Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.     

Cumulative Incidence and Predictors of Progression in Corticosteroid-Na?ve Patients with Sarcoidosis

Background

Assessment of the clinical course of sarcoidosis requires long-term observation. However, the appropriate period of follow-up for sarcoidosis remains unclear, especially in patients without indication of corticosteroid therapy at the time of diagnosis.

Objective

This study aimed to clarify the cumulative incidence and identify risk factors for disease progression in corticosteroid-naïve sarcoidosis patients.

Methods

The clinical courses of 150 Japanese patients with sarcoidosis, who were followed for more than 2 years and had no indication for corticosteroid therapy at diagnosis, were retrospectively reviewed. Disease progression was defined as worsening of pulmonary sarcoidosis, development of new organ involvement, or extrapulmonary organ damage. The cumulative incidence of progression was estimated by generating a cumulative incidence curve with the Fine and Gray method.

Results

The median follow-up duration was 7.7 years (interquartile range, 4.7–13.6 years). Thirty-two (21%) patients experienced disease progression. New organ involvement appeared in 16 patients (11%). The 6-month, and 1-, 5-, 10-, and 15-year cumulative incidence of progression was 2%, 5%, 15%, 28%, and 31%, respectively. The number of organs involved at diagnosis was an independent predictor for progression with a multifactorial adjusted hazard ratio of 1.71 (95% confidence interval, 1.11–2.62). The optimal cut-off of the number of organs involved at diagnosis to identify future progression was three.

Conclusions

In corticosteroid-naïve sarcoidosis patients, the risks of disease progression are comparable from 0–5 years and 5–10 years after diagnosis. The number of organs involved at diagnosis is a useful predictor for progression of sarcoidosis.     

Homoepiboxidines: further potent agonists for nicotinic receptors

Homoepiboxidine (3) and the corresponding N-methyl (4) and N-benzyl (5) derivatives were prepared from a 6beta-carbomethoxynortropane (8). Affinities and functional activities at neuromuscular, central neuronal and ganglionic-type nicotinic receptors were compared to those of epibatidine 1, and epiboxidine 2. Homoepiboxidine had equivalent affinity/activity to epiboxidine at neuromuscular, neuronal alpha4beta2, and most alpha3-containing ganglionic-type nicotinic receptors. The N-substituted derivatives showed reduced affinity/activity at most receptor subtypes. Replacement of the methylisoxazole moiety of 3 and 4 with a methyloxadiazole moiety provided analogues 6 and 7, which had greatly reduced affinity/activity in virtually all assays at nicotinic receptors. Marked analgetic activity in mice occurred at the following ip doses: epibatidine 10 microg/kg; epiboxidine 25 microg/kg; homoepiboxidine 100 microg/kg; N-methylhomoepiboxidine 100 microg/kg; the methyloxadiazole (6) 100 microg/kg. The time course at such ip doses was significantly longer for homoepiboxidine 3 with marked analgesia still manifest at 30 min post-injection. Epiboxidine and the homoepiboxidines were less toxic than epibatidine.     

A lipid peroxidation-derived inflammatory mediator: identification of 4-hydroxy-2-nonenal as a potential inducer of cyclooxygenase-2 in macrophages

Cyclooxygenases (COXs) catalyze the conversion of arachidonic acid to eicosanoids, which mediate a variety of biological actions involved in vascular pathophysiology. In the present study, we investigated the role of lipid peroxidation products in the up-regulation of COX-2, an inducible isoform responsible for high levels of prostaglandin production during inflammation and immune responses. COX-2 was found to colocalize with 4-hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived aldehyde, in foamy macrophages within human atheromatous lesions, suggesting that COX-2 expression may be associated with the accumulation of lipid peroxidation products within macrophages. To test the hypothesis that lipid peroxidation products might be involved in the regulation of prostanoid biosynthesis, we conducted a screen of oxidized fatty acid metabolites and found that, among the compounds tested, only HNE showed inducibility of the COX-2 protein in RAW264.7 macrophages. In addition, intraperitoneal administration of HNE resulted in an increase in cell numbers in the peritoneal cavity that was associated with significant increases in the peritoneal and tissue levels of COX-2 in mice. To understand the possible signaling mechanism underlying the inducing effect of HNE on COX-2 up-regulation, we examined the phosphorylation events that may lead to COX-2 induction and found that HNE did not stimulate the induction of nitric oxide synthase and activation of NF-kappaB but significantly activated p38 mitogen-activated protein kinase and its upstream kinase in RAW264.7 macrophages. Tyrosine kinases, such as the epidermal growth factor-like and Src family tyrosine kinases, appeared to mediate the stabilization of COX-2 mRNA via the p38 mitogen-activated protein kinase pathway. These findings suggest that HNE accumulated in macrophages/foam cells may represent an inflammatory mediator that plays a role in stimulation of the inflammatory response and contributes to the progression of atherogenesis.     

