Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.     

Species Diversity of Japanese Clusiidae (Diptera: Acalyptrata) with description of 12 new species

The Japanese Clusiidae are revised and 23 species, including 12 species new to science, are recognized and keyed. Clusiodes angulosus n. sp., Clusiodes discostylus n. sp., Clusiodes tobi n. sp., Clusiodes usikumuri n. sp., Craspedochaeta varicolor n. sp., Heteromeringia crenulata n. sp., Heteromeringia quadrispinosa n. sp., Heteromeringia sexramifera n. sp., Heteromeringia yamata n. sp., Phylloclusia quadrivittata n. sp., Sobarocephala uncinata n. sp., and Tetrameringia borealis n. sp. were described as new. Distribution records were compiled and mapped. Transitional climatic zone between the warm and cool temperate zones is possibly suggested as one of the most richest temperate area in terms of Japanese clusiid species richness. Hendelia beckeri Czerny 1903, Paraclusia japonica Sasakawa 1957, and Clusiodes plumosus Sasakawa 1964 are broadly distributed over all the Japanese temperate forests, but these are not always most abundant species. The clusiid assemblage was heterogeneous in the species composition among various forest types. The femalebiased sample caught by Malaise traps and distinction of clusiid species composition between temperate and subtropical zones are also pointed out.     

Top-down motif engineering of a cytokine receptor for directing ex vivo expansion of hematopoietic stem cells

The technique to expand hematopoietic stem cells (HSCs) ex vivo is eagerly anticipated to secure an enough amount of HSCs for clinical applications. Previously we developed a scFv-thrombopoietin receptor (c-Mpl) chimera, named S-Mpl, which can transduce a proliferation signal in HSCs in response to a cognate antigen. However, a remaining concern of the S-Mpl chimera may be the magnitude of the cellular expansion level driven by this molecule, which was significantly less than that mediated by endogenous wild-type c-Mpl. In this study, we engineered a tyrosine motif located in the intracellular domain of S-Mpl based on a top-down approach in order to change the signaling properties of the chimera. The truncated mutant (trunc.) and an amino-acid substitution mutant (Q to L) of S-Mpl were constructed to investigate the ability of these mutants to expand HSCs. The result showed that the truncated and Q to L mutants gave higher and considerably lower number of the cells than unmodified S-Mpl, respectively. The proliferation level through the truncated mutant was even higher than that of non-transduced HSCs with the stimulation of a native cytokine, thrombopoietin. Moreover, we analyzed the signaling properties of the S-Mpl mutants in detail using a pro-B cell line Ba/F3. The data indicated that the STAT3 and STAT5 activation levels through the truncated mutant increased, whereas activation of the Q to L mutant was inhibited by a negative regulator of intracellular signaling, SHP-1. This is the first demonstration that a non-natural artificial mutant of a cytokine receptor is effective for ex vivo expansion of hematopoietic cells compared with a native cytokine receptor.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC₅₀ = 8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED₅₀ = 4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment     

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.     

Chemical Synthesis and Characterization of α-N-Octanoyl and Other α-N-Acyl Colistin Nonapeptide Derivatives

Eight α-N-acyl colistin nonapeptide derivatives including three aliphatic, four aromatic and one alicyclic derivatives were synthesized by the reaction of colistin nonapeptide with corresponding acid chlorides. This acylation reaction was carried out under the condition kept restrictedly at pH 5,0 in order to introduce an acyl group only to α-amino group but not to γ-amino group existing in a colistin nonapeptide molecule. Synthetic method and several physico-chemical natures of these acyl colistin nonapeptide derivatives are given in this paper.

All of the acylated derivatives thus synthesized exhibited characteristic antimicrobial activities. Antimicrobial spectra were substantially based upon a gram-negative type and not so much altered by chemical structures of acyl groups which were considerably differentiated from each other such as cyclic or chain form. Thus, more possible response of carbon size than its structure to the antimicrobial effectiveness was inferred. In spite of almost no toxicity and feeble antimicrobial activity of colistin nonapeptide itself, these acylated colistin nonapeptide derivatives showed a toxicity against mice at a dose of 16.9~70 mg/kg in LD₅₀, which, however, was inferior to the toxicity of colistin sulfate, possibly correspondent to their much weaker antimicrobial activities, as a whole. Hence, it seems likely that acyl part of these acylated colistin nonapeptide derivatives including that of colistin is seriously responsible for the biological activities.     

Development of a Host-vector System for Gluconobacter suboxydans

A shuttle vector for Gluconobacter suboxydans and Escherichia coli was constructed by ligation of a cryptic plasmid, pMV201, found in G. suboxydans IFO 3130 to E. coli plasmid pACYC177. The chimeric plasmid named pMGlOl carries the ampicillin resistance gene derived from pACYC177 and transforms G. suboxydans var. α IFO 3254 as well as E. coli. The transformation conditions for G. suboxydansvar. α IFO 3254 were examined using pMGlOl DNA. Competent cells were induced efficiently by treatment with LiCl or RbCl CaCl₂ which induced the competency of Acetobacter was much less effective. Addition of polyethylene glycol enhanced the transformation efficiency significantly. An efficiency of approximately 10² transformants per μg DNA was finally obtained.     

Separation and Purification of Molecular Species of Galactolipids by High Performance Liquid Chromatography

The NaCl concentration of the growth medium affected hydrogen production by Lyngbya sp. (No. 108) strain. Cells grown in medium containing 3% NaCl produced the most hydrogen. The carbohydrate content of this strain also increased with increasing NaCl concentration of the growth medium up to 720 fig/mg cells at 5 % NaCl. In the presence of 20 finlol/ml MFA (monofluoroacetic acid), inhibition of hydrogen production was observed. We extracted the glycogen from this nonheterocystous filamentous cyanobacterium, Lyngbya sp. (No. 108), and observed that glycogen and carbohydrate consumption of this strain is coincident with hydrogen production.

These results led us to the conclusion that the reserve glycogen or other carbohydrate were used as sources of electron donors for hydrogen production, and that the NaCl concentration of the medium affected the hydrogen production by this strain.     

Enzymatic Synthesis of the Crithidia Factors in Cell-free Extracts of Serratia indica

The concomitant production of formic acid and pterin compounds from guanosine-5′-triphosphate (GTP) has been found in cell-free extracts of Serratia indica. Among the pterin compounds, l-threo-neopterin–the major Crithidia factor in S. indica–, a cyclic phosphate of neopterin (cNP), d-erythro-neopterin and 6-hydroxymethyl pterin were detected and isolated. Formate-¹⁴C elimination from GTP-8-¹⁴C was quantitatively distributed in the ethyl acetate layer in the ehyl acetate-hydrochloric acid partition system. Carbon 8 of GTP was released as formic acid. Enzymatic production of formate and cNP was linear for 2 hr at 37°C. Formate production was proportional to the enzyme concentration. The optimum pH for formate elimination was observed around pH 8.6. Optimum temperature for the production of formate and cNP was 50°C. The apparent Km value of GTP for formate production was 6.2×10^?bm. Formate eliminating activity was activated by disodium phosphate but was inhibited by Mg²⁺ or AMP. Incorporation of GTP-U-¹⁴C into pterin compounds was also regulated with disodium phosphate. Effective incorporation into cNP and d-erythro-neopterin occurred in the presence of phosphate. When phosphate was omitted from the system, however, effective incorporation into 6-hydroxymethyl pterin was observed. The biosynthetic process of the Crithidia factors, i.e. l-threo-neopterin and cNP, from GTP in S. indica is also discussed.