Mechanism for manganese enhancement of dopamine-induced oxidative DNA damage and neuronal cell death

Although the cause of dopaminergic cell death in Parkinson's disease is still poorly understood, there is accumulating evidence suggesting that metal ions can be involved in the processes. We investigated the effect of manganese on cell death and DNA damage in PC12 cells treated with dopamine. Mn(II) enhanced cell death induced by dopamine. Mn(II) also increased the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) contents of DNA in PC12 cells treated with dopamine. To clarify the mechanism of cellular DNA damage, we investigated DNA damage induced by dopamine and Mn(II) using (32)P-labeled DNA fragments. Mn(II) enhanced Cu(II)-dependent DNA damage by dopamine. The Mn(II)-enhanced DNA damage was greatly increased by NADH. Piperidine and formamidopyrimidine-DNA glycosylase treatment induced cleavage sites mainly at T and G of the 5'-TG-3' sequence, respectively. Bathocuproine, a Cu(I) chelator, and catalase inhibited the DNA damage. Oxygen consumption and UV-visible spectroscopic measurements showed that Mn(II) enhanced autoxidation of dopamine with H(2)O(2) formation. These results suggest that reactive species derived from the reaction of H(2)O(2) with Cu(I) participates in Mn(II)-enhanced DNA damage by dopamine plus Cu(II). Therefore, it is concluded that oxidative DNA damage induced by dopamine in the presence of Mn(II), NADH, and Cu(II) is possibly linked to the degeneration of dopaminergic neurons.     

Anoikis-resistant MDCK cells carrying susceptibilities to TNF-α and verotoxin that are suitable for influenza virus cultivation

Madin-Darby canine kidney (MDCK) cells were originally anchorage-dependent epithelial cells. Here, we have isolated a novel MDCK-derived cell population, termed 6 M-4, by means of culturing MDCK cells in suspension for nearly 6 months in the presence of Streptomyces griseus metalloendopeptidase (MEP). The isolated cells showed unique proliferation characteristics, which differed from parental MDCK cells. They proliferated adherently on a polystyrene matrix, but proliferated non-adherently both in the presence of MEP and on a non-adhesive matrix coated with poly 2-methacryloyloxyethyl phosphorylcholine (MPC). The 6 M-4 cells consisted of at least two cell types. One type, termed 6 M-4-TR7, would not grow in soft agar and showed a novel phenotype in that the cells were susceptible to both TNF-α and verotoxin 1 (VT1). In addition, the isolated adhesion-independent cells sustained epithelial traits of parental MDCK cells. We further show that these MDCK-derivative cells are suitable for influenza virus cultivation. Hemagglutination (HA) titers of influenzaviruses A and B were increased in the suspension culture of 6 M-4-TR7 cells supplemented with the MEP in comparison to adherently growing cells in the presence of trypsin.     

Visual search for a target changing in synchrony with an auditory signal

We examined whether the detection of audio-visual temporal synchrony is determined by a pre-attentive parallel process, or by an attentive serial process using a visual search paradigm. We found that detection of a visual target that changed in synchrony with an auditory stimulus was gradually impaired as the number of unsynchronized visual distractors increased (experiment 1), whereas synchrony discrimination of an attended target in a pre-cued location was unaffected by the presence of distractors (experiment 2). The effect of distractors cannot be ascribed to reduced target visibility nor can the increase in false alarm rates be predicted by a noisy parallel processing model. Reaction times for target detection increased linearly with number of distractors, with the slope being about twice as steep for target-absent trials as for target-present trials (experiment 3). Similar results were obtained regardless of whether the audio-visual stimulus consisted of visual flashes synchronized with amplitude-modulated pips, or of visual rotations synchronized with frequency-modulated up-down sweeps. All of the results indicate that audio-visual perceptual synchrony is judged by a serial process and are consistent with the suggestion that audio-visual temporal synchrony is detected by a 'mid-level' feature matching process.     

