Small off-shore islands can serve as important refuges for endemic beetle conservation

Although large islands generally support a richer insect fauna than small islands, many large islands, which are more often inhabited, have lost numerous species because of human activities and introduced organisms. To clarify the consequences of endemic insect conservation on small islands near inhabited islands, we compared the species richness, abundance, and composition of two beetle groups (Coleoptera: Cerambycidae and Mordellidae) captured using Malaise traps among three islands (Chichijima, 24.0 km²; Anijima, 7.85 km²; Nishijima, 0.49 km²) in the oceanic Ogasawara (Bonin) Island group in the northwestern Pacific during June–July 2006 and 2007. Chichijima, the largest island, is inhabited, while Anijima and Nishijima are not. The numbers of cerambycid and mordellid species previously recorded were positively correlated with island area. However, the total numbers of cerambycid and mordellid species we captured in Malaise traps were not correlated with island area because we were unable to collect many species previously documented on Chichijima. The numbers of cerambycid and mordellid species per trap did not differ significantly among islands and years, although the deviance was well explained by the island variable. We captured greater numbers of cerambycid and mordellid individuals on Chichijima than on Anijima and Nishijima, and the numbers of cerambycid and mordellid individuals per trap significantly differed among islands and between years. Redundancy analysis (RDA) showed that the species composition of cerambycids and mordellids differed among the three islands. Whereas endangered species were rarely captured on Chichijima, alien or non-endemic species were frequently collected. Cerambycid and mordellid beetles on Chichijima may have been deleteriously affected by recent forest disturbance and introduced organisms. Therefore, conserving insect fauna on uninhabited island “refugia” is important for preserving the insect diversity of the Ogasawara Islands.     

Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1

We report the whole DNA sequence of two plasmids, pPI-1 (30.2 kb) and pPI-2 (2.8 kb). These plasmids are from Staphylococcus warneri ISK-1, which produces a lantibiotic, nukacin ISK-1. Curing of pPI-1 resulted in a loss of bactericidal activity in the culture supernatant and the host's immunity to nukacin ISK-1, suggesting that the biosynthetic genes of the bacteriocin are encoded by pPI-1. Based on the results of a homology search of each open reading flame, pPI-1 is comprised of the following four distinct regions: (1) the nukacin ISK-1 biosynthesis and immunity gene cluster, (2) the thioredoxin gene cluster, (3) the replication region, and (4) a region of Staphylococcus epidermidis ATCC 12228, highly homologous to pSE-12228-05. Gene organization in the nukacin ISK-1 biosynthesis and immunity gene cluster is different from that in other lacticin-481 type gene clusters. The features of the replication protein encoded in the replicating region are somewhat different from other staphylococcus theta-replicating plasmids. pPI-2 comprised a disinfectant resistant gene, qacC, and the whole DNA sequence showed significant similarity to those of other qacC plasmids such as pSK108, suggesting that pPI-2 belongs to the qacC plasmid group.     

Adiponectin gene therapy of streptozotocin-induced diabetic mice using hydrodynamic injection

BACKGROUND: Adiponectin (Adipo), an adipocyte hormone involved in the regulation of glucose and lipid metabolism, has already been identified as a potential therapeutic target for the treatment of diabetes. However, successful delivery of Adipo to the receptors is difficult due to their peptide characteristics. Receptors for Adipo are abundantly expressed in the liver and skeletal muscle. METHODS: Uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) in hepatoblastoma HepG2 cells expressing Adipo was examined. Adipo-expressing plasmid DNA (10-50 microg) in saline solution (0.1 ml/g body weight) was rapidly injected into the tail vein of 4-week-old diabetic mice after 4-6 weeks of treatment with streptozotocin (STZ). Uptake of glucose in diabetic mice also was measured using a planar positron imaging system featuring 18-fluorodeoxyglucose. RESULTS: HepG2 cells expressing Adipo exhibited significantly increased 2-NBDG uptake compared with cells transfected with control plasmid even in the absence of insulin. STZ-induced diabetic mice showed decreased serum Adipo levels compared with non-diabetic mice. A single hydrodynamic injection of 10-50 microg Adipo-expressing plasmid DNA into diabetic mice led to approximately 10-15-fold elevation in serum Adipo levels, and resulted in decreased serum levels of glucose and triglyceride. As well as exhibiting higher levels of Adipo expression, diabetic mice also had higher hepatic glucose uptake than similar mice injected with control plasmid. CONCLUSIONS: We report that STZ-induced diabetic mice exhibited decreased Adipo levels and hyperglycemia which may be alleviated by hydrodynamic injection of the Adipo gene. This type of gene delivery system to the liver offers a different approach in developing novel treatments for type 1 and 2 diabetes.     

Structural analysis of gene loci for rat U1 small nuclear RNA.

Four phage clones which hybridize with U1 small nuclear RNA were obtained from a rat gene library. Two clones contain a presumed pseudogene. A third clone includes two gene candidates that are co-linear with the rat U1-RNA, 3.6kb apart and in the opposite orientation. The two genes are surrounded by identical sequences of 491bp upstream and 178bp downstream. The upstream sequences do not contain a TATA box, but share many block homologies with those for the human U1-RNA gene(1-3). A 101bp "identifier (ID) sequence", which was reported to be specifically expressed in rat brain (4), is inserted immediately after the shared sequence downstream of one of the genes. In the fourth clone, there are two putative pseudogenes, which have one or three nucleotide changes, 3kb apart and in the same orientation. Southern blot analysis of total rat DNA reveals about 50 U1-RNA genes/pseudogenes in the genome.     

Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach

The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(ε-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/μkat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.     

Molecular characterization of the protease from Clostridium botulinum serotype C responsible for nicking in botulinum neurotoxin complex

A protease was purified from the culture medium of Clostridium botulinum serotype C strain Stockholm (C-St). The purified protease belonged to the cysteine protease family based on assays for enzyme inhibitors, activators and kinetic parameters. The protease formed a binary complex consisting of 41- and 17-kDa proteins held together non-covalently. The DNA sequence encoding the protease gene was shown to be a single open reading frame of 1593 nucleotides, predicting 530 amino acid residues including a signal peptide. The N-terminal region of the native enzyme underwent further proteolytic modification after processing by a signal peptidase. The protease introduced intermolecular cleavage into an intact single chain botulinum neurotoxin (BoNT) at a specific site. Homology modeling and docking simulation of C-St BoNT and C-St protease demonstrated that the specific nicking-site of the BoNT appears to fit into the deep pocket in the active site of the protease.     

Serum-free culture of murine primordial germ cells and embryonic germ cells

Fetal calf serum (FCS) has usually been used for culture of embryonic stem (ES) cell as a component of the culture medium. However, FCS contains undefined factors, which promote cell proliferation and occasionally stimulate differentiation of ES cells. Recently, a chemically-defined serum replacement, Knockout Serum Replacement (KSR), was developed to maintain ES cells in an undifferentiated state. In this experiment, we examined the effects of KSR on the growth and differentiation of primordial germ cells (PGCs) and embryonic germ (EG) cells. PGCs were collected 8.5 days postcoitum (dpc) from B6D2F1 (C57BL/6JxDBA/2J) female mice mated with B6D2F1 males. Most of the PGCs that were cultured in FCS-supplemented medium (FCS medium) had alkaline phosphatase (AP) activity and acquired a fibroblast cell shape. In contrast, PGCs in KSR-supplemented medium (KSR medium) proliferated, maintaining round and stem cell-like morphology. In addition, EG cells were established more easily from PGCs cultured in KSR medium than from PGCs cultured in FCS medium. The percentage of undifferentiated colonies of EG cells was significantly higher in KSR medium than in FCS medium. The germ line chimera was also produced from EG cells established in KSR medium. These results suggest that KSR can be used for sustaining an undifferentiated state of PGCs and EG cells in vitro.     

Serum heat shock protein 47 levels are elevated in acute exacerbation of idiopathic pulmonary fibrosis

Little is known about the pathophysiology of acute exacerbation (AE) of idiopathic pulmonary fibrosis (IPF). Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules. Previous studies in experimental animal fibrosis models have shown that downregulation of HSP47 expression reduces collagen production and diminishes fibrosis progression. In this study, serum HSP47 levels were evaluated to elucidate pathogenic differences involving HSP47 between AE-IPF and stable (S)-IPF. Subjects comprised 20 AE-IPF and 33 S-IPF patients. Serum levels of HSP47, Krebs von den Lungen-6 (KL-6), surfactant protein (SP)-A, SP-D, and lactate dehydrogenase (LDH) were measured. Immunohistochemical analysis of lung HSP47 expression was determined in biopsy and autopsy tissues diagnosed as diffuse alveolar damage (DAD) and usual interstitial pneumonia (UIP). Serum levels of HSP47 were significantly higher in AE-IPF than in S-IPF patients, whereas serum levels of KL-6, SP-A, and SP-D did not differ significantly. Receiver operating characteristic curves revealed that HSP47 was superior for discriminating AE-IPF and S-IPF. The cutoff for HSP47 resulting in the highest diagnostic accuracy was 559.4 pg/mL; sensitivity, specificity, and diagnostic accuracy were 100.0 %, 93.9 %, and 96.2 %, respectively. Immunohistochemical analysis revealed that pulmonary HSP47 expression was greater in DAD than UIP tissues. Serum HSP47 was significantly higher in AE-IPF than in S-IPF patients, suggesting that underlying fibrogenic mechanisms involving HSP47 differ in the two conditions.     

Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. The keloid matrix contains excess collagen and glycosaminoglycans (GAGs), but lacks elastic fiber. However, the roles of these matrix components in the pathogenesis of keloid are largely unknown. Here, we show that elastin and DANCE (also known as fibulin-5), a protein required for elastic fiber formation, are not deposited in the extracellular matrix of keloids, due to excess accumulation of chondoitin sulfate (CS), although the expression of elastin and DANCE is not affected. Amount of CS accumulated in the keloid legion was 6.9-fold higher than in normal skin. Fibrillin-1, a scaffold protein for elastic fiber assembly, was abnormally distributed in the keloid matrix. Addition of purified CS to keloid fibroblast culture resulted in abnormal deposition of fibrillin-1, concomitant with significantly decreased accumulation of elastin and DANCE in the extracellular matrix. We propose that CS plays a crucial role in the development of keloid lesions through inhibition of elastic fiber assembly.