Resolution of racemic carboxylic acids via the lipasecatalyzed irreversible transesterification using vinyl esters; Effects of alcohols as nucleophiles and organic solvents on enantioselectivity

Summary The lipase-catalyzed irreversible transesterification procedure using vinyl esters has been applied to the resolution of racemic 2-phenoxypropanoic acids. The enantioselectivity was found to be affected profoundly by both the alcohol as a nucleophile and the organic solvent used.     

Correction to: Quantification of the contrasting root systems of Pinus thunbergii in soils with different groundwater levels in a coastal forest in Japan

Plant and Soil - The original version of this article unfortunately contained a mistake. Table 3 was published erroneously. This Table has now been corrected.     

Synthesis and Evaluation of DMPO-Type Spin Traps

The spin traps substituted with some groups at the 4-position of dimethyl-1-pyrroline N-oxide(DMPO) were compared with DMPO itself regarding their abilities as spin traps and their physical properties. 4,5,5-Trimethyl-l-pyroHine N-oxide (4MDMPO) and 5,5-dimethyl- 4-phenyl-l-pyrolline N-oxide (4PDMPO) were synthesized by the Bonnett method, and 5,5-dimethyl-4-hydroxymethyl-l-pyrolline N-oxide (4HMDMPO) was made by a unique method from 2(5H)-furanone. The melting points of 4MDMPO, 4PDMPO and 4HMDMPO were higher than that of DMPO. The magnitude of hydrophilicity was in the order of 4HMDMPO, DMPO, 4MDMPO, and 4PDMPO based on the partition coefficient experiments in a 1-octanol-water system. Several radicals, O₂, HO-, -CH₃, -CH₂OH, -CH(CH₃)OH, (CH₃)₃ CO and H radicals, were trapped with these DMPO derivatives for comparison with the trapping by DMPO itself. Spin adducts of O J with the three DMPO derivatives showed ESR spectra similar to that of DMPO. In spite of the formation of diastereomers arising from spin trapping, the line-width enlargement was very small. The intensities and the decay rates of the spectra of 4MDMPO-O₂^-, 4PDMPO-O₂^- 4HMDMPO-O₂^- and DMPO-O₂^- were almost equal. In the trapping of the OH radical by 4MDMPO, 4PDMPO and 4HMDMPO, the eight-line ESR spectra observed were different from the well-known four-line spectrum of DMPO-OH.     

Interleukin 2 induces rapid phosphorylation of cellular proteins in murine T-lymphocytes

When quiescent murine T-lymphocyte cells were stimulated by the addition of interleukin 2 (IL-2), they reinitiated DNA synthesis after a lag period of 5 h. Under these conditions, rapid but transient phosphorylation of two cellular proteins with Mr values of 27 000 and 26 000 was detected; maximal phosphorylation occurred within 10-15 min after the addition of IL-2. The protein of Mr 27 000 contained phosphoserine, while the protein of Mr 26 000 contained phosphothreonine.     

Conformation of diastereomeric peptide sequences: structural analysis of Z-D-Val-Ac6c-Gly-L-Phe-OMe.

We have recently undertaken a systematic structural analysis of fully protected tetrapeptides containing at the N- and C-terminus either homo- or heterochiral amino acids, spaced by an achiral dipeptide segment. The interest for this class of peptides derives from the observation that, on reverse-phase (HPLC), the homo- and heterochiral sequences have a markedly different retention times. The diastereomeric sequences, namely Z-(L/D)-Val-X-Y-L-Phe-OMe (X = Sar, Gly, Ac3c, Aib, Ac5c, Ac6c, Deg, Dpg, Dbu, Dip, Dph; Y = Sar, Gly, Ac3c, Aib, Ac5c, Ac6c) show different overall hydrophobicity attributed to a different three-dimensional structure that also depends on the X-Y segment. Therefore, following preliminary studies in solution, we report here the detailed x-ray analysis of the tetrapeptide Z-D-Val-Ac6c-Gly-L-Phe-OMe in order to understand the structural features governing the overall hydrophobicity of linear fully protected tetrapeptides.     

Electron spin relaxation of synthetic melanin and melanin-containing human tissues as studied by electron spin echo and electron spin resonance

Electron spin lattice relaxation times (T1) and the phase memory times (Tm) were obtained for the synthetic melanin system from 3-hydroxytyrosine (dopa) by means of electron spin echo spectroscopy at 77 degrees K. Saturation behavior of the ESR spectra of melanins in melanin-containing tissue and of the synthetic melanin was also determined at the same temperature. The spin lattice relaxation time and the spectral diffusion time of the synthetic melanin are very long (4.3 ms and 101 microseconds, respectively, in the solid state), and the ESR signal saturates readily at low microwave powers. On the other hand, ESR spectra of natural melanins from the tissues chosen for this study, as well as those of synthetic melanins which contain Fe3+ of g = 4.3 and Mn2+ of g = 2, are relatively difficult to saturate compared with samples without such metal ions. These results show clearly that a large part of those two metal ions in sites responsible for the ESR spectral components with these particular g values are coordinated to melanin in melanin-containing tissue, and modify the magnetic relaxation behavior of the melanin. Accumulations of these metal ions in melanins are different from system to system, and they increase in the order: hair (black), retina and choroid (brown), malignant melanoma of eye and skin, and lentigo and nevus of skin.     