A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity

Proteins capable of selective and specific inhibition of cysteine protease have been identified as cystatins and are isolated from a variety of microbes and tissues of animals and plants. The physiological function of these proteins has been proposed to be the regulation of protein turnover and defense against pathogens as well as the balance of the host-parasite immune relationship. Genes encoding cystatins have been found in several species of ticks, but the function of cystatin in ticks is not understood. We cloned a gene encoding cystatin from tick H. longicornis and designated it as Hlcyst-2 (H. longicornis cystatin-2). Its full-length cDNA is 569 bp, and it encodes a putative 133 amino acid protein with an obvious signal peptide. Sequence analysis demonstrated that it has significant homology with the known cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain, cathepsin L, and cathepsin B was identified by fluorogenic substrate analysis. Cystatin was mostly expressed in the tick midgut and hemocyte. Blood feeding induced significantly increased expression in the midgut. Real-time PCR confirmed that LPS-injected adult ticks expressed Hlcyst-2 1.6 more times than the PBS-injected control; Babesia gibsoni-infected larvae ticks expressed Hlcyst-2 1.8 more times than normal larvae ticks. The recombinant protein also showed a significant growth-inhibitory effect on Babesia bovis cultured in vitro. These results indicated this cystatin Hlcyst-2 is involved in tick innate immunity.     

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.     

Protein kinase A–dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin

Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during β-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca²⁺ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca²⁺ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca²⁺ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca²⁺ sensitivity, but also abolished the Ca²⁺-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca²⁺ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca²⁺ sensitivity, and PKA further decreased Ca²⁺ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca²⁺ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.     

Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation

Brome mosaic virus (BMV) packages its genomic RNAs (RNA1, RNA2, and RNA3) and subgenomic RNA4 into three different particles. However, since the RNAs in the virions have distinct lengths and electrostatic charges, we hypothesize that subsets of the virions should have distinct properties. A glutamine to cysteine substitution at position 120 of the capsid protein (CP) was found to result in a mutant virus named QC that exhibited a dramatically altered ratio of the RNAs in virions. RNA2 was far more abundant than the other RNAs, although the ratios could be affected by the host plant species. RNAs with the QC mutation were competent for replication early in the infection, suggesting that they were either selectively packaged or degraded after packaging. In support of the latter idea, low concentrations of truncated RNA1 that co-migrated with RNA2 were found in the QC virions. Spectroscopic analysis and peptide fingerprinting experiments showed that the QC virus capsid interacted with the encapsidated RNAs differently than did the wild type. Furthermore, wild-type BMV RNA1 was found to be more susceptible to nuclease digestion relative to RNA2 as a function of the buffer pH. Other BMV capsid mutants also had altered ratios of packaged RNAs.     

Regulation of De Novo-Initiated RNA Synthesis in Hepatitis C Virus RNA-Dependent RNA Polymerase by Intermolecular Interactions

