A new nuclear protease with cathepsin L properties is present in HeLa and Caco‐2 cells

Recently many authors have reported that cathepsin L can be found in the nucleus of mammalian cells with important functions in cell‐cycle progression. In previous research, we have demonstrated that a cysteine protease (SpH‐protease) participates in male chromatin remodeling and in cell‐cycle progression in sea urchins embryos. The gene that encodes this protease was cloned. It presents a high identity sequence with cathepsin L family. The active form associated to chromatin has a molecular weight of 60 kDa, which is higher than the active form of cathepsin L described until now, which range between 25 and 35 kDa. Another difference is that the zymogen present in sea urchin has a molecular weight of 75 and 90 kDa whereas for human procathepsin L has a molecular weight of 38–42 kDa. Based on these results and using a polyclonal antibody available in our laboratory that recognizes the active form of the 60 kDa nuclear cysteine protease of sea urchin, ortholog to human cathepsin L, we investigated the presence of this enzyme in HeLa and Caco‐2 cells. We have identified a new nuclear protease, type cathepsin L, with a molecular size of 60 kDa, whose cathepsin activity increases after a partial purification by FPLC and degrade in vitro histone H1. This protease associates to the mitotic spindle during mitosis, remains in the nuclei in binuclear cells and also translocates to the cytoplasm in non‐proliferative cells. J. Cell. Biochem. 111: 1099–1106, 2010. © 2010 Wiley‐Liss, Inc.     

Aryldiazenido derivatives: A new entry to the functionalization of Keggin polyoxometalates

A series of aryldiazenido polyoxomolybdates of the type (nBu₄N)₂[Mo₅O₁₃(OMe)₄(NNAr){Na(MeOH)}] (Ar = C₆F₅, 1; Ar = O₂N-o-C₆H₄, 2; Ar = O₂N-m-C₆H₄, 3; Ar = O₂N-p-C₆H₄, 4a; Ar = (O₂N)₂-o,p-C₆H₃, 5) have been obtained by controlled degradation of the parent compounds (nBu₄N)₃[Mo₆O₁₈(NNAr)] with NaOH in methanol. They have been characterized by elemental analysis and UV-Vis and IR spectroscopy. In addition, 4a has been characterized by ⁹⁵Mo NMR spectroscopy and the crystal structure of (nBu₄N)₂[Mo₅O₁₃(OMe)₄(NNC₆H₄-p-NO₂){Na(H₂O))]·H₂O (4b) has been determined by X-ray diffraction. The molecular structure of the anion of 4b features a lacunary Lindqvist-type anion [Mo₅O₁₃(OMe)₄(NNC₆H₄-p-NO₂)]³⁻ interacting with a sodium cation through the four terminal axial oxygen atoms. The 1:1 sodium complexes react with BaCl₂ and BiCl₃ to yield 2:1 complexes which have been isolated as (nBu₄N)₄[Ba{Mo₅O₁₃(OMe)₄(NNAr)}₂] (Ar = C₆F₅, 6; Ar = O₂N-p-C₆H₄, 7) and (nBu₄N)₃[Bi{Mo₅O₁₃(OMe)₄(NNAr)}₂] (Ar = C₆F₅, 8; Ar = O₂N-p-C₆H₄, 9). X-ray crystallography analysis of 9·Me₂CO has shown that the tetradentate [Mo₅O₁₃(OMe)₄(N₂C₆H₄-p-NO₂)]³⁻ anions provide a square-antiprismatic environment for Bi. In contrast, IR spectroscopy provides evidence for a square-prismatic environment of Ba in 6 and 7. In acetonitrile-methanol mixed solvent, [Mo₅O₁₃(OMe)₄(NNAr)]³⁻ and [PW₁₁O₃₉]⁷⁻, generated in situ by alkaline degradation of their respective parents, [Mo₆O₁₈(NNAr)]³⁻ and [PW₁₂O₄₀]³⁻, react together to give the Keggin-type diazenido compounds (nBu₄N)₄[PW₁₁O₃₉(MoNNAr)] (Ar = O₂N-o-C₆H₄, 10; Ar = O₂N-m-C₆H₄, 11; Ar = O₂N-p-C₆H₄, 12), which have been characterized by ³¹P and ¹⁸³W NMR spectroscopy.     

