Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin

The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.     

An efficient and expedient method for the synthesis of 11C-labeled α-aminoisobutyric acid: a tumor imaging agent potentially useful for cancer diagnosis

We describe the synthesis of ¹¹C-labeled α-aminoisobutyric acid 2 from iodo[¹¹C]methane and methyl N-(diphenylmethylen)-d,l-alaniate (5). The tetrabutylammonium fluoride (TBAF)-promoted α-[¹¹C]methylation of sterically hindered analog 5 was a key step in our synthesis process. Total radiochemical conversion of 2 was high and a remote-controlled synthesis was carried out. A comparative tumor positron emission tomography (PET) imaging study using the same model mouse showed higher uptake of 2 than with ¹¹C-labeled methionine and [¹⁸F] fluorodeoxyglucose (FDG).     

5a-Carba-β-D-glucopyranose derivatives as novel sodium-dependent glucose cotransporter 2 (SGLT2) inhibitors for the treatment of type 2 diabetes

5a-Carba-β-D-glucopyranose derivatives were synthesized and identified as novel SGLT2-selective inhibitors. These inhibitors exhibited potent SGLT2 inhibition with high selectivity over SGLT1. Among the tested compounds, 6f indicated the most potent hSGLT2 inhibition and the highest selectivity over hSGLT1. Moreover, the pharmacokinetics data also showed that 6h, which had the same aglycon structure as sergliflozin-active (3-active), had a threefold longer half-life time (T(1/2)) than sergliflozin (3) with a high distribution volume in db/db mice. Subsequently, 6h lowered blood glucose levels as much as 3 and showed longer hypoglycemic action than 3 in db/db mice.     

Per-N-methylated analogues of an antitumor bicyclic hexapeptide RA-VII

Penta-N-methyl and hexa-N-methyl analogues of RA-VII, an antitumor bicyclic hexapeptide of plant origin, were prepared. In the former, the nitrogens of d-Ala-1 and Ala-4 and in the latter, those of d-Ala-1, Ala-2, and Ala-4 were methylated under the phase-transfer catalysis conditions. Their solution structures were established by NOESY experiments and the crystal structures by X-ray crystallography. Those two methylated analogues showed much weaker cytotoxicity against P-388 leukemia cells than the parent RA-VII.     

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.     

Functional and genetic survey of all known single-nucleotide polymorphisms within the human deoxyribonuclease I gene in wide-ranging ethnic groups

The single-nucleotide polymorphisms (SNPs) in the human DNase I gene (DNASE1) might be involved in susceptibility to some common diseases; however, only limited population data are available. Further, the effects of these SNPs on in vivo DNase I activity remain unknown. The genotype and haplotype of all the SNPs in DNASE1 were determined in 3 ethnic groups including 14 populations using newly developed methods. Together with our previous data on the nonsynonymous SNPs, two major haplotypes based on the five exonic SNPs were identified; genetic diversity in the Asian population was low. Among 10 SNPs, other than exonic SNPs in the gene, only 3 were polymorphic among all the populations. Haplotype distribution, based on all the polymorphic SNPs, was clarified to be generally varied in an ethnic-dependent manner. Thus, the genetic aspects of DNASE1 with regard to all the SNPs in wide-ranging ethnic groups could be first demonstrated. Further, there was no correlation of all the polymorphic SNPs other than nonsynonymous ones with serum DNase I activity levels. Polymorphic SNPs other than the exonic SNPs might not be directly related to common diseases through alterations in in vivo levels of the activity.     

Generation of novel cationic antimicrobial peptides from natural non-antimicrobial sequences by acid-amide substitution

Background

The accessory gene regulator (agr) is a quorum sensing cluster of genes which control colonization and virulence in Staphylococcus aureus. We evaluated agr function in community- (CA) and healthcare-associated (HA) MRSA, to compare the pharmacodynamics and bactericidal activity of vancomycin against agr functional and dysfunctional HA-MRSA and CA-MRSA.

Methods

40 clinical isolates of MRSA from the Canadian Nosocomial Infection Surveillance Program were evaluated for delta-haemolysin production, as a surrogate marker of agr function. Time kill experiments were performed for vancomycin at 0 to 64 times the MIC against an initial inoculum of 10⁶ and 10⁸ cfu/ml of agr functional and dysfunctional CA-MRSA and HA-MRSA and these data were fit to a hill-type pharmacodynamic model.

