Preservation of boar semen at 18 degrees C induces lipid peroxidation and apoptosis like changes in spermatozoa

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (DeltaPsi(m)) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 degrees C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, DeltaPsi(m), and membrane permeability. The lipid peroxidation status of the sperm was assessed based on the malonaldehyde (MDA) levels. Detection of DeltaPsi(m) was done using 3,3'-dihexyloxacarbocyanine iodide [DiOC(6)(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p<0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/10(9) sperm) were low in sperm (99.83+/-2.69) and seminal plasma (191.98+/-11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (-0.97), live percent (-0.97), acrosomal integrity (-0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (-0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r=0.98). There was a significant (p<0.05) increase in the sperm cells with low DeltaPsi(m) from 0 to 96 h of preservation. Before preservation, 14.85+/-4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00+/-6.25%. The apoptotic sperm population was 8.33+/-2.31% in fresh semen, while this population was 25.19+/-4.25% at 96 h of preservation and the difference was significant (p<0.05). The findings of the present study revealed that liquid preservation of boar semen at 18 degrees C induces lipid peroxidation, decrease mitochondrial membrane potential and increase the plasma membrane permeability.     

Detecting the form of selection in the outer membrane protein C of Enterobacter aerogenes strains and Salmonella species

The types of selective pressure operating on the outer membrane protein C (ompC) of Enterobacter aerogenes strains, the causative agent for nosocomial infections, and Salmonella sp., the hazardous pathogen are investigated using the maximum likelihood-based codon substitution models. Although the rate of amino acid replacement to the silent substitution (omega) across the entire codon sites of ompC of E. aerogenes (omega=0.3194) and Salmonella sp. (omega=0.2047) indicate that the gene is subjected to purifying selection (i.e. omega<1), approximately 3.7% of ompC codon sites in E. aerogenes (omega=21.52) are under the influence of positive Darwinian selection (i.e. omega>1). Such contrast in the intensity of selective pressures in both pathogens could be associated with the differential response to the adverse environmental changes. In E. aerogenes, majority of the positively selected sites are located in the hypervariable cell-surface-exposed domains whereas the trans-membrane domains are functionally highly constrained.     

Efficient synthesis of 3,3-diheteroaromatic oxindole analogues and their in vitro evaluation for spermicidal potential

Syntheses of 3,3-diheteroaromatic oxindole derivatives has been achieved by coupling indole-2,3-dione (isatin) with differently substituted indoles and pyrrole in presence of I₂ in i-PrOH. The in vitro spermicidal potentials and the mode of spermicidal action of the synthesized analogues were evaluated and the derivative, 3,3-bis (5-methoxy-1H-indol-3-yl) indolin-2-one (3d) exhibited most significant activity.     

Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins

The human cytomegalovirus (HCMV) US12 gene family is a group of predicted seven-transmembrane, G-protein-coupled receptor-related proteins, about which little is known. Specific rabbit polyclonal antibodies detected US17 and US18 beginning 54 and 36 h after infection, respectively, with expression of both proteins dependent on viral DNA synthesis. While US14 and US18 are expressed exclusively in the cytoplasm, we unexpectedly found abundant expression of US17 in both the cytoplasm and nucleoplasm. N- and C-terminally tagged versions of US17 were readily detected in the cytoplasm of transfected mammalian cells, but not in nuclei, suggesting that nuclear localization involves other viral proteins or an infection-triggered cellular process. There was no specific colocalization between US17 and other nuclear expressed HCMV-encoded proteins (IE-2, DNA polymerase processivity factor, and pp28/UL99). To determine whether the observed nuclear localization might be the product of a process by which a soluble C-terminal segment of the full-length protein is expressed, we constructed a recombinant virus that incorporates a synthetic epitope at its N terminus, which in conjunction with the antipeptide antibody that targets its predicted cytoplasmic C-terminal segment, enables simultaneous independent detection of both termini. In cells infected with the recombinant, the US17 N and C termini had limited colocalization, with the N-terminal segment not detected in nuclei, supporting the segmentation hypothesis. Consistent with this, a fragment with an apparent molecular size of 10 kDa was detected by immunoblotting. We have identified the first viral example of a seven-transmembrane protein that is either segmented or expressed in nuclei. Further study will be required to learn the mechanism by which this occurs and the function of the nuclear localizing segment. This likely represents yet another mechanism by which a virus has hijacked or modified cellular regulatory pathways for its benefit.     

A synthetic S6 segment derived from KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.