Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells

Dendritic cells (DCs) are central to T cell immunity, and many strategies have been used to manipulate DCs to modify immune responses. We investigated the effects of antioxidants ascorbate (vitamin C) and alpha-tocopherol (vitamin E) on DC phenotype and function. Vitamins C and E are both antioxidants, and concurrent use results in a nonadditive activity. We have demonstrated that DC treated with these antioxidants are resistant to phenotypic and functional changes following stimulation with proinflammatory cytokines. Following treatment, the levels of intracellular oxygen radical species were reduced, and the protein kinase RNA-regulated, eukaryotic translation initiation factor 2alpha, NF-kappaB, protein kinase C, and p38 MAPK pathways could not be activated following inflammatory agent stimulation. We went on to show that allogeneic T cells (including CD4(+)CD45RO, CD4(+)CD45RA, and CD4(+)CD25(-) subsets) were anergized following exposure to vitamin-treated DCs, and secreted higher levels of Th2 cytokines and IL-10 than cells incubated with control DCs. These anergic T cells act as regulatory T cells in a contact-dependent manner that is not dependent on IL-4, IL-5, IL-10, IL-13, and TGF-beta. These data indicate that vitamin C- and E-treated DC might be useful for the induction of tolerance to allo- or autoantigens.     

Phe-Gly-Leu-amide allatostatin in the termite Reticulitermes flavipes: content in brain and corpus allatum and effect on juvenile hormone synthesis

In the subterranean termite Reticulitermes flavipes, allatostatins (ASTs) with the C-terminus Phe-Gly Leu-amide were localized by immunocytochemistry with antibody against a cockroach AST, Dippu AST-7. AST-immunoreactivity occurred in the corpus cardiacum and corpus allatum and in the lateral and medial neurosecretory cells of the brain that innervate these organs as well as in many other nerve cells of the brain. This was observed in workers, nymphs, soldiers and secondary reproductives. A radioimmunoassay, using anti-Dippu AST-11, demonstrated about 40 fmole equivalents of AST in brains of soldiers and secondary reproductives. The product of the corpora allata in this species was determined to be juvenile hormone III. Its synthesis by corpora allata of secondary reproductives, determined by in vitro radiochemical assay, was inhibited in a dose-dependent fashion by two cockroach allatostatins, Dippu AST-7 and Dippu AST-11. Thus, as in cockroaches and crickets, allatostatin-containing nerves innervate the corpora allata of this termite species and their production of juvenile hormone is inhibited by these neuropeptides.     

Endothelial lipase modulates HDL but has no effect on atherosclerosis development in apoE-/- and LDLR-/- mice

Endothelial lipase (EL) is a determinant of high density lipoprotein-cholesterol (HDL-C) level, which is negatively correlated with atherosclerosis susceptibility. We found no difference in aortic atherosclerotic lesion areas between 26-week-old EL+/+ apolipoprotein E-deficient (apoE-/-) and EL-/- apoE-/- mice. To more firmly establish the role of EL in atherosclerosis, we extended our study to EL-/- and EL+/+ low density lipoprotein receptor-deficient (LDLR-/-) mice that were fed a Western diet. Morphometric analysis again revealed no difference in atherosclerosis lesion area between the two groups. Compared with EL+/+ mice, we found increased HDL-C in EL-/- mice with apoE-/- or LDLR-/- background but no difference in macrophage content between lesions of EL-/- and EL+/+ mice in apoE-/- or LDLR-/- background. EL inactivation had no effect on hepatic mRNAs of proteins involved in reverse cholesterol transport. A survey of lipid homeostasis in EL+/+ and EL-/- macrophages revealed that oxidized LDL-induced ABCA1 was attenuated in EL-/- macrophages. This potentially proatherogenic change may have nullified any minor protective increase of HDL in EL-/- mice. Thus, although EL modulated lipoprotein profile in mice, there was no effect of EL inactivation on atherosclerosis development in two hyperlipidemic atherosclerosis-prone mouse models.     

Human and animal hepatocytes in vitro with extrapolation in vivo

Human and animal hepatocytes are now being used as an in vitro technique to aid drug discovery by predicting the in vivo metabolic pathways of drugs or new chemical entities (NCEs), identifying drug-metabolizing enzymes and predicting their in vivo induction. Because of the difficulty of establishing whether the cytotoxic susceptibility of human hepatocytes to xenobiotics/drugs in vitro could be used to predict in vivo human hepatotoxicity, a comparison of the susceptibility of the hepatocytes of human and animal models to six chemical classes of drugs/xenobiotics in vitro have been related to their in vivo hepatotoxicity and the corresponding activity of their metabolizing enzymes. This study showed that the cytotoxic effectiveness of 16 halobenzenes towards rat hepatocytes in vitro using higher doses and short incubation times correlated well with rat hepatotoxic effectiveness in vivo with lower doses/longer times. The hepatic/hepatocyte xenobiotic metabolizing enzyme activities of various animal species and human have been reviewed for use by veterinarians and research scientists. Where possible, recommendations have been made regarding which animal hepatocyte model is most applicable for modeling the susceptibility to xenobiotic induced hepatotoxicity of those humans with slow versus rapid metabolizing enzyme polymorphisms. These recommendations are based on the best human fit for animal drug/xenobiotic metabolizing enzymes in terms of activity, kinetics and substrate/inhibitor specificity. The use of human hepatocytes from slow versus rapid metabolizing individuals for drug metabolism/cytotoxicity studies; and the research use of freshly isolated rat hepatocytes and "Accelerated Cytotoxicity Mechanism Screening" (ACMS) techniques for identifying drug/xenobiotic reactive metabolites are also described. Using these techniques the molecular hepatocytotoxic mechanisms found in vitro for seven classes of xenobiotics/drugs were found to be similar to the rat hepatotoxic mechanisms reported in vivo.     

