Tartrate-resistant acid phosphatase 5B circulates in human serum in complex with alpha2-macroglobulin and calcium

Tartrate-resistant acid phosphatase (TRACP) is an enzyme with unknown biological function. In addition to its acid phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS) at neutral pH. Two forms of TRACP circulate in human serum, macrophage-derived TRACP 5a and osteoclast-derived TRACP 5b. Here we have studied the circulating forms of the osteoclast-derived TRACP 5b in rat and human serum. In human serum, TRACP 5b circulates in a large complex that contained α₂M and calcium. On the contrary, rat serum TRACP 5b circulates as a free molecule. Formation of the TRACP 5b complex in vitro decreased significantly the ROS generating activity of TRACP 5b without affecting its phosphatase activity. These results suggest that the complex formation may be necessary to eliminate the formation of the harmful ROS in the neutral pH of serum.     

Molecular dynamics simulation studies of cytochrome b5 from outer mitochondrial and microsomal membrane

Two forms of cytochrome b(5) have been identified, associated with the outer membrane of liver mitochondria (OM cyt b(5)) and with the membrane of the endoplasmic reticulum (microsomal, Mc cyt b(5)). These proteins have very similar structures, but differ significantly in physical properties, with the OM cyt b(5) exhibiting a more negative reduction potential, higher stability, and stronger interactions with the heme. We perform molecular dynamics simulations to probe the structures and fluctuations of the two proteins in solution, to help explain the observed physical differences. We find that the structures of the two proteins, highly similar in the crystal, differ in position of a surface loop involving residues 49-51 in solution. Hydrophobic residues Ala-18, Ile-32, Leu-36, and Leu-47 tend to cluster together on the surface of rat OM cyt b(5), blocking water access to the protein interior. In bovine Mc cyt b(5), two of these positions, Ser-18 and Arg-47, are occupied by hydrophilic residues. This leads to breaking the hydrophobic cluster and allowing the protein to occupy a more open conformation. A measure of this structural transition is the opening of a cleft on the protein surface, which is 5 A wider in the OM cyt b(5) simulation compared to the Mc form. The OM protein also appears to have a more compact hydrophobic core in its beta-sheet region. These effects may be used to explain observed stability differences between the two proteins.     

Potential mechanisms of leukemia cell resistance to TRAIL-induced apopotosis

There are many factors contributing to the resistance to TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis. However, it is not clear whether the mechanism of resistance to TRAIL is constitutive or inductive. Therefore, the purpose of this study was to investigate the resistant mechanisms to TRAIL at different levels in the apoptotic pathway. The human T-lymphoblastic leukemic CEM cell line showed more resistant to TRAIL-induced apoptosis compared with the human chronic myeloid leukemic K562 cell line. Lower level of constitutive caspase-8 expression in the CEM cell line led to a poor response to both TRAIL-induced activation of caspase-3 and reduction in the mitochondrial membrane potential (m). There was no significant difference in the constitutive levels of NF-B in CEM and K562 cell lines. However, CEM cells showed a faster response to TRAIL-induced NF-B activation than K562 cells. TRAIL-induced regulation of Bcl-2 family of proteins included an up-regulation in Bcl-2/Bcl-XL and a down-regulation in Bax. IAPs, such as XIAP, cIAP-1, cIAP-2 and Survivin were all up-regulated during the treatment with TRAIL. In summary, our data suggest that the leukemic cells resistance to TRAIL-induced apoptosis might be due to the deficiency in the constitutive caspase-8 expression. Development of potential resistance to apoptosis by TRAIL can occur in both TRAIL-resistant and TRAIL-sensitive leukemic cells.     

Synthesis and induction of apoptosis in B cell chronic leukemia by diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride and its derivatives

2-Acetamido-2-deoxy-D-glucose hydrochloride (D-glucosamine hydrochloride) has been used for the preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-trifluoroacetamido-beta- (4) and 2-tetrachlorophthalimido-alpha,beta-D-glucopyranose (6), which have been transformed into the appropriate bromides and the chloride. Both bromo and chloro sugars were used as a glycosyl donors for the glycosylation of diosgenin [(25R)-spirost-5-en-3beta-ol]. These condensations were conducted under mild conditions, using silver triflate as a promoter, and gave diosgenyl glycosides 9 and 12. Each of them was converted into diosgenyl 2-amino-2-deoxy-beta-D-glucopyranoside hydrochloride (11) and N-acylamido derivatives. The structures of all new glycosides were established by 1H and 13C NMR spectroscopy. These diosgenyl glycosides are the first saponins containing the D-glucosamine residue that have been synthesized. These compounds show promising antitumor activities. The synthetic saponins increase the number of apoptotic B cells, in combination with cladribine (2-CdA), that are isolated from chronic lymphotic leukemia (B-CLL) patients.     

