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.     

Transpiration integrated model for root ion absorption under salinized condition

Salinization of crop fields is a pressing matter for sustainable agriculture under desertification and is largely attributed to root absorptive functions of the major crops such as maize. The rates of water and ion absorption of intact root system of maize plants were measured under the salinized condition, and the salt absorptive function of maize roots was analyzed by applying different two kinetic models of root ion absorption (i.e. the concentration dependent model and the transpiration integrated model). The absorption rates for salinization ions (Na⁺, Cl^?, Ca²⁺ and Mg²⁺) were found to depend on ion mass flow through roots driven by the transpiration, and therefore the transpiration integrated model represented more accurately rates of root ion absorption. The root absorption of salinization ions was characterized quantitatively by two model parameters of Q′_max and K′_M involved in the transpiration integrated model, which are considered to relate to the potential absorbing power and the ion affinity of transport proteins on root cell membranes, respectively.     

Synthesis and evaluation of N-alkyl-S-[3-(piperidin-1-yl)propyl]isothioureas: High affinity and human/rat species-selective histamine H3 receptor antagonists

S-Alkyl-N-alkylisothiourea compounds containing various cyclic amines were synthesized in the search for novel nonimidazole histamine H₃ receptor (H₃R) antagonists. Among them, four N-alkyl S-[3-(piperidin-1-yl)propyl]isothioureas 18, 19, 22, and 23 were found to exhibit potent and selective H₃R antagonistic activities against in vitro human H₃R, but were inactive against in vitro human H₄R. Furthermore, three alkyl homologs 18–20 showed inactivity for histamine release in in vivo rat brain microdialysis, suggesting differences in antagonist affinities between species. In addition, in silico docking studies of N-[4-(4-chlorophenyl)butyl]-S-[3-piperidin-1-yl)propyl]isothiourea 19 and a shorter homolog 17 with human/rat H₃Rs revealed that structural differences between the antagonist-docking cavities of rat and human H₃Rs were likely caused by the Ala122/Val122 mutation.     

Structural and dynamics characterization of norovirus protease

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.     

Discovery and structure–activity relationship of thienopyridine derivatives as bone anabolic agents

A cell-based assay was performed for the discovery of novel bone anabolic agents. Alkaline phosphatase (ALPase) activity of ST2 cells was utilized as an indicator of osteoblastic differentiation, and thienopyridine derivative 1 was identified as a hit compound. 3-Aminothieno[2,3-b]pyridine-2-carboxamide was confirmed to be a necessary core structure for the enhancement of ALPase activity, and then optimization of the C4-substituent on the thienopyridine ring was carried out. Introduction of cyclic amino groups to the C4-position of the thienopyridine ring improved the activity. Especially, N-phenyl-homopiperazine derivatives were found to be strong enhancers of ALPase among this new series. Furthermore, 3-amino-4-(4-phenyl-1,4-diazepan-1-yl)thieno[2,3-b]pyridine-2-carboxamide (15k) was orally administered to ovariectomized (OVX) rats over 6 weeks for evaluating the effects on areal bone mineral density (aBMD), and statistically significant improvements in aBMD were observed from the dosage of 10 mg/kg/day.     

Mutagen from the Gaseous Phase of Protein Pyrolyzate

A mutagenic fraction was separated by gas chromatography (gc) from the gaseous phase of protein pyrolyzate and a compound in the fraction was identified as hydrogen cyanide (hcn). Authentic hcn shows mutagenicity. It is proposed that most of the mutagenic activity in the gaseous phase of protein pyrolyzate is due to hcn.     

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.     

Post-proline Dipeptidyl Amino-peptidase from Flavobacterium

Nitrogenase catalyzes not only the reduction of N₂ to NH₃ but also the reduction of C₂H₂ to C₂H₄ and H⁺ ion to H₂ gas, etc. The detailed mechanism of the nitrogenase reaction is not clear. We have prepared monoclonal antibodies against Component I nitrogenase of A. vinelandii and examined the effects of antibodies on the nitrogenase reactions. A monoclonal antibody designated MA-1 inhibited C₂H₂ reduction activity strongly but did not inhibit H₂ evolution activity. MA-2, on the contrary, inhibited only H₂ evolution activity. MA-8 inhibited both C₂H₂ reduction and H₂ evolution activity to the same extent.     

Pyrolytic Yields of 2-Amino-9H-pyrido[2,3-b]indole and 3-Amino-1-methyl-5H-pyrido[4,3-b]indole as Matagens from Proteins

An extracellular exo-maltohexaohydrolase [EC 3.2.1.98] from a Klebsiella pneumoniae (Aerobacter aerogenes) mutant produced about 40% maltohexaose (G₆) from short-chain amylose ( =23). Mostly G₆ was produced from maltooligosaccharides larger than G₆ by an exo-mechanism action. It also hydrolyzed G₆ and shorter maltooligosaccharides to give smaller maltooligosaccharides. Its position specificity of action on G₃ through G₈ was studied with maltodextrins specifically labeled at the reducing-end glucose unit with ¹⁴C. The highest frequency of cleavage was at the second bond from the reducing end in G₃ through G₆. For G₇ and G₈, the sixth bond from the nonreducing end of the substrate was cleaved with absolute specificity by the exo-mechanism action.

Kinetic parameters of the exo-maltohexaohydrolase on various substrates were also studied. The Michaelis constant (Km) for short-chain amylose was the smallest among the various substrates examined.

G₆ was also formed from G₄ by a transfer action of the enzyme, with an action pattern dependent on the substrate concentration.     

Inhibitory Effect of Glycine on the Production of Amylase and Proteinase by Bacillus subtilis

It was found that the production of amylase and proteinase by washed cells of Bacillus subtilis var. amyloliquefaciens was inhibited by glycine and its peptides but not by glycine derivatives, in which the free amino group was protected with various groups. Incorporation experiments of glycine-C¹⁴ revealed that about 60 per cent of the radioactivity which had been incorporated into the cells was found in the free amino acid fraction of the bacteria. The inhibitory effect of glycine was easily reversed by the addition of amino acid such as alanine, methionine and glutamic acid. Spermine also caused the reversal of inhibition of the enzyme production by glycine.