Structure and stability of hereditary spherocytosis mutants of the cytosolic domain of the erythrocyte anion exchanger 1 protein

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Its 52 kDa C-terminal domain functions as a chloride-bicarbonate exchanger, while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the cytoskeleton to the membrane. Several proteins bind to cdb3, including protein 4.2, a cytoskeletal protein. Three mutations in cdb3 are associated with hereditary spherocytosis (HS) and decreased levels of protein 4.2 in erythrocytes. In this study, these cdb3 mutants (E40K, G130R, and P327R) were expressed in and purified from Escherichia coli. Sedimentation experiments showed that the wild-type and mutant proteins are dimers. No difference in secondary structure between mutant and wild-type proteins was detected using circular dichroism (CD) analysis. The wild-type and mutant proteins underwent similar pH-dependent conformational changes when monitored by intrinsic tryptophan fluorescence. Urea denaturation of proteins monitored by intrinsic fluorescence showed no significant differences in the sensitivity of the proteins to this chemical denaturant. Thermal denaturation monitored by CD and by calorimetry revealed that only the P327R mutant had a significantly lower midpoint of transition (approximately 5 degrees C) than the wild-type protein, suggesting a modest decrease in stability. The results show that the HS mutant cdb3 proteins do not differ to any great extent in structure from the wild-type protein, suggesting that the HS mutations may directly affect protein 4.2 binding.     

Antibacterial activity of wine phenolic compounds and oenological extracts against potential respiratory pathogens

Aims: To investigate the effect of seven wine phenolic compounds and six oenological phenolic extracts on the growth of pathogenic bacteria associated with respiratory diseases (Pseudomonas aeruginosa, Staphylococcus aureus, Moraxella catarrhalis, Enterococcus faecalis, Streptococcus sp Group F, Streptococcus agalactiae and Streptococcus pneumoniae). Methods and Results: Antimicrobial activity was determined using a microdilution method and quantified as IC₅₀. Mor. catarrhalis was the most susceptible specie to phenolic compounds and extracts. Gallic acid and ethyl gallate were the compounds that showed the greatest antimicrobial activity. Regarding phenolic extracts, GSE (grape seed extract) and GSE‐O (oligomeric‐rich fraction from GSE) were the ones that displayed the strongest antimicrobial effects. Conclusions: Results highlight the antimicrobial properties of wine phenolic compounds and oenological extracts against potential respiratory pathogens. The antimicrobial activity of wine phenolic compounds was influenced by the type of phenolic compounds. Gram‐negative bacteria were more susceptible than Gram‐positive bacteria to the action of phenolic compounds and extracts; however, the effect was species‐dependent. Significance and Impact of Study: The ability to inhibit the growth of respiratory pathogenic bacteria as shown by several wine phenolic compounds and oenological extracts warrants further investigations to explore the use of grape and wine preparations in oral hygiene.     

Diamine and aminoalcohol derivatives active against Trypanosoma brucei

Twenty compounds selected as representative members of three series of long-chain 1,2-diamines, 2-amino-1-alkanols and 1-amino-2-alkanols structurally related to dihydrosphingosin, were synthesized and tested in vitro for their ability to inhibit the sleeping sickness parasites Trypanosoma bruceirhodesiense and Trypanosoma brucei gambiense. Eight compounds showed EC(50) values in the submicromolar range, with selectivity indexes up to 39 related to the respective cytotoxicity values for Vero cells. The parasite phenotype detected after treatment with the most potent compounds showed irreversible cell morphology alterations of the flagellar pocket that lead to inhibition of cell growth and parasite death.     

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.     

Involvement of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in response to oxidative stress

Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Because non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (NP-Ga3PDHase) is involved in the production of reductive power (NADPH) in the cytosol, its behavior under oxidative stress conditions was analyzed. The specific activity of the enzyme was found to increase up to 2-fold after oxidative conditions imposed by methylviologen in wheat and maize seedlings. Under moderate oxidant concentration, lack of mRNA induction was observed. The increase in specific activity would thus be a consequence of a significant stability of NP-Ga3PDHase. Our results suggest that the enzyme could be modified by oxidation of cysteine residues, but formation of disulfide bridges is dependent on levels of divalent cations and 14-3-3 proteins. The latter differential effect could be critical to relatively maintain energy and reductant levels in the cytoplasm of plant cells under oxidative stress.     

Cicada acoustic communication: potential sound partitioning in a multispecies community from Mexico (Hemiptera: Cicadomorpha: Cicadidae)

Multispecies cicada communities in neotropical rainforests produce a complex and intense acoustic environment. In a fragment of a Mexican rainforest (Veracruz, Mexico), a cicada community at the end of the dry season consisted of nine species ( Daza montezuma; Pacarina schumanni; Miranha imbellis; Dorisiana sutori; Fidicinoides picea; Fidicinoides pronoe; Quesada gigas; one species of the genus Neocicada and one uncaught canopy species). Seven of the nine species formed dense choruses at dawn and at dusk. Each species showed preferences in the height of calling sites. Males of the species were solitary or gregarious, and followed a 'call-fly' or a 'call-stay' calling strategy. Acoustic signals of each species had particular time and frequency patterns. All these specific features appear to separate the nine species acoustically and lead to a partitioning of the acoustic environment. The acoustic partitioning might decrease the risk of heterospecific courting and mating.© 2002 The Linnean Society of London, Biological Journal of the Linnean Society , 2002, 75 , 379–394.