Convenient preparation of L-arabino-hexos-5-ulose derivatives from lactose

2,6-di-O-benzyl- (9), 2-O-benzyl-3,4-O-isopropylidene- (19), and 2-O-benzyl-6-O-m-chlorobenzoyl-L-arabino-hexos-5-ulose (20) have been prepared using 4'-deoxy-4'-eno- and 6'-deoxy-5'-eno lactose dimethyl acetal derivatives 7 and 14 as key intermediates. The synthesis of enol ethers 7 and 14 has been performed with good yields by base-promoted elimination of acetone or p-toluenesulfonic acid from 2',6'-di-O-benzyl-, and 6'-O-p-toluenesulfonyl-2,3:5,6:3',4'-tri-O-isopropylidenelactose dimethyl acetal, respectively. The epoxidation with MCPBA of 7 and 14 in methanol or dichloromethane furnishes C-5'-methoxy and C-5'-m-chlorobenzoyloxy derivatives, easily transformed with good yields into L-arabino 5-ketoaldohexoses 9, 19 and 20.     

Role for laminin-alpha5 chain LG4 module in epithelial branching morphogenesis

Laminin-alpha5 chain was localized in all epithelial basement membranes (BMs) of mouse submandibular gland (SMG) from the onset of branching morphogenesis and became restricted to BMs of epithelial ducts in the adult. To investigate whether the laminin-alpha5 chain plays a role in branching morphogenesis, a set of cell-adhesive peptides from the C-terminal globular domains (LG1-5) was tested for their effects in SMG organ cultures. One peptide, LVLFLNHGH (A5G77f), which represents a sequence located in the connecting loop between strands E and F of LG4, perturbed branching morphogenesis and resulted in irregularities in the contours of epithelial structures, with formation of deep clefts. The data suggest a role for the laminin-alpha5 LG4 module in the development of the duct system, rather than in the bifurcation of epithelial clusters. The epithelial BM of A5G77f-peptide-treated explants was continuous, which was in contrast to our previous finding of impaired epithelial BM assembly in explants treated with the laminin-alpha1 LG4 module peptide, or with a monoclonal antibody against this domain. A5G77f also perturbed in vitro development of lung and kidney. These results suggest a crucial role for the LG4 module of laminin-alpha5 in epithelial morphogenesis that is distinct from that of the laminin-alpha1 LG4.     

Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore

The presumed protective effect of folic acid on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of folic acid. Peroxynitrite (PON) scavenging activity and inhibition of lipid peroxidation (LPO) of the physiological forms of folate and of structurally related compounds were tested. It was found that the fully reduced forms of folate, i.e. tetrahydrofolate (THF) and 5-methyltetrahydrofolate (5-MTHF), had the most prominent antioxidant activity. It appeared that their protection against LPO is less pronounced than their PON scavenging activity. The antioxidant activity of these forms of folic acid resides in the pterin core, the antioxidant pharmacophore is 4-hydroxy-2,5,6-triaminopyrimidine. It is suggested that an electron donating effect of the 5-amino group is of major importance for the antioxidant activity of 4-hydroxy-2,5,6-triaminopyrimidine. A similar electron donating effect is probably important for the antioxidant activity of THF and 5-MTHF.     

Anatomical and functional evidence for a stress-responsive,monoamine-accumulating area in the dorsomedial hypothalamus of adult rat brain

The dorsomedial hypothalamus (DMH) plays an important role in relaying information to neural pathways mediating neuroendocrine, autonomic, and behavioral responses to stress. Evidence suggests that the DMH is a structurally and functionally diverse integrative structure that contributes to both facilitation and inhibition of the hypothalamo-pituitary-adrenal axis, depending on the nature of the stimulus and the specific neural circuits involved. Previous studies have determined that stress or stress-related stimuli elevate tissue concentrations of serotonin (5-hydroxytryptamine; 5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine, and noradrenaline selectively within the DMH. In order to determine the specific region of the rat DMH involved, we used high-performance liquid chromatography with electrochemical detection to measure tissue concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline within five different subregions of the DMH in adult female Lewis and Fischer rats immediately or 4 h following a 30-min period of restraint stress. Compared to unrestrained control rats, restrained rats had elevated concentrations of 5-HT, 5-HIAA, dopamine, and noradrenaline immediately after a 30-min period of restraint and had elevated concentrations of 5-HT 4 h following the onset of a 30-min period of restraint stress. These effects were confined to a specific region that included medial portions of the dorsal hypothalamic area and dorsal ependymal, subependymal, and neuronal components of the periventricular nucleus. Furthermore, these effects were observed in Lewis rats, but not Fischer rats, two closely related rat strains with well-documented differences in neurochemical, neuroendocrine, autonomic, and behavioral responses to stress. These data provide support for the existence of a stress-responsive, amine-accumulating area in the DMH that may play an important role in the differential stress responsiveness of Lewis and Fischer rats.     

5-Azido neuraminic acid thioglycoside as sialylation donor

5-Azido neuraminic acid thioglycoside with O-benzyl protecting groups was synthesized. The sialylations of this new donor type showed good alpha-selectivities for certain primary hydroxyls.