Inhibitory effects of Ge-132 (carboxyethyl germanium sesquioxide) derivatives on enkephalin-degrading enzymes

Twenty-eight species of carboxyethyl germanium sesquioxide (Ge-132) derivatives were examined for their inhibitory effects on enkephalin-degrading enzymes that were purified from monkey brain, the longitudinal muscle layer of bovine small intestine, and human cerebrospinal fluid (CSF). A series of the sulfurized Ge-132 derivatives showed strong inhibition of these purified enzymes. The most effective ones were Ge-014 and Ge-107, which showed IC50 values of 60 and 100 micrograms/ml, respectively, for dipeptidylcarboxypeptidase from the longitudinal muscle layer. They also exhibited inhibitory activity against aminopeptidase from human CSF, the IC50 values being 450 and 440 micrograms/ml, respectively. Furthermore, these compounds showed inhibition of dipeptidylaminopeptidase from monkey brain and the longitudinal muscle layer of bovine small intestine. These compounds are expected to have analgesic effects due to their inhibition of the degradation of endogenous opioid peptides.     

A geographical model of high species diversity

Several cases of high species diversity, for example in tropical rain forests, imply that speciation has been frequent or rapid. However, how speciation could proceed so frequently as to generate extraordinary diversity still remains unsolved, despite recent advancements of diverse theories of allopatric and sympatric speciation. This paper presents a theoretical model that demonstrates the process of frequent speciation by means of geographical fragmentation. We focus on allopatric speciation and explore the evolutionary effect of fragmentation and extinction of demes (subpopulations) in a widespread species or species group. After a large contagious population of a single species is fragmented into demes, extinction of some demes could result in isolation of multiple demes. Thus, several demes could become good species simultaneously through the process of allopatric speciation. We apply the random extinction method to this fragmentation process where demes become randomly extinct. The present model illustrates that frequent speciation could occur in communities where large environmental changes frequently take place.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Essential structure of orexin 1 receptor antagonist YNT-707, part III: Role of the 14-hydroxy and the 3-methoxy groups in antagonistic activity toward the orexin 1 receptor in YNT-707 derivatives lacking the 4,5-epoxy ring

Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX₁R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX₁R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX₁R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives.     

A Subclone of HuH-7 with Enhanced Intracellular Hepatitis C Virus Production and Evasion of Virus Related-Cell Cycle Arrest

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.     

Identification of MK-1925: A selective,orally active and brain-penetrable opioid receptor-like 1 (ORL1) antagonist

Structure–activity relationship studies directed toward improving the metabolic stability of compound 1 resulted in the identification of 3-[5-(3,5-difluorophenyl)-3-({[(1S,3R)-3-fluorocyclopentyl]amino}methyl)-4-methyl-1H-pyrazol-1-yl]propanenitrile 39 (MK-1925) as a selective, orally available and brain-penetrable opioid receptor-like 1 (ORL1) antagonist. The compound also showed in vivo efficacy after oral dosing. Therefore, compound 39 was selected to undergo further studies as a clinical candidate.     

Stereochemical Configuration of 4-Hydroxy-2-nonenal-Cysteine Adducts and Their Stereoselective Formation in a Redox-regulated Protein