Detection of biosphere frontier by using phosphatase activity.

A wide variety of methods have been proposed to detect microbial activities, but most of them can be applied to limited categories of terrestrial organisms. We propose here to use phosphatase activity, which seems to be an essential catalytic activity for all the terrestrial organisms, and possibly for extraterrestrial organisms. We determined phosphatase activity in core samples, chimney samples, and sea water samples obtained in submarine hydrothermal systems located at Suiyo Seamount, Izu-Bonin Arc, and South Mariana. It was shown that phosphatase activity is one of possible biomarkers for extant life.     

Molecular Characterization of a Deep-Sea Methanotrophic Mussel Symbiont that Carries a RuBisCO Gene

In our previous investigation on the genes of 1,5-ribulose bisphosphate carboxylase/oxygenase (RuBisCO; EC 4.1.1.39) in deep-sea chemoautotrophic and methanotrophic endosymbioses, the gene encoding the large subunit of RuBisCO form I (cbbL) had been detected in the gill of a mussel belonging to the genus Bathymodiolus from a western Pacific back-arc hydrothermal vent. This study further examined the symbiont source of the RuBisCO cbbL gene along with the genes of 16S ribosomal RNA (16S rDNA) and particulate methane monooxygenase (EC 1.14.13.25; pmoA) and probed for the presence of the ATP sulfurylase gene (EC 2.7.7.4; sopT). The 16S rDNA sequence analysis indicated that the mussel harbors a monospecific methanotrophic Gammaproteobacterium. This was confirmed by amplification and sequencing of the methanotrophic pmoA, while thiotrophic sopT was not amplified from the same symbiotic genome DNA. Fluorescence in situ hybridization demonstrated simultaneous occurrence of the symbiont-specific 16S rDNA, cbbL and pmoA, but not sopT, in the mussel gill. This is the first molecular and visual evidence for a methanotrophic bacterial endosymbiont that bears the RuBisCO cbbL gene relevant to autotrophic CO₂ fixation.     

Feasibility of ex vivo gene therapy for neurological disorders using the new retroviral vector GCDNsap packaged in the vesicular stomatitis virus G protein

Neuronal progenitor cells (NPC) are particularly suited as the target population for genetic and cellular therapy of neurological disorders such as Parkinson's disease or stroke. However, genetic modification of these cells using retroviral vectors remains a great challenge because of the low transduction rate and the need for fetal calf serum (FCS) during the transduction process that induces the cell differentiation to mature neurons. To overcome these problems, we developed a new retrovirus production system in which the simplified retroviral vector GCDNsap engineered to be resistant to denovo methylation was packaged in the vesicular stomatitis virus G protein (VSV-G), concentrated by centrifugation, and resuspended in serum-free medium (StemPro-34 SFM). In transduction experiments using enhanced green fluorescent protein (EGFP) as a marker, the concentrated FCS-free virus supernatant infected NPC at a high rate, while maintaining the ability of these cells to self-renew and differentiate in vitro. When such cells were grafted into mouse brains, EGFP-expressing NPC were detected in the region around the injection site at 8 weeks post transplantation. These findings suggest that the gene transfer system described here may provide a useful tool to genetically modify NPC for treatments of neurological disorders.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.