within species. Thus, sperm survival within the female reproductive tract likely plays a key role in how mating systems evolve. In the fruit fly, Drosophila melanogaster, however, the extent and cause of sperm death has been the subject of recent debate. Here, we assess sperm death within the female reproductive tract of D. melanogaster following single and multiple matings in order to elucidate the extent of death and its potential mechanisms, including an acute female response to mating, female age and/or sperm senescence. We found no evidence that sperm viability was influenced by an acute female response or female age. We also found that rival ejaculates did not influence viability, supporting recent work in the system. Instead, the majority of death appears to be due to the aging of male gametes within the female, and that at least some dead resident sperm remain in the female after multiple mating. In contrast to earlier in vivo work, we found that overall sperm death was minimal (8.7%), indicating viability should have a negligible influence on female remating rates.     

Interaction of calmodulin with L-selectin at the membrane interface: implication on the regulation of L-selectin shedding

The calmodulin (CaM) hypothesis of ectodomain shedding stipulates that CaM, an intracellular Ca²⁺-dependent regulatory protein, associates with the cytoplasmic domain of l-selectin to regulate ectodomain shedding of l-selectin on the other side of the plasma membrane. To understand the underlying molecular mechanism, we have characterized the interactions of CaM with two peptides derived from human l-selectin. The peptide ARR18 corresponds to the entire cytoplasmic domain of l-selectin (residues Ala317-Tyr334 in the mature protein), and CLS corresponds to residues Lys280-Tyr334, which contains the entire transmembrane and cytoplasmic domains of l-selectin. Monitoring the interaction by fluorescence spectroscopy and other biophysical techniques, we found that CaM can bind to ARR18 in aqueous solutions or the l-selectin cytoplasmic domain of CLS reconstituted in the phosphatidylcholine bilayer, both with an affinity of approximately 2 μM. The association is calcium independent and dynamic and involves both lobes of CaM. In a phospholipid bilayer, the positively charged l-selectin cytoplasmic domain of CLS is associated with anionic phosphatidylserine (PS) lipids at the membrane interface through electrostatic interactions. Under conditions where the PS content mimics that in the inner leaflet of the cell plasma membrane, the interaction between CaM and CLS becomes undetectable. These results suggest that the association of CaM with l-selectin in the cell can be influenced by the membrane bilayer and that anionic lipids may modulate ectodomain shedding of transmembrane receptors.     

Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.     

A Natural Genetic Variant of Granzyme B Confers Lethality to a Common Viral Infection

Many immune response genes are highly polymorphic, consistent with the selective pressure imposed by pathogens over evolutionary time, and the need to balance infection control with the risk of auto-immunity. Epidemiological and genomic studies have identified many genetic variants that confer susceptibility or resistance to pathogenic micro-organisms. While extensive polymorphism has been reported for the granzyme B (GzmB) gene, its relevance to pathogen immunity is unexplored. Here, we describe the biochemical and cytotoxic functions of a common allele of GzmB (GzmB^W) common in wild mouse. While retaining ‘Asp-ase’ activity, GzmB^W has substrate preferences that differ considerably from GzmB^P, which is common to all inbred strains. In vitro, GzmB^W preferentially cleaves recombinant Bid, whereas GzmB^P activates pro-caspases directly. Recombinant GzmB^W and GzmB^P induced equivalent apoptosis of uninfected targets cells when delivered with perforin in vitro. Nonetheless, mice homozygous for GzmB^W were unable to control murine cytomegalovirus (MCMV) infection, and succumbed as a result of excessive liver damage. Although similar numbers of anti-viral CD8 T cells were generated in both mouse strains, GzmB^W-expressing CD8 T cells isolated from infected mice were unable to kill MCMV-infected targets in vitro. Our results suggest that known virally-encoded inhibitors of the intrinsic (mitochondrial) apoptotic pathway account for the increased susceptibility of GzmB^W mice to MCMV. We conclude that different natural variants of GzmB have a profound impact on the immune response to a common and authentic viral pathogen.     