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys⁷³ and, to the lesser extent, the active site Cys³². More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys⁷³, whereas Cys³² exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids and has been implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, post-ischemic reoxygenation injury, and aging (1). The peroxidative breakdown of polyunsaturated fatty acids has also been implicated in the pathogenesis of many types of liver injury and especially in the hepatic damage induced by several toxic substances. Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them is a variety of different aldehydes (2). The primary products of lipid peroxidation, lipid hydroperoxides, can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals giving rise to the formation of short chain, unesterified aldehydes, or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including protein, leading to disruption of important cellular functions. The important agents that give rise to the modification of protein may be represented by α,β-unsaturated aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2-alkenals, and 4-oxo-2-alkenals (3, 4).4-Hydroxy-2-nonenal (HNE),² among the reactive aldehydes, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress (2, 5). HNE exerts these effects because of its facile reactivity with biological materials, particularly the sulfhydryl groups of proteins. The reaction of HNE with sulfhydryl groups leads to the formation of thioether adducts that further undergo cyclization to form cyclic hemiacetals (2). Although HNE also forms Michael adducts with the imidazole moiety of histidine residues and the ϵ-amino group of lysine residues (5), the formation of thiol-derived Michael adducts, stabilized as the cyclic hemiacetal, is considered to constitute the main reactivity of HNE, because of the nucleophilic potential of the sulfhydryl group compared with those of the imidazole and amine groups. However, because of the lack of specific and reliable methods for the determination of HNE-cysteine adducts, no study has so far quantitatively demonstrated their formation in proteins.Because HNE generated in lipid peroxidation is a racemic mixture of 4R- and 4S-enantiomers (6), the HNE Michael adducts, possessing three chiral centers at C-2, C-4, and C-5 in the tetrahydrofuran moiety (Fig. 1A), are composed of at least eight isomers. In our previous study (7), we characterized the configurational isomers of an HNE-histidine adduct by NMR spectroscopy and by molecular orbital calculations, and we found that the configuration of the tetrahydrofuran ring could affect the electron delocalization features, which contribute to the stability of the adduct. Moreover, we raised monoclonal antibodies against (R)-HNE- and (S)-HNE-histidine adducts and observed differential cellular distributions of these adducts in vivo. Balogh et al. (8) recently characterized the stereochemical configurations of the HNE-glutathione adduct by NMR experiments in combination with simulated annealing structure determinations. Despite these studies, however, the stereoselectivity of the HNE Michael addition adducts generated in proteins remains to be fully explored. In this study, to gain further structural insight into sulfhydryl modification by the lipid peroxidation product, we characterized the stereochemical configuration of the HNE-N-acetylcysteine adducts by NMR spectroscopy. In addition, we adapted a pyridylamination-based method for fluorescent labeling of the HNE-cysteine adducts, using 2-aminopyridine (2-AP) and sodium cyanoborohydride (NaCNBH₃), and successfully analyzed the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Furthermore, using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin (Trx).Open in a separate windowFIGURE 1.Reaction of cysteine residue with HNE. A, formation of the HNE-cysteine Michael adduct, possessing three chiral centers (asterisks). B, reaction of N-acetylcysteine with enantioisomeric HNE. The reactions were performed as described under “Experimental Procedures.” AU, absorbance units.     

Dicer Is Required for Maintaining Adult Pancreas

Dicer1, an essential component of RNA interference and the microRNA pathway, has many important roles in the morphogenesis of developing tissues. Dicer1 null mice have been reported to die at E7.5; therefore it is impossible to study its function in adult tissues. We previously reported that Dicer1-hypomorphic mice, whose Dicer1 expression was reduced to 20% in all tissues, were unexpectedly viable. Here we analyzed these mice to ascertain whether the down-regulation of Dicer1 expression has any influence on adult tissues. Interestingly, all tissues of adult (8–10 week old) Dicer1-hypomorphic mice were histologically normal except for the pancreas, whose development was normal at the fetal and neonatal stages; however, morphologic abnormalities in Dicer1-hypomorphic mice were detected after 4 weeks of age. This suggested that Dicer1 is important for maintaining the adult pancreas.     

