Effect of mutation and phosphorylation of type I keratins on their caspase-mediated degradation

Type I keratins K18 and K19 undergo caspase-mediated degradation during apoptosis. Two known K18 caspase cleavage sites are aspartates in the consensus sequences VEVDA and DALDS, located within the rod domain and tail domain, respectively. Several K14 (another type I keratin) mutations within the caspase cleavage motif have been described in patients with epidermolysis bullosa simplex. Here we use extensive mutational analysis to show that K19 and K14 are caspase substrates and that the ability to undergo caspase-mediated digestion of K18, K19, or K14 is highly dependent on the location and nature of the mutation within the caspase cleavage motif. Caspase cleavage of K14 occurs at the aspartate of VEMDA, a consensus sequence found in type I keratins K12-17 with similar but not identical sequences in K18 and K19. For K18, apoptosis-induced cleavage occurs sequentially, first at (393)DALD and then at (234)VEVD. Hyperphosphorylation of K18 protects from caspase-3 in vitro digestion at (234)VEVD but not at (393)DALD. Hence, keratins K12-17 are likely caspase substrates during apoptosis. Keratin hyperphosphorylation, which occurs early in apoptosis, protects from caspase-mediated K18 digestion in a cleavage site-specific manner. Mutations in epidermolysis bullosa simplex patients could interfere with K14 degradation during apoptosis, depending on their location.     

Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-alpha-treated human aortic endothelial cells

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 microg/ml), the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2 +/- 3.2% and 80.0 +/- 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 microg/ml), 84.5 +/- 1.9%, 78.8 +/- 1.2%, 58.9 +/- 0.4%, 58.7 +/- 0.9%, and 57.4 +/- 0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 microg/ml) or Sal B (5 microg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (45.7 +/- 2.5% and 55.8 +/- 1.2%, respectively). SME or Sal B significantly inhibited TNF-alpha-induced activation of nuclear factor kappa B (NF-kappaB) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis.     

DEATH OF STEPPE CRYPTOGAMS UNDER THE ASH FROM MOUNT ST. HELENS

Terrestrial mosses and lichens in the steppe of Washington were buried under volcanic ash from the 18 May 1980 eruption of Mount St. Helens. Unlike adult vascular plants in these communities, these cryptogams died following the deposition of silt-size ash. Death within as little as four months was demonstrated by anatomical deterioration and these organisms' inability to fix CO₂, to fluoresce in IR radiation, and to absorb vital stains. Although cryptogams in the steppe have probably been destroyed repeatedly by ash falls during the Holocene, this recent destruction is the first since the arrival of alien plants in the nineteenth century. Death of cryptogams may allow the initiation of further colonization by alien plants because a cryptogamic crust forms a barrier to seedling establishment.     

Determination of bilayer thickness and lipid surface area in unilamellar dimyristoylphosphatidylcholine vesicles from small-angle neutron scattering curves: a comparison of evaluation methods

Small-angle neutron scattering (SANS) experiments were performed on unilamellar 1,2-dimyristoylphosphatidylcholine (DMPC) vesicles prepared in heavy water by extrusion through polycarbonate filters with 500 Å pores. The data obtained at 30±0.1 °C were evaluated using a five-strip function model of the bilayer coherent neutron scattering length density, three different approximate form factors describing scattering from vesicles, and different methods of evaluation of the experimental data. It is shown that the results obtained from the SANS data in the range of scattering vector values 0.0316 Å^–1<q<0.0775 Å^–1 are not sensitive to the vesicle form factor, nor to the evaluation method. Using the hollow sphere model of vesicles convoluted with the Gaussian distribution of their sizes, a constrained bilayer polar region thickness of 9 Å and a DMPC headgroup volume of 325.5 ų, it was possible to obtain from the experimental data the DMPC surface area as 58.9±0.8 Ų, the bilayer thickness as 44.5±0.3 Å and the number of water molecules as 6.8±0.2 per DMPC located in the bilayer polar region.     

Toxicity and antineoplastic effect of (24R)-1,24-dihydroxyvitamin D3 (PRI-2191)

Many efforts have been made to obtain active and less toxic Vitamin D analogs for new clinical applications. The results of previous studies demonstrated the efficacy and safety of topical treatment of psoriasis with one of these analogs, 1,24-dihydroxyvitamin D(3), tacalcitol (1,24-(OH)(2)D(3)). In the present study, we evaluated the toxicity and antitumor effect of this analog. Lethal toxicity of 1,24-(OH)(2)D(3) after s.c. injection was significantly lower than that of calcitriol. No significant differences were observed in the toxicity of the analogs when administered p.o. Calcium levels in the serum of mice treated with calcitriol were significantly higher (111%) than those in mice treated with 1,24-(OH)(2)D(3) (89%) at 5 day after the first s.c. (10 microg/kg/day) administration in comparison to the control (healthy, untreated animals). Oral administration increased the calcium level by 78% for calcitriol and only to 47% over the control for 1,24-(OH)(2)D(3). Parallel administration of clodronate prevented the calcitriol- and 1,24-(OH)(2)D(3)-induced lethal toxicity and also prevented increase in calcium levels. Single therapy with calcitriol did not affect tumor growth in the 16/C mouse mammary cancer model. In contrary, 1,24-(OH)(2)D(3) alone reduced tumor volume to 41% of control. Cisplatin alone did not affect growth of 16/C tumor in these conditions. The growth of tumors in the presence of cisplatin was inhibited by 1,24-(OH)(2)D(3) but not by calcitriol. Interestingly, the inhibition of tumor growth in cisplatin-treated mice by 1,24-(OH)(2)D(3) was greater, than that observed in mice treated with this analog alone. In conclusion, 1,24-(OH)(2)D(3) revealed higher antitumor and lower calcemic activity and toxicity than calcitriol. Application of biphosphonates along with Vitamin D analogs is sufficient to overcome the calcemic and toxic side effects of the proposed treatment.     

DNA binding and degradation by the HNH protein ColE7

The bacterial toxin ColE7 bears an HNH motif which has been identified in hundreds of prokaryotic and eukaryotic endonucleases, involved in DNA homing, restriction, repair, or chromosome degradation. The crystal structure of the nuclease domain of ColE7 in complex with a duplex DNA has been determined at 2.5 A resolution. The HNH motif is bound at the minor groove primarily to DNA phosphate groups at and beyond the 3' side of the scissile phosphate, with little interaction with ribose groups and bases. This result provides a structural basis for sugar- and sequence-independent DNA recognition and the inhibition mechanism by inhibitor Im7, which blocks the substrate binding site but not the active site. Structural comparison shows that two families of endonucleases bind and bend DNA in a similar way to that of the HNH ColE7, indicating that endonucleases containing a "betabetaalpha-metal" fold of active site possess a universal mode for protein-DNA interactions.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development

Cyclin dependent kinases (CDKs) play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. To understand the regulatory network involving the CDKs, we have examined the role of ACK1, a gene that has significant homology to known ICKs (inhibitors of CDKs), but occupies a distinct branch of the ICK phylogenetic tree. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. ACK1 transgenic plants also differed morphologically from control Arabidopsis plants, and the cells of ACK1 transgenics were more irregular than the corresponding cells of control plants. These results suggest that ACK1 acts as a CDK inhibitor in Arabidopsis, and that the alterations in leaf shape may be the result of restricted cell division.     

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.