Biosynthesis of shikonin in callus cultures of Lithospermum erythrorhizon

Administration of various supposed precursors to the callus cultures of Lithospermum erythrorhizon grown on the Linsmaier—Skoog medium supplemented with IAA and kinetin established that the constituent shikonin is formed via shikimic acid, p-hydroxybenzoic acid, m-geranyl-p-hydroxybenzoic acid and geranylhydroquinone. In a strain of callus culture lacking the capacity to synthesize shikonin and in callus cultures which have had this capacity but lost it due to cultivation on a medium supplemented with 2,4-D, substances up to m-geranyl-p-hydroxybenzoic acid in the biosynthetic sequence have been detected. Although illumination with white light also arrested shikonin production, traces of pigment were still formed presumably because light did not reach the innermost part of the callus cultures.     

Presence of Thrombin-Activatable Fibrinolysis Inhibitor in Helicobacter pylori-Associated Gastroduodenal Disease

Thrombin-activatable fibrinolysis inhibitor (TAFI) plays a role in the regulation of coagulation and inflammation. In addition to inhibiting the fibrinolytic system, TAFI may also regulate the bradykinin and complement systems. We hypothesized that TAFI also plays a role in defense mechanisms of the gastric mucosa during Helicobacter pylori infection. This study comprised 65 patients with gastroduodenal disorders: 41 patients with H. pylori infection, 13 without, and 11 patients with cured H. pylori infection. The gastric intramucosal concentrations of TAFI were measured by enzyme immunoassay. The gastric levels of TAFI and plasminogen activator inhibitor-1 were significantly increased in patients with H. pylori compared to those without infection or cured H. pylori . The presence of TAFI was detected in gastric mucosal epithelial cells. The concentration of TAFI was correlated with the degree of gastric mucosal atrophy, inflammation, and disease activity. These results show that TAFI is present in the gastric mucosa and that it may play a role in the pathogenesis of H. pylori infection-associated gastroduodenal disorders.     

Effects of acidic pH on the formation of vinblastine-induced paracrystals in Chinese hamster ovary cells

Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10^?6 M and 10^?5 M). At higher concentrations of VLB (5×10^?5 M and 10^?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH.     

Production,Purification and Crystallization of 3α-Hydroxysteroid Dehydrogenase of Pseudomonas putida

The ability of formation of 3α-hydroxysteroid dehydrogenase was studied in bacteria and actinomycetes. The enzyme activity was found in several bacteria belonging to the genera Pseudomonas, Bacillus and Corynebacterium, when they were grown on cholic acid as a sole source of carbon. Of these bacteria, Pseudomonas putida NRRL B–11064 isolated from soil, showed the highest activity of 3α-hydroxysteroid dehydrogenase. The enzyme was purified from the cell-free extract by procedure including fractionation with ammonium sulfate and column chromatographies on DEAE-celluIose, Sephadex G–100 and hydroxylapatite. Crystals of the enzyme were obtained by the addition of ammonium sulfate to the purified enzyme in the presence of glycerol or polyethylene glycol. The overall purification was about 550-fold with an yield of 18.5%. The crystalline enzyme was homogeneous on polyacrylamide disc electrophoresis and analytical ultracentrifugation (s_20,w=3.2).     

Inhibition of TACE activity enhances the susceptibility of myeloma cells to TRAIL

Background

TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) selectively induces apoptosis in various cancer cells including myeloma (MM) cells. However, the susceptibility of MM cells to TRAIL is largely low in most of MM cells by yet largely unknown mechanisms. Because TNF-α converting enzyme (TACE) can cleave some TNF receptor family members, in the present study we explored the roles of proteolytic modulation by TACE in TRAIL receptor expression and TRAIL-mediated cytotoxicity in MM cells.

Methodology/Principal Findings

MM cells preferentially expressed death receptor 4 (DR4) but not DR5 on their surface along with TACE. Conditioned media from RPMI8226 and U266 cells contained a soluble form of DR4. The DR4 levels in these conditioned media were reduced by TACE inhibition by the TACE inhibitor TAPI-0 as well as TACE siRNA. Conversely, the TACE inhibition restored surface levels of DR4 but not DR5 in these cells without affecting DR4 mRNA levels. The TACE inhibition was able to restore cell surface DR4 expression in MM cells even in the presence of bone marrow stromal cells or osteoclasts, and enhanced the cytotoxic effects of recombinant TRAIL and an agonistic antibody against DR4 on MM cells.

Conclusions/Significance

These results demonstrate that MM cells post-translationally down-modulate the cell surface expression of DR4 through ectodomain shedding by endogenous TACE, and that TACE inhibition is able to restore cell surface DR4 levels and the susceptibility of MM cells to TRAIL or an agonistic antibody against DR4. Thus, TACE may protect MM cells from TRAIL-mediated death through down-modulation of cell-surface DR4. It can be envisaged that TACE inhibition augments clinical efficacy of TRAIL-based immunotherapy against MM, which eventually becomes resistant to the present therapeutic modalities.     