Protective Role of Fas-FasL Signaling in Lethal Infection with Herpes Simplex Virus Type 2 in Mice

Herpes simplex virus type 2 (HSV-2) induces acute local infection followed by latent infection in the nervous system and often leads to the development of lethal encephalitis in immunocompromised hosts. The mechanisms of immune protection against lethal HSV-2 infection, however, have not been clarified. In this study, we examined the roles of Fas-Fas ligand (FasL) signaling in lethal infection with HSV-2 by using mice with mutated Fas (lpr) or FasL (gld) in C57BL/6 background. Both lpr and gld mice exhibited higher mortality than wild-type (WT) C57BL/6 mice after infection with virulent HSV-2 strain 186 and showed significantly increased viral titers in the spinal cord compared with WT mice 9 days after infection, just before the mice started to die. There were no differences in the numbers of CD4⁺ and CD8⁺ T cells infiltrated in the spinal cord or in the levels of HSV-2-specific gamma interferon produced by those cells in a comparison of lpr and WT mice 9 days after infection. Adoptive transfer studies demonstrated that CD4⁺ T cells from WT mice protected gld mice from lethal infection by HSV-2. Furthermore, CD4⁺ T cells infiltrated in the spinal cord of HSV-2-infected WT mice expressed functional FasL that induced apoptosis of Fas-expressing target cells in vitro. These results suggest that FasL-mediated cytotoxic activity of CD4⁺ T cells plays an important role in host defense against lethal infection with HSV-2.Fas-Fas ligand (FasL) signaling-induced apoptotic cell death has pleiotropic roles in T-cell-mediated host defense mechanisms. First, Fas and FasL are expressed on activated T cells and thereby limit their number by inducing suicide or fratricide. It is generally accepted that Fas-mediated activation-induced cell death plays a predominant role during chronic infection, whereas starvation-induced cell death mediated by the proapoptotic BH3-only subgroup of the Bcl-2 protein family is the main mechanism for T-cell death during termination of immune responses in acute infection (30). Fas-FasL signaling might also play a role in T-cell development, as suggested by an accumulation of T-cell receptor αβ-positive (TCR αβ⁺) CD4⁻ CD8⁻ T cells expressing B220 in lymphoid organs of mice with mutated Fas (lpr) or FasL (gld) although the origin and functions of such double-negative T cells are still a matter of debate (21). Lastly, Fas-FasL interaction can be directly involved in host defense by inducing apoptosis of infected cells to facilitate pathogen clearance (23). Therefore, the roles of Fas-FasL signaling in immune responses for host defense might vary depending on the pathogen.Herpes simplex virus type 2 (HSV-2) is an alphaherpesvirus that causes genital herpes, the most common viral sexually transmitted disease (29). After initial infection in the vaginal epithelium, HSV-2 invades local nerve termini, travels via retrograde axonal transport to neuronal cell bodies in sensory ganglia, and establishes latent infection (13). However, especially in neonates and immunocompromised hosts, HSV-2 can cause lethal central nervous system (CNS) infection, which indicates the importance of immune systems in limiting the pathogenicity of HSV-2. Immune responses against HSV-2 have been studied in various murine models using different strains of virus and routes of inoculation, with or without vaccination with an attenuated strain of HSV-2. In such vaccination models, CD4⁺ T cells producing gamma interferon (IFN-γ) predominantly conferred protection against challenge with a virulent strain of HSV-2 (11, 19), whereas various subsets of lymphocytes, including NK cells, NK T cells, and TCR γδ T cells as well as CD4⁺ T cells were reported to be involved in host defense against primary infection with virulent HSV-2 (3, 15, 24), in which IFN-γ also played an important role (9). Fas-FasL signaling was shown to be dispensable for the clearance of an attenuated strain of HSV-2, which lacks thymidine kinase and causes only transient mild vaginal pathologies but not neurologic diseases (6, 16). Similarly Fas-mediated apoptosis was not involved in the vaccination effect of the attenuated HSV-2 (11). However, the roles of Fas-FasL signaling in host defense against a virulent strain of HSV-2 have not been clarified.In this study, we examined the roles of Fas-FasL signaling in a murine model of HSV-2 infection by using a highly virulent HSV-2 strain 186 with lpr and gld mice. We found that FasL-Fas signaling plays an important role in host defense against lethal HSV-2 infection.