Expression of p57 in mouse and human testes

The expression of cyclin-dependent kinases inhibitors, p57kip2, was investigated during the postnatal development of mouse testis, and in adult human testis. Expression of p57kip2 mRNA was higher in immature than pubertal or adult mouse testes. In postnatal day 7 (PND7) testes, moderate p57kip2 immunoreactivity was found in spermatogonia, but signal was heterogeneous among the spermatogonia. In PND14 testes onward, strong immunoreactivity of p57kip2 was found in the nuclei of early spermatocytes but not in the late pachytene stage onward. In PND28 and PND50 testes, p57kip2 immunoreactivity was varying among the seminiferous tubules. There was no visible signal in late pachytene stage onward. In Leydig cells, heterogeneous immunoreactivity of p57kip2 was found in immature testis and the signal intensity was higher in adult testis than immature ones. In Sertoli cells, weak or negligible immunoreactivity of p57kip2 was found. In human seminiferous tubule, strong immunoreactivity of p57kip2 was found in the nucleus of early spermatocytes, but not in the late pachytene spermatocytes onward and Sertoli cells. These results suggest the possible role of p57kip2 in the regulation of early spermatogonial proliferation, meiotic progression of early spermatocytes and differentiation of Leydig cells in testis.     

The conserved carboxy-terminus of the MscS mechanosensitive channel is not essential but increases stability and activity

The Escherichia coli MscS mechanosensitive channel protein has a distinct domain structure that terminates in a conserved seven-strand beta barrel. This distinctive feature suggested it could be a critical determinant of channel stability and activity. Measurements on a protein deleted for the base of the vestibule and the beta barrel (residues 266-286) suggested that the modified channel had reduced activity. However, induction of the mutant protein resulted in membrane protein accumulation equivalent to wild type and a physiologically functional channel. In patch clamp analysis the activity profile was similar to wild type but reduced numbers of channel were seen per patch, suggesting reduced assembly or stability of the mutant protein. The mutant channel exhibited a subtle change in character - channels did not re-open after full desensitization. Thus the immediate carboxy-terminus (residues 266-286) is not essential for MscS gating but improves stability and activity and is required for recovery of channel activity after desensitization.     

Synthesis, photophysical and electrochemical properties, and biological labelling studies of luminescent cyclometallated iridium(III) bipyridine-aldehyde complexes

We report the synthesis, characterisation, and photophysical and electrochemical properties of a series of luminescent cyclometallated iridium(III) bipyridine-aldehyde complexes [Ir(N-C)₂(bpy-CHO)](PF₆) (HN-C=2-phenylpyridine, Hppy (1); 2-(4-methylphenyl)pyridine, Hmppy (2); 1-phenylpyrazole, Hppz (3); 3-methyl-1-phenylpyrazole, Hmppz (4); 7,8-benzoquinoline, Hbzq (5); 2-phenylquinoline, Hpq (6); bpy-CHO=4-formyl-4^′-methyl-2,2^′-bipyridine). The X-ray crystal structures of complexes 1 and 4 have been determined. On the basis of the photophysical data, the emission of these complexes is assigned to an excited state of predominantly triplet metal-to-ligand charge-transfer (³MLCT) (dπ(Ir) → (bpy-CHO)) character. For complex 6, the excited state is also mixed with substantial (³IL) () (pq⁻) character. The protein bovine serum albumin has been labelled with these complexes to produce luminescent bioconjugates. The photophysical properties of the luminescent conjugates have also been investigated.     

Testing for common structures in a panel of threshold models

We consider the problem of examining the extent of (partial) similarity in the dynamics of a panel of independent threshold autoregressive processes. We develop some tests for common structure via Wald's approach and by checking whether the parameter estimates of the unconstrained threshold models satisfy the constraints defining the common structure. One test concerns the equality of independent ratios of normal means, which is shown to have nonstandard asymptotic null distribution. These tests are illustrated with a modern panel of Canadian lynx data; our analysis suggests that the lynx data over Canada share similar dynamics in the decrease phase, but they appear to be different in the increase phase.     

Crystal structure of the apo forms of psi 55 tRNA pseudouridine synthase from Mycobacterium tuberculosis: a hinge at the base of the catalytic cleft

The three-dimensional structure of the RNA-modifying enzyme, psi55 tRNA pseudouridine synthase from Mycobacterium tuberculosis, is reported. The 1.9-A resolution crystal structure reveals the enzyme, free of substrate, in two distinct conformations. The structure depicts an interesting mode of protein flexibility involving a hinged bending in the central beta-sheet of the catalytic module. Key parts of the active site cleft are also found to be disordered in the substrate-free form of the enzyme. The hinge bending appears to act as a clamp to position the substrate. Our structural data furthers the previously proposed mechanism of tRNA recognition. The present crystal structure emphasizes the significant role that protein dynamics must play in tRNA recognition, base flipping, and modification.