The charge effects of the self-diffusion constant of bovine mercaptalbumin

The charge effect on the translational self-diffusion constant, D, of polyelectrolytes has been quantitatively analyzed based on dynamic light scattering experiments. Perfectly monodisperse bovine mercaptalbumin has been used at low pH as a positively charged polyelectrolyte sample. Completely linear plots of log{g2(t)-1} vs. time t have been obtained for uncharged states of the protein, for the cor relation function of the scattered light intensity, g2(t). The plots deviate from linearity as polyions bear the charges. The D values for various ionic states, obtained from the initial slopes of the plots, have been analyzed using the simple theory of Imai and Mandel (N. Imai and M. Mandel, Macromolecules 15 (1982) 1562) derived based on the Onsager-Navier-Stokes equation for solvent flow with counterion distribution around a polyion. It has turned out that the experimental D values coincide well with the theory and that the characteristic nature of D can be elucidated principally from the charge effect.     

Uptake of Oxidized Low Density Lipoprotein by CD36 Occurs by an Actin-dependent Pathway Distinct from Macropinocytosis

The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated.Uptake and storage of cholesterol by macrophages are key contributors to the formation of atherosclerotic plaque. Endothelial cells, seemingly activated by the deposition of modified forms of low-density lipoprotein (LDL),³ release chemokines that recruit macrophages into the vascular intima. Infiltrated macrophages can then readily oxidize and take up the modified LDL. Accumulation of lipids derived from oxidized LDL (oxLDL) transforms macrophages into foam cells, which release excess cytokines, triggering an inflammatory cascade. In addition, foam cells express proteases and other factors that contribute to plaque rupture and subsequent thrombosis.OxLDL particles are recognized by a variety of receptors, including the class A scavenger receptor SR-A and the class B scavenger receptor CD36. CD36 is thought to be responsible for ∼50% of oxLDL uptake by murine and human macrophages (1–3). In addition, this protein mediates cholesterol uptake from high density lipoprotein (4) and is also a receptor for internalization of oxidized high density lipoprotein (5).CD36 encodes a protein with two transmembrane domains located near the N and C termini, leaving only short cytoplasmic tails at each end. Despite having small intracellular domains, engagement of CD36 by its cognate ligands triggers signaling reactions that lead to the internalization of the resulting complex. However, the precise pathways that are activated and the specific mode of internalization remain unclear.Jones and Willingham (6) demonstrated that, in macrophages, modified LDL stimulates ruffling activity and the formation of phase-bright macropinosomes. By transmission electron microscopy they found that gold-conjugated modified LDL associated with ruffles and was present within macropinosomes. These observations underlie the widely accepted view that uptake of modified LDL occurs by macropinocytosis. However, Zeng et al. (7) showed that internalized DiI-oxLDL and CD36 were found in moderately sized cytoplasmic structures that co-localized with a glycosylphosphatidylinositol-anchored protein, suggesting uptake via lipid raft endocytosis. Moreover, Sun et al. (8) reported that uptake of oxLDL by CD36 was independent of actin but dependent on dynamin. The results of these two studies are not easily reconciled with mediation by macropinocytosis, an actin-dependent process that generates large vacuoles, and they suggest instead that CD36 is internalized by a more conventional endocytic pathway.It is not clear whether the apparent discrepancy stems from the engagement of different receptors in the different biological systems used in these studies. Jones and Willingham used macrophages, whereas Zeng et al. and Sun et al. studied, respectively, Chinese hamster ovary and COS cells heterologously transfected with CD36. The types and abundance of receptors capable of binding modified LDL in all likelihood differ greatly in these systems, and heterologous (over)expression in immortalized cells is liable to produce results of questionable biological relevance.In view of this uncertainty and considering the important and versatile roles of CD36, we set out to reexamine the mode of internalization of this receptor. We used both primary and cultured macrophages and selectively targeted CD36 using specific antibodies. The responses triggered by selective CD36 cross-linking were also compared with those elicited by oxLDL. We show that clustering CD36 initiates a signaling cascade that results in the activation of both macropinocytosis and internalization of CD36. Remarkably, however, CD36 internalization is largely independent of macropinocytosis and occurs by a novel dynamin-independent, actin-driven process that requires activation of Src family and c-Jun N-terminal kinases.     

Ligand-induced recruitment of a histone deacetylase in the negative-feedback regulation of the thyrotropin beta gene.

We have investigated ligand-dependent negative regulation of the thyroid-stimulating hormone beta (TSHbeta) gene. Thyroid hormone (T3) markedly repressed activity of the TSHbeta promoter that had been stably integrated into GH(3 )pituitary cells, through the conserved negative regulatory element (NRE) in the promoter. By DNA affinity binding assay, we show that the NRE constitutively binds to the histone deacetylase 1 (HDAC1) present in GH(3 )cells. Significantly, upon addition of T3, the NRE further recruited the thyroid hormone receptor (TRbeta) and another deacetylase, HDAC2. This recruitment coincided with an alteration of in vivo chromatin structure, as revealed by changes in restriction site accessibility. Supporting the direct interaction between TR and HDAC, in vitro assays showed that TR, through its DNA binding domain, strongly bound to HDAC2. Consistent with the role for HDACs in negative regulation, an inhibitor of the enzymes, trichostatin A, attenuated T3-dependent promoter repression. We suggest that ligand-dependent histone deacetylase recruitment is a mechanism of the negative-feedback regulation, a critical function of the pituitary-thyroid axis.