Synthesis and Characterization of Polysialic Acid–Uricase Conjugates for the Treatment of Hyperuricemia

Uricase, an enzyme used for the treatment of hyperuricemia, is conjugated with polysialic acid (PSA) of average molecular weight of 10 kDa by reductive amination in presence of NaCNBH3 in order to improve its pharmacological properties. Polysialylation with 50-,100-,150- and 200-fold molar excess of PSA increased the percentage substitution of the free amino groups on enzyme surface (46, 66, 78 and 80 % respectively). The SDS-PAGE is used to visualize the conjugates with increased molecular weight and it retained almost 65 % of their initial specific activity after conjugation. The stability studies at physiological condition reveals improved stability and activity than the native enzyme. The apparent KM of the enzyme has increased slightly from 4.18 × 10^?5 M to 5.46 × 10^?5 M suggesting that the affinity of the substrate to the enzyme has not been altered to a higher extent. The conjugates, when probed against anti-uricase antibodies generated in rabbit, showed a clean decline in the affinity by 35 % and also have retained double the catalytic activity than that of the native enzyme after exposure to antiserum. The results suggest that uricase-PSA conjugates can be used as an alternative to the conventional synthetic polymer-enzyme conjugates.     

Ca2+–Calmodulin regulates SNARE assembly and spontaneous neurotransmitter release via v-ATPase subunit V0a1

Most chemical neurotransmission occurs through Ca²⁺-dependent evoked or spontaneous vesicle exocytosis. In both cases, Ca²⁺ sensing is thought to occur shortly before exocytosis. In this paper, we provide evidence that the Ca²⁺ dependence of spontaneous vesicle release may partly result from an earlier requirement of Ca²⁺ for the assembly of soluble N-ethylmaleimide–sensitive fusion attachment protein receptor (SNARE) complexes. We show that the neuronal vacuolar-type H⁺-adenosine triphosphatase V0 subunit a1 (V100) can regulate the formation of SNARE complexes in a Ca²⁺–Calmodulin (CaM)-dependent manner. Ca²⁺–CaM regulation of V100 is not required for vesicle acidification. Specific disruption of the Ca²⁺-dependent regulation of V100 by CaM led to a >90% loss of spontaneous release but only had a mild effect on evoked release at Drosophila melanogaster embryo neuromuscular junctions. Our data suggest that Ca²⁺–CaM regulation of V100 may control SNARE complex assembly for a subset of synaptic vesicles that sustain spontaneous release.     

RPE65, Visual Cycle Retinol Isomerase, Is Not Inherently 11-cis-specific: SUPPORT FOR A CARBOCATION MECHANISM OF RETINOL ISOMERIZATION*

The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C₁₁ of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to ∼75% of wild type, W331F to ∼50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-π carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C₁₅ oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11- and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal.     

In Silico Analysis of Natural Resistance-Associated Macrophage Protein (NRAMP) Family of Transporters in Rice

In Oryza sativa (rice) there are seven members in the NRAMP (natural resistance- associated macrophage protein) family of transporter proteins. They have been identified as OsNRAMP1, OsNRAMP2, OsNRAMP3, OsNRAMP4, OsNRAMP5, OsNRAMP6 and OsNRAMP7. Several metal ions like Zn²⁺, Mn²⁺, Fe²⁺, Cd²⁺ etc. have been studied to be transported via NRAMP transporter proteins in rice plant. In spite of this, very little information is available regarding these transporters. Hence it is important to computationally predict and characterize the OsNRAMP family of transporters for studying and understanding their molecular insights in future studies. For this purpose, various in silico methods and tools were used for the characterization of OsNRAMP family of transporter proteins. Physico-chemical properties of the protein sequences were calculated, putative transmembrane domains (TMDs) and conserved motif signatures were determined and their interaction partners were predicted. 3D models of all the members of OsNRAMP transporters were generated using online structure prediction tool followed by their analysis. In silico microarray analysis was done to understand the expression pattern of these transporters in rice plant. Currently, only limited knowledge is available about the structural and functional aspects of these transporters, hence this study would provide more theoretical information about them.