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been proposed to change conformations in association with RNA synthesis and to interact with cellular proteins. In vitro, the RdRp can initiate de novo from the ends of single-stranded RNA or extend a primed RNA template. The interactions between the Δ1 loop and thumb domain in NS5B are required for de novo initiation, although it is unclear whether these interactions are within an NS5B monomer or are part of a higher-order NS5B oligomeric complex. This work seeks to address how polymerase conformation and/or oligomerization affects de novo initiation. We have shown that an increasing enzyme concentration increases de novo initiation by the genotype 1b and 2a RdRps while primer extension reactions are not affected or inhibited under similar conditions. Initiation-defective mutants of the HCV polymerase can increase de novo initiation by the wild-type (WT) polymerase. GTP was also found to stimulate de novo initiation. Our results support a model in which the de novo initiation-competent conformation of the RdRp is stimulated by oligomeric contacts between individual subunits. Using electron microscopy and single-molecule reconstruction, we attempted to visualize the low-resolution conformations of a dimer of a de novo initiation-competent HCV RdRp.Polymerases undergo a series of conformational changes at different stages of nucleic acid synthesis (14). Of the template-dependent polymerases, the RNA-dependent RNA polymerases (RdRps) are the least understood in terms of their mechanism of action. RdRps are of increasing interest since cellular RdRps play important roles in the defense against nonself RNAs (44). In addition, virus-encoded RdRps are important targets for the development of antivirals. A better understanding of RNA-dependent RNA polymerases is thus important for both basic and applied science.Several model systems for biochemical study of viral RNA-dependent RNA synthesis exist (4, 19, 20, 25, 37, 42). Well-characterized RdRps include those from the hepatitis C virus (HCV) and poliovirus (5, 17). In the host, the RdRps are complexed with other viral and/or cellular proteins that are usually associated with membranous intracellular structures. The replicases are usually difficult to study biochemically, but the catalytic RdRp subunits of several viruses can be purified for functional and structural analyses (53). These recombinant proteins can reproduce some of the activities of the replicases, including the ability to initiate RNA synthesis by a de novo mechanism (22, 47-49). Furthermore, recombinant RdRps can affect the activities of other replicase subunits in vitro, suggesting that the recombinant RdRp is useful for an in-depth understanding of RNA synthesis by HCV (45, 60).RdRps form a right-hand-like structure with thumb, finger, and palm subdomains. The metal-coordinating residues important for nucleotide binding are positioned within the palm subdomain (26). An interesting feature of viral RdRps is that they tend to exist in a closed conformation, even in the absence of template, in contrast to DNA-dependent RNA polymerases, which transition from open to closed complexes upon template recognition (13). The closed form of the phage φ6 RdRp has been proposed to allow specific recognition of the single-stranded viral RNA (7). The template channel formed by the closed structure, however, is too narrow to accommodate the partially duplexed RNA that forms during RNA synthesis, and hence, the closed conformation needs to undergo significant rearrangements in the ternary complex. Biswal et al. (3) have captured an X-ray crystallographic structure of a partially open conformation of the HCV RdRp. Bovine viral diarrhea virus (BVDV) RdRp was also shown to exist in a partially open conformation (11). Ranjith-Kumar and Kao (49) demonstrated that the HCV RdRp could initiate RNA synthesis from a circular RNA template, and thus, the threading of a single-stranded RNA into the template channel is not required for de novo-initiated RNA synthesis. Altogether, these results raise the possibility that the HCV RdRp can undergo rearrangements from the closed conformation seen in the crystal structure prior to de novo initiation.A secondary structure that extends from the finger to the thumb subdomains, named the Δ1 loop, has been proposed to serve as a gate to cover the template channel and regulate the switch from de novo initiation to elongation (5, 10). Mutations that affect the interaction between the Δ1 loop and the mostly hydrophobic residues that it contacts have resulted in polymerases that are defective for de novo initiation but can bind to partially duplexed RNA and can extend from the 3′ terminus of an RNA primer (10).Two general models for RNA synthesis by the HCV RdRp can be proposed (Fig. ​(Fig.1).1). The first posits that the HCV RdRp functions as a monomer at least during de novo initiation because the closed template channel is needed for specific recognition of the template (5, 7, 10). It was presumed that the Δ1 loop and thumb domain interaction in the HCV RdRp is stable and mutations that disrupted this interaction would render the enzyme catalytically inactive (5, 24). However, a deletion of five residues in the tip of the Δ1 loop did not prevent RNA synthesis from a primed template by the polymerase (10). Furthermore, a genotype 2a RdRp was crystallized in a form with altered interaction between the Δ1 loop and thumb domain in comparison to the 1b RdRp (3). Interestingly, a low-affinity GTP binding site exists on the thumb domain close to the base of the Δ1 loop binding pocket. GTP binding at this site has been proposed to stabilize the Δ1 loop and thumb domain interactions, favoring the closed monomer model (6). A second model is based on the reports that HCV RdRp can oligomerize and that oligomerization increases its activity (12, 16, 46, 54). The dimer could be active due to either the second subunit increasing the stability of the Δ1 loop and thumb interactions in the first subunit to increase de novo initiation or the two subunits forming a common template-binding domain (Fig. ​(Fig.1).1). Here we have attempted to determine whether monomers or oligomers of the HCV RdRp can better perform de novo initiation using biochemical and biophysical analyses.Open in a separate windowFIG. 1.Models for RNA synthesis by the HCV RdRp. The monomer model is based on the central tenet that intramolecular interactions within an RdRp molecule regulate the modes of RNA synthesis. The curved arrow represents the possible orientation of the template RNA. The oligomer model is an adaptation from the dimer model of the norovirus RdRp (18). T, P, and F represent the thumb, palm, and finger domains, respectively, in different shades of gray, and the thick black line connecting the thumb and finger domains represents the Δ1 loop.     

Synthesis and evaluation of opioid receptor-binding affinity of elaeocarpenine and its analogs

Both enantiomers of elaeocarpenine (1) and its analogs, 21, 22, 25, and 27, were synthesized from bicyclic aldehydes 8–10 via a flexible route previously established for total synthesis of grandisines, and their binding affinities for μ-, κ- and δ-opioid receptor subtypes were evaluated. We found that (9R)-1 exhibited higher affinity than (9S)-1 for all the subtypes, but the enantiomers showed little subtype selectivity. Analogs 21 having a pyrrolizidine skeleton and 27 having a stemona-type skeleton in place of the indolizidine unit of (9S)-1 showed μ-selective and μ-, κ-selective binding, respectively.