Chronic exercise enhances insulin secretion ability of pancreatic islets without change in insulin content in non-diabetic rats

We evaluated the effect of chronic exercise on insulin secretion in response to high-glucose by using a perifusion method with isolated pancreatic islets from normal rats. Male Wistar rats were assigned to one of two groups: a sedentary group and a trained group. Running exercise was carried out on a treadmill for one hour per day, five days per week, for six, nine, or 12 weeks. The chronic exercise significantly enhanced the insulin secretion ability of pancreatic islets in response to the high-glucose stimulation upon nine and 12 weeks of exercise. The insulin content in the pancreas and the weight of the pancreas did not change upon nine weeks of exercise. Potassium-stimulated insulin secretion was also increased in the islets isolated from rats that trained for nine weeks compared with that in sedentary rats, suggesting that insulin secretion events downstream of membrane depolarization are involved in targets of the exercise effect. These findings suggest that chronic exercise could be a useful strategy not only for the maintenance of peripheral insulin sensitivity but also for the promotion of islet function to secrete insulin in non-diabetics.     

Structural characterization of the MIT domain from human Vps4b

The microtubule interacting and trafficking (MIT) domain is a small protein module of unknown function that is conserved in proteins of diverse function, such as Vps4, sorting nexin 15 (SNX15), and spastin. One non-synonymous single nucleotide polymorphism was reported, which results in a Ile58-to-Met (I58M) substitution in hVps4b. Here, we have determined the solution structure of the MIT domain isolated from the NH(2)-terminus of human Vps4b, an AAA-ATPase involved in multivesicular body formation. The MIT domain adopts an 'up-and-down' three-helix bundle. Comparison with the sequences of other MIT domains clearly shows that the residues involved in inter-helical contacts are well conserved. The Ile58-to-Met substitution resulted a substantial thermal instability. In addition, we found a shallow crevice between helices A and C that may serve as a protein-binding site. We propose that the MIT domain serves as a putative adaptor domain for the ESCRT-III complex involved in endosomal trafficking.     

Both mammalian PIG-M and PIG-X are required for growth of GPI14-disrupted yeast

GPI mannosyltransferase I (GPI-MT-I) transfers the first mannose to a GPI-anchor precursor, glucosamine-(acyl)phosphatidylinositol [GlcN-(acyl)PI]. Mammalian GPI-MT-I consists of two components, PIG-M and PIG-X, which are homologous to Gpi14p and Pbn1p in Saccharomyces cerevisiae, respectively. In the present study, we disrupted yeast GPI14 and analysed the phenotype of gpi14 yeast. The gpi14 haploid cells were inviable and accumulated GlcN-(acyl)PI. We cloned PIG-M homologues from human, Plasmodium falciparum (PfPIG-M) and Trypanosoma brucei (TbGPI14), and tested whether they could complement gpi14-disrupted yeast. None of them restored GPI-MT-I activity and cell growth in gpi14-disrupted yeast. However, gpi14-disrupted yeast cells with human PIG-M, but not with PfPIG-M or TbGPI14, grew slowly but significantly when they were supplemented with rat PIG-X. This suggests that the association of PIG-X and PIG-M for GPI-MT-I activity is not interchangeable between mammals and the other lower eukaryotes.     

The role played by cell-substrate interactions in the pathogenesis of osteoclast-mediated peri-implant osteolysis

Prosthetic wear debris-induced peri-implant osteolysis is a major cause of aseptic loosening after total joint replacement. In this condition, wear particles released from the implant components induce a granulomatous inflammatory reaction at the interface between implant and adjacent bone, leading to progressive bone resorption and loss of fixation. The present study was undertaken to characterize definitively the phenotype of osteoclast-like cells associated with regions of peri-implant focal bone resorption and to compare the phenotypic features of these cells with those of mononucleated and multinucleated cells associated with polyethylene wear particles. Peri-implant tissues were obtained from patients undergoing hip revision surgery for aseptic loosening after total joint replacement. Cells were examined for the expression of several markers associated with the osteoclast phenotype using immunohistochemistry, histochemistry, and/or in situ hybridization. CD68 protein, a marker expressed by multiple macrophage lineage cell types, was detected in mononucleated and multinucleated cells associated with polyethylene particles and the bone surface. Cathepsin K and tartrate-resistant acid phosphatase were expressed highly in both mononucleated and multinucleated cells associated with the bone surface. Levels of expression were much lower in cells associated with polyethylene particles. High levels of β₃ integrin protein were detected in cells in contact with bone. Multinucleated cells associated with polyethylene particles exhibited faint positive staining. Calcitonin receptor mRNA expression was detected solely in multinucleated cells present in resorption lacunae on the bone surface and was absent in cells associated with polyethylene particles. Our findings provide further evidence that cells expressing the full repertoire of osteoclast phenotypic markers are involved in the pathogenesis of peri-implant osteolysis after total joint replacement. They also demonstrate that foreign body giant cells, although believed to be phenotypically and functionally distinct from osteoclasts, express many osteoclast-associated genes and gene products. However, the levels and patterns of expression of these genes in the two cell types differ. We speculate that, in addition to the role of cytokines and growth factors, the substrate with which these cells interact plays a critical role in their differential phenotypic and functional properties.