Neuroprotective Mechanisms of Antiparkinsonian Dopamine D2-Receptor Subfamily Agonists

Numerous studies have shown that endogenous and/or environmental neurotoxins and oxidative stress may participate in the pathogenesis of Parkinson's disease (PD), but the detailed mechanisms are still unclear. While dopamine (DA) replacement therapy with L-DOPA (levodopa) improves PD symptoms, it does not inhibit the degeneration of DA neurons in the substantia nigra. Recently, bromocriptine, pramipexole and several other agonists of the dopamine D₂-receptor subfamily (including D₂, D₃ and D₄-subtypes) have been shown to have neuroprotective effects in parkinsonian models in vitro and in vivo. Their neuroprotective effects may be mediated directly and/or indirectly by antioxidant effects, mitochondrial stabilization or induction of the antiapoptotic Bcl-2 family.     

Synthesis and structure-activity relationship for new series of 4-phenoxyquinoline derivatives as specific inhibitors of platelet-derived growth factor receptor tyrosine kinase

We discovered a new series of 4-phenoxyquinoline derivatives as potent and selective inhibitors of the platelet-derived growth factor receptor (PDGFr) tyrosine kinase. We researched the highly potent and selective inhibitors on the basis of both PDGFr and epidermal growth factor receptor (EGFr) inhibitory activity. First, we found a compound, Ki6783 (1), which inhibited PDGFr autophosphorylation at 0.13 microM, but it did not inhibit EGFr autophosphorylation at 100 microM. After extensive explorations, we found the two desired compounds, Ki6896 (2) and Ki6945 (3), which are substituted by benzoyl and benzamide at the 4-position of the phenoxy group on 4-phenoxyquinoline, respectively. These inhibitory activities were 0.31 and 0.050 microM, respectively, but neither of them inhibited EGFr autophosphorylation at 100 microM. We further investigated the profile of both compounds toward various tyrosine and serine/threonine kinases. The three compounds specifically inhibited PDGFr rather than the other kinases.     

Synthesis of amide compounds of ferulic acid,and their stimulatory effects on insulin secretion in vitro

We prepared amide compounds which were derived from ferulic acid using various amines, and investigated their stimulatory effects on insulin secretion using rat pancreatic RIN-5F cells. Most of these compounds exhibited significant promotion of the insulin-release at a concentration of 10 microM and in particular, the amides having n-butyl, n-pentyl, pyrrolidine, and piperidine groups showed high activity.     

Synthesis of the cyclic and acyclic acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine,a potent antitumor nucleoside. Design of prodrugs to be selectively activated in tumor tissues via the bio-reduction-hydrolysis mechanism

We have designed and synthesized the acetal derivatives of 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd, 1), the 2',3'-O-nitrobenzylidene derivatives 2 and 3 and the 5'-O-(alkoxy)(nitrophenyl)methyl derivatives 6-10 as potential prodrugs of ECyd. These prodrugs can be selectively activated in tumor tissues via a bio-reduction-hydrolysis mechanism owing to the characteristic properties of tumor tissues, such as hypoxia and lower pH. Although the 2',3'-O-(4-nitrobenzylidene) derivatives 2 and 3 were converted bio-reductively into the corresponding 4-aminobenzylidene derivatives by rat S-9 mix, the reduction products, that is, the corresponding amino congeners 4 and 5, proved to be rather stable in an aqueous solution at pH 6.5 used as a pH model for acidic tumor tissues. In contrast, the 5'-O-(alkoxy)(4-nitropheny)methyl derivatives 6-8 were also reduced by rat S-9 mix to the corresponding amino congeners 11-13, which were hydrolyzed to release ECyd more effectively at pH 6.5 than at pH 7.4. Accordingly, the acyclic acetals 6-8 may be efficient prodrugs of ECyd, that are effectively reduced under physiological conditions releasing ECyd in acidic tumor tissues.     

An inverse correlation between expression of a preprocathepsin B-related protein with cysteine-rich sequences and steroid 11beta -hydroxylase in adrenocortical cells

A cDNA encoding a secretory protein hitherto unknown was cloned from mouse adrenocortical cells by subtractive hybridization between the cells without and with expressing steroid 11beta-hydroxylase (Cyp11b-1), a marker for the functional differentiation of cells in the zonae fasciculata reticularis (zFR). The deduced protein consisting of 466 amino acids contained a secretory signal, epidermal growth factor-like repeats, and a proteolytically inactive cathepsin B-related sequence. The amino acid sequence was 89% identical with that of human tubulointerstitial nephritis antigen-related protein. Among the mouse organs examined, adrenal glands prominently expressed its mRNA. The mRNA and its encoded protein were detected in the outer adrenocortical zones that do not express Cyp11b-1, i.e. the zona glomerulosa and the undifferentiated cell zone, while being undetectable in zFR that express Cyp11b-1. The new protein was designated as adrenocortical zonation factor 1 (AZ-1). Clonal lines with different levels of AZ-1 expression were established from Y-1 adrenocortical cells that originally express Cyp11b-1 but little AZ-1. Analyses of the clonal lines revealed that Cyp11b-1 is detected in the clonal lines maintaining little AZ-1 expression and becomes undetectable in those expressing AZ-1. On the other hand, irrespective of the AZ-1 expression, all clones expressed cholesterol side-chain cleavage enzyme, which occurs throughout the cortical zones. These results demonstrated that adrenocortical cells expressing AZ-1 do not express Cyp11b-1, whereas those with little AZ-1 express this zFR marker in vitro and in vivo, implying a putative role of AZ-1 in determining the zonal differentiation of adrenocortical cells.     

