BackgroundOptineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys) is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process.ResultsWe show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N) in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking.ConclusionsOur results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for the pathogenesis of glaucoma caused by the E50K mutation because endocytic recycling is vital for maintaining homeostasis.

Curcumin, the active ingredient of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. Although its precise mode of action remains elusive, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis in cancer cells. Curcumin was shown to be responsible for the inhibition of AK-5 tumor (a rat histiocytoma) growth by inducing apoptosis in AK-5 tumor cells via caspase activation. This study was designed to investigate the mechanism leading to the induction of apoptosis in AK-5 tumor cells. Curcumin treatment resulted in the hyperproduction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (v vi i m ) and cytochrome c release to the cytosol, with the concomitant exposure of phosphatidylserine (PS) residues on the cell surface. This study suggests redox signalling and caspase activation as the mechanisms responsible for the induction of curcumin mediated apoptosis in AK-5 tumor cells.z 1999 Federation of European Biochemical Societies.

With a view to understand the molecular basis of sperm motility, we have tried to establish the human sperm proteome by two-dimensional PAGE MALDI MS/MS analysis. We report identification of 75 different proteins in the human spermatozoa. Comparative proteome analysis was carried out for asthenozoospermic and normozoospermic patients to understand the molecular basis of sperm motility. Analysis revealed eight proteins (including one unidentified) with altered intensity between the groups. Differential proteins distributed into three functional groups: 'energy and metabolism' (triose-phosphate isomerase, glycerol kinase 2, testis specific isoform and succinyl-CoA:3-ketoacid co-enzyme A transferase 1, mitochondrial precursor); 'movement and organization' (tubulin beta 2C and tektin 1) and 'protein turnover, folding and stress response' (proteasome alpha 3 subunit and heat shock-related 70 kDa protein 2). It was interesting to note that although the proteins falling in the functional group of 'energy and metabolism' are higher in the asthenozoospermic patients, the other two functional groups contain proteins, which are higher in the normozoospermic samples. Validation of results carried out for proteasome alpha 3 subunit by immunoblotting and confocal microscopy, confirmed significant changes in intensity of proteasome alpha 3 subunit in asthenozoospermic samples when compared with normozoospermic controls. Significant positive correlation too was found between proteasome alpha 3 subunit levels and rapid, linear progressive motility of the spermatozoa. In our understanding, this data would contribute appreciably to the presently limited information available about the proteins implicated in human sperm motility.

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The activities of defensins HBD-1, HBD-2, and HBD-3 and their C-terminal analogs Phd1, Phd2, and Phd3 against Candida albicans were investigated. Phd1 to Phd3 showed lower-level activities than HBD-1 to HBD-3, although metabolic inhibitors did not render Phd1 to Phd3 inactive. Their activities were also less salt sensitive than those of HBD-1 to HBD-3. Confocal microscope images indicated that the initial site of action was the fungal membrane.Mammalian defensins comprising the alpha and beta families are important components of the innate immune system (1,8,17,18,24,25,29,30). HBD-1 and HBD-2 are active against gram-negative bacteria. Their activities are attenuated by increasing concentrations of NaCl (2, 9, 10). HBD-3 is active against both gram-negative and gram-positive bacteria and is not affected by NaCl (3, 11). The findings of extensive studies have indicated that native disulfide bridges are not essential for antibacterial activity and that segments of HBD-1 to HBD-3 shorter than the full-length defensins also exhibit antibacterial activities (12-16, 21, 22, 26, 28, 35, 36). In recent years, there has been considerable interest in the antifungal activities of beta-defensins, as Candida albicans is responsible for causing oral candidiasis, particularly in patients infected with human immunodeficiency virus (5, 20). HBD-1 to HBD-3 have been detected previously in salivary glands and salivary secretions (4,6,7,23,27). The killing of C. albicans by HBD-2 and HBD-3 is salt sensitive and energy dependent (33). We have shown that single disulfide peptides spanning the C-terminal segments of HBD-1 to HBD-3, i.e., Phd1 (ACPIFTKIQGTY RGKAKCK), Phd2 (FCPRRYKQIGTGLPGTKCK), and Phd3 (SCLPKEEQIGKSTRGRKCRRKK) (disulfide bridges indicated by underlining), exhibit antibacterial activities (16). In this report, we describe their activities against C. albicans and compare the effects of salts and metabolic inhibitors on these peptides with the effects on HBD-1 to HBD-3.HBD-1, HBD-2, and HBD-3 were purchased from Peptides International (Louisville, KY). Phd1, Phd2, and Phd3 were synthesized as described earlier using 4-(hydroxymethyl)phenoxyacetamidomethyl resin and 9-fluorenylmethoxy carbonyl chemistry (16). The formation of disulfide bonds was accomplished by air oxidation at a peptide concentration of 0.5 mg/ml for 24 h at room temperature. Purified peptides were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry on an ABI Voyager DE STR matrixassisted laser desorption ionization-time of flight mass spectrometer (PerSeptive Biosystems) using recrystallized ␣-cyano-4-hydroxycinnamic acid as a matrix (16). Peptide labeling with carboxyfluorescein (CF) at a free amino group of the N-terminal amino acid was carried out by treating 10 mg of resinbound peptide with 0.8 ml of dimethylformamide containing CF and activating agents as described earlier (34). The deprotection of CF-labeled peptides (CF-Phd1 to CF-Phd3) from the resin, purification, and characterization by mass spectromet...

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. We have used enhanced green fluorescent protein-tagged X. oryzae pv. oryzae cells in conjunction with confocal microscopy to monitor the role of several adhesin-like functions in bacterial adhesion to leaf surface and early stages of leaf entry. Mutations in genes encoding either the Xanthomonas adhesin-like protein A (XadA) or its paralog, Xanthomonas adhesin-like protein B (XadB), as well as the X. oryzae pv. oryzae homolog of Yersinia autotransporter-like protein H (YapH), exhibit deficiencies in leaf attachment or entry. A mutation in the X. oryzae pv. oryzae pilQ gene, which is predicted to encode the type IV pilus secretin, appears to have no effect on leaf attachment or entry. The xadA- mutant is deficient in the ability to cause disease following surface inoculation while the XadB, YapH, and PilQ functions are less important than XadA for this process. The xadA- and xadB- mutants have no effect on virulence following wound inoculation whereas the yapH- and pilQ- mutants are always virulence deficient following wound inoculation. Overall, these results indicate that multiple adhesin-like functions are involved in promoting virulence of X. oryzae pv. oryzae, with preferential involvement of individual functions at different stages of the disease process.