Cloning and sequencing of the gene for a Pseudomonas paucimobilis enzyme that cleaves beta-aryl ether.

We isolated Pseudomonas paucimobilis SYK-6, which was able to degrade various dimeric lignin compounds (Y. Katayama, S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N. Morohoshi, and T. Haraguchi, Mokuzai Gakkaishi 33:77-79, 1987). This metabolic process is a distinct characteristic of this bacterium, which is equipped with an enzymatic modification system for various dimeric lignin compounds involved in the tricarboxylic acid cycle. Cleavage of the beta-aryl ether linkage is essential in this process, because this linkage is the most abundant (approximately 50%) in lignin. Here, we report the isolation and characterization of the beta-etherase gene, which contains an open reading frame of 843 bp and which we call ligE. This gene was expressed in Escherichia coli, and the enzyme had the same kinetic properties as the P. paucimobilis SYK-6 enzyme.     

Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT).

Methods

Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed.

Results

Among the 4 probes examined in this study, ¹²⁵I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, ¹²³I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft.

Conclusion

¹²³I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.     

DNA polymerase IV mediates efficient and quick recovery of replication forks stalled at N2-dG adducts

Escherichia coli DNA polymerase IV (Pol IV, also known as DinB) is a Y-family DNA polymerase capable of catalyzing translesion DNA synthesis (TLS) on certain DNA lesions, and accumulating data suggest that Pol IV may play an important role in copying various kinds of spontaneous DNA damage including N²-dG adducts and alkylated bases. Pol IV has a unique ability to coexist with Pol III on the same β clamp and to positively dissociate Pol III from β clamp in a concentration-dependent manner. Reconstituting the entire process of TLS in vitro using E. coli replication machinery and Pol IV, we observed that a replication fork stalled at (−)-trans-anti-benzo[a]pyrene-N²-dG lesion on the leading strand was efficiently and quickly recovered via two sequential switches from Pol III to Pol IV and back to Pol III. Our results suggest that TLS by Pol IV smoothes the way for the replication fork with minimal interruption.     

DNA-like class R inhibitory oligonucleotides (INH-ODNs) preferentially block autoantigen-induced B-cell and dendritic cell activation in vitro and autoantibody production in lupus-prone MRL-Fas lpr/lpr mice in vivo

Introduction  

B cells have many different roles in systemic lupus erythematosus (SLE), ranging from autoantigen recognition and processing to effector functions (for example, autoantibody and cytokine secretion). Recent studies have shown that intracellular nucleic acid-sensing receptors, Toll-like receptor (TLR) 7 and TLR9, play an important role in the pathogenesis of SLE. Dual engagement of rheumatoid factor-specific AM14 B cells through the B-cell receptor (BCR) and TLR7/9 results in marked proliferation of autoimmune B cells. Thus, strategies to preferentially block innate activation through TLRs in autoimmune B cells may be preferred over non-selective B-cell depletion.     

Blockade of CD26-mediated T cell costimulation with soluble caveolin-1-Ig fusion protein induces anergy in CD4T cells

CD26 binds to caveolin-1 in antigen-presenting cells (APC), and that ligation of CD26 by caveolin-1 induces T cell proliferation in a TCR/CD3-dependent manner. We report herein the effects of CD26-caveolin-1 costimulatory blockade by fusion protein caveolin-1-Ig (Cav-Ig). Soluble Cav-Ig inhibits T cell proliferation and cytokine production in response to recall antigen, or allogeneic APC. Our data hence suggest that blocking of CD26-associated signaling by soluble Cav-Ig may be an effective approach as immunosuppressive therapy.     

Bacterial Growth Stimulation with Exogenous Siderophore and Synthetic N-Acyl Homoserine Lactone Autoinducers under Iron-Limited and Low-Nutrient Conditions

The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for Pelagiobacter sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 μM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM N-acyl-octanoylhomoserine lactone (C₈-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C₈-HSL; however, C₈-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.     

Establishment of a shortened annual cycle system; a tool for the analysis of annual re-translocation of phosphorus in the deciduous woody plant (Populus alba L.)

The supply of phosphorus, the essential element for plant growth and development, is often limited in natural environments. Plants employ multiple physiological strategies to minimize the impact of phosphate deficiency. In deciduous trees, phosphorus is remobilized from senescing leaves in autumn and stored in other tissues for reuse in the following spring. We previously monitored the annual changes in leaf phosphate content of white poplar (Populus alba) growing under natural conditions and found that about 75 % of inorganic and 60 % of organic leaf phosphates observed in May were remobilized by November. In order to analyze this process (such annual events), we have established a model system, in which an annual cycle of phosphate re-translocation in trees can be simulated under laboratory conditions by controlling temperature and photoperiod (=‘shortened annual cycle’). This system evidently allowed us to monitor the annual changes in leaf color, phosphate remobilization from senescent leaves, and bud break in the next spring within five months. This will greatly facilitate the analysis of cellular and molecular mechanisms of annual phosphate re-translocation in deciduous trees.     

Rearing the 28-spotted ladybird beetle,Henosepilachna vigintioctopunctata (Coleoptera: Coccinelidae), with a switchover from host plant leaves to artificial diet

In this study, I tested an artificial diet, Insecta LFS, for rearing the 28-spotted ladybird beetle, Henosepilachna vigintioctopunctata (Fabricius). H. vigintioctopunctata larvae could not be reared through all of the larval stages on the artificial diet. However, initially they could be reared on tomato leaves up to the second or third instar, and thereafter exclusively on the artificial diet. The larval and pupal periods were not significantly different from those reared only on tomato leaves. For females reared by the diet-switching method, the preoviposition period was significantly longer and the number of eggs laid significantly lower than for those on tomato leaves. However, these differences did not become a hindrance to laboratory rearing. This rearing method saves labor involving obtaining fresh plant leaves and produces a reliable supply of the insects.     

Role of TAPP1 and TAPP2 adaptor binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis

Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P(3) second messenger. PtdIns(3,4,5)P(3) can be broken down to PtdIns(3,4)P(2) through the action of the SHIPs (Src-homology-2-domain-containing inositol phosphatases). As PtdIns(3,4)P(2) levels peak after those of PtdIns(3,4,5)P(3), it has been proposed that PtdIns(3,4)P(2) controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P(2) through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P(2). These homozygous TAPP1(R211L/R211L) TAPP2(R218L/R218L) double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1(R211L/R211L) TAPP2(R218L/R218L) knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P(3) and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adap-tors to PtdIns(3,4)P(2) function as negative regulators of the insulin and PI3K signalling pathways.