Human leukocyte-derived arginine aminopeptidase. The third member of the oxytocinase subfamily of aminopeptidases

In this study we report the cloning and characterization of a novel human aminopeptidase, which we designate leukocyte-derived arginine aminopeptidase (L-RAP). The sequence encodes a 960-amino acid protein with significant homology to placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase. The predicted L-RAP contains the HEXXH(X)18E zinc-binding motif, which is characteristic of the M1 family of zinc metallopeptidases. Phylogenetic analysis indicates that L-RAP forms a distinct subfamily with placental leucine aminopeptidase and adipocyte-derived leucine aminopeptidase in the M1 family. Immunocytochemical analysis indicates that L-RAP is located in the lumenal side of the endoplasmic reticulum. Among various synthetic substrates tested, L-RAP revealed a preference for arginine, establishing that the enzyme is a novel arginine aminopeptidase with restricted substrate specificity. In addition to natural hormones such as angiotensin III and kallidin, L-RAP cleaved various N-terminal extended precursors to major histocompatibility complex class I-presented antigenic peptides. Like other proteins involved in antigen presentation, L-RAP is induced by interferon-gamma. These results indicate that L-RAP is a novel aminopeptidase that can trim the N-terminal extended precursors to antigenic peptides in the endoplasmic reticulum.     

Ca2+-dependent modulation of intracellular Mg2+ concentration with amiloride and KB-R7943 in pig carotid artery

It has long been recognized that magnesium is associated with several important diseases, including diabetes, hypertension, cardiovascular, and cerebrovascular diseases. In the present study, we measured the intracellular free Mg2+ concentration ([Mg2+]i) using 31P nuclear magnetic resonance (NMR) in pig carotid artery smooth muscle. In normal solution, application of amiloride (1 mm) decreased [Mg2+]i by approximately 12% after 100 min. Subsequent washout tended to further decrease [Mg2+]i. In contrast, application of amiloride significantly increased [Mg2+]i (by approximately 13% after 100 min) under Ca2+-free conditions, where passive Mg2+ influx is facilitated. The treatments had little effect on intracellular ATP and pH (pHi). Essentially the same Ca2+-dependent changes in [Mg2+]i were produced with KB-R7943, a selective blocker of reverse mode Na+-Ca2+ exchange. Application of dimethyl amiloride (0.1 mM) in the presence of Ca2+ did not significantly change [Mg2+]i, although it inhibited Na+-H+ exchange at the same concentration. Removal of extracellular Na+ caused a marginal increase in [Mg2+]i after 100-200 min, as seen in intestinal smooth muscle in which Na+-Mg2+ exchange is known to be the primary mechanism of maintaining a low [Mg2+]i against electrochemical equilibrium. In Na+-free solution (containing Ca2+), neither amiloride nor KB-R7943 decreased [Mg2+]i, but they rather increased it. The results suggest that these inhibitory drugs for Na+-Ca2+ exchange directly modulate Na+-Mg2+ exchange in a Ca2+-dependent manner, and consequently produce the paradoxical decrease in [Mg2+]i in the presence of Ca2+.     

Transgenerational carcinogenesis: induction and transmission of genetic alterations and mechanisms of carcinogenesis

Parental exposure, i.e. germ cell exposure to radiation and chemicals, increased the incidence of tumors and malformations in the offspring, and the germ-line alterations that cause cancer are transmissible to further generations. However, tumor incidences were 100-fold higher than those of ordinary mouse mutations and there were apparent strain differences in the types of induced tumors. In human, higher risk of leukemia is reported in the children of fathers who had been exposed to radionuclides at the nuclear reprocessing plants or to diagnostic doses of radiation. However, these findings in mice and men have not been confirmed in the children of atomic bomb survivors in Hiroshima and Nagasaki. Another important finding was that germ-line exposure was very weakly tumorigenic by itself. However, the transmissible alterations caused persistent hypersensitivity to tumor induction in the offspring, e.g. enhanced by postnatal treatment with tumor promoting/carcinogenic agents. The above results suggest that transmissible alterations might be imprinted in germ cells for the future development of cancer by the postnatal environment. Many gene loci concerning immunological, biochemical and physiological function might be involved, and the cumulative changes in such genes may slightly elevate or enhance tumor incidences, although mutations of tumor suppressor genes such as p53 were also detected in some offspring and genomic instability may modify tumor occurrence in transgenerational manner. In fact, Gene Chip analysis showed suppression and/or over-expression of many functional genes rather than cancer-related genes in the preconceptionally irradiated cancer prone progeny.     

The role of the MxaD protein in the respiratory chain of Methylobacterium extorquens during growth on methanol

The largest of the gene clusters coding for proteins involved in methanol oxidation is the cluster mxaFJGIR(S)ACKLDEHB. Disruption of most of these genes leads to lack of growth on methanol. The previous results showed that the mutant lacking MxaD grows on methanol although at a low rate. This is explained by the low rate of methanol oxidation by whole cells. The specific activity of methanol dehydrogenase (MDH) is higher in the mutant but its electron acceptor (cytochrome c(L)) is unchanged. Using the purified proteins, it was shown that the rate of interaction of MDH and cytochrome c(L) was higher in the wild-type MDH containing some MxaD proteins, which was absent in the mutant MDH. It is suggested that the gene mxaD codes for the 17-kDa periplasmic protein that directly or indirectly stimulates the interaction between MDH and cytochrome c(L); its absence leads to a lower rate of respiration with methanol and therefore a lower growth rate on this substrate.     

A point mutation in ribosomal protein L7/L12 reduces its ability to form a compact dimer structure and to assemble into the GTPase center

An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.