Results

15% isolates were agr dysfunctional, which was higher among HA-MRSA (26.3%) versus CA-MRSA (4.76%). Against a low initial inoculum of 10⁶ cfu/ml of CA-MRSA, vancomycin pharmacodynamics were similar among agr functional and dysfunctional strains. However, against a high initial inoculum of 10⁸ cfu/ml, killing activity was notably attenuated against agr dysfunctional CA-MRSA (USA400) and HA-MRSA (USA100). CA-MRSA displayed a 20.0 fold decrease in the maximal reduction in bacterial counts (Emax) which was 3.71 log₁₀ CFU/ml for agr functional vs. 2.41 log₁₀ CFU/ml for agr dysfunctional MRSA (p = 0.0007).

Conclusions

Dysfunction in agr was less common among CA-MRSA vs. HA-MRSA. agr dysfunction demonstrated an impact on vancomycin bactericidal activity and pharmacodynamics against a high initial inoculum of CA-MRSA and HA-MRSA, which may have implications for optimal antimicrobial therapy against persistent, difficult to treat MRSA infections.     

Ultrafine cellulose fibers produced by Asaia bogorensis, an acetic acid bacterium

The ability to synthesize cellulose by Asaia bogorensis, a member of the acetic acid bacteria, was studied in two substrains, AJ and JCM. Although both strains have identical 16S rDNA sequence, only the AJ strain formed a solid pellicle at the air-liquid interface in static culture medium, and we analyzed this pellicle using a variety of techniques. In the presence of cellulase, glucose and cellobiose were released from the pellicle suggesting that it is made of cellulose. Field emission electron microscopy allowed the visualization of a 3D knitted structure with ultrafine microfibrils (approximately 5-20 nm in width) in cellulose from A. bogorensis compared with the 40-100 nm wide microfibrils observed in cellulose isolated from Gluconacetobacter xylinus, suggesting differences in the mechanism of cellulose biosynthesis or organization of cellulose synthesizing sites in these two related bacterial species. Identifying these differences will lead to a better understanding of cellulose biosynthesis in bacteria.     

Strategy for shoot meristem proliferation in plants

Shoot apical meristem (SAM) of plants harbors stem cells capable of generating the aerial tissues including reproductive organs. Therefore, it is very important for plants to control SAM proliferation and its density as a survival strategy. The SAM is regulated by the dynamics of a specific gene network, such as the WUS-CLV interaction of A. thaliana. By using a mathematical model, we previously proposed six possible SAM patterns in terms of the manner and frequency of stem cell proliferation. Two of these SAM patterns are predicted to generate either dichotomous or axillary shoot branch. Dichotomous shoot branches caused by this mechanism are characteristic of the earliest vascular plants, such as Cooksonia and Rhynia, but are observed in only a small minority of plant species of the present day. On the other hand, axillary branches are observed in the majority of plant species and are induced by a different dynamics of the feedback regulation between auxin and the asymmetric distribution of PIN auxin efflux carriers. During evolution, some plants may have adopted this auxin-PIN system to more strictly control SAM proliferation.     

A trial of production of the plant-derived high-value protein in a plant factory: Photosynthetic photon fluxes affect the accumulation of recombinant miraculin in transgenic tomato fruits

One of the ultimate goals of plant science is to test a hypothesis obtained by basic science and to apply it to agriculture and industry. A plant factory is one of the ideal systems for this trial. Environmental factors affect both plant yield and the accumulation of recombinant proteins for industrial applications within transgenic plants. However, there have been few reports studying plant productivity for recombinant protein in closed cultivation systems called plant factories.To investigate the effects of photosynthetic photon flux (PPF) on tomato fruit yield and the accumulation of recombinant miraculin, a taste-modifying glycoprotein, in transgenic tomato fruits, plants were cultivated at various PPFs from 100 to 400 (µmol m⁻² s⁻¹) in a plant factory. Miraculin production per unit of energy used was highest at PPF100, although miraculin production per unit area was highest at PPF300. The commercial productivity of recombinant miraculin in transgenic tomato fruits largely depended on light conditions in the plant factory. Our trial will be useful to consider the trade-offs between the profits from production of high-value materials in plants and the costs of electricity.Key words: light, molecular farming, recombinant miraculin, taste-modifying protein, transgenic tomato