Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide by 3-chloro-D-alanine hydrogen chloride-lyase (deaminating) of Pseudomonasputida

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide is catalyzed by highly purified 3-chloro-D-alanine hydrogen chloride-lyase from $\text{Pseudomonas}\text{putida}$. The synthetic reaction proceeds optimally at pH 8.5, as a function of enzyme concentration and incubation time. The enzymatically synthesized D-cysteine was isolated from the large scale reaction mixture and identified by physicochemical means.     

Mapping protein interactions of sodium channel NaV1.7 using epitope‐tagged gene‐targeted mice

The voltage‐gated sodium channel Na_V1.7 plays a critical role in pain pathways. We generated an epitope‐tagged Na_V1.7 mouse that showed normal pain behaviours to identify channel‐interacting proteins. Analysis of Na_V1.7 complexes affinity‐purified under native conditions by mass spectrometry revealed 267 proteins associated with Nav1.7 in vivo. The sodium channel β3 (Scn3b), rather than the β1 subunit, complexes with Nav1.7, and we demonstrate an interaction between collapsing‐response mediator protein (Crmp2) and Nav1.7, through which the analgesic drug lacosamide regulates Nav1.7 current density. Novel Na_V1.7 protein interactors including membrane‐trafficking protein synaptotagmin‐2 (Syt2), L‐type amino acid transporter 1 (Lat1) and transmembrane P24‐trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated by co‐immunoprecipitation (Co‐IP) from sensory neuron extract. Nav1.7, known to regulate opioid receptor efficacy, interacts with the G protein‐regulated inducer of neurite outgrowth (Gprin1), an opioid receptor‐binding protein, demonstrating a physical and functional link between Nav1.7 and opioid signalling. Further information on physiological interactions provided with this normal epitope‐tagged mouse should provide useful insights into the many functions now associated with the Na_V1.7 channel.     

Identification of novel genes expressed highly and selectively in the corpora allata of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Applied Entomology and Zoology - Juvenile hormone (JH) has crucial roles in insect physiology, including development, reproduction, and polyphenism. JH is synthesized in the corpora allata (CA)...     

Vanillin production using <Emphasis Type="Italic">Escherichia coli</Emphasis> cells over-expressing isoeugenol monooxygenase of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.     

Quinolinate dehydrogenase and 6-hydroxyquinolinate decarboxylase involved in the conversion of quinolinic acid to 6-hydroxypicolinic acid by<Emphasis Type="Italic"> Alcaligenes</Emphasis> sp. strain UK21

In the conversion of quinolinic acid to 6-hydroxypicolinic acid by whole cells of Alcaligenes sp. strain UK21, the enzyme reactions involved in the hydroxylation and decarboxylation of quinolinic acid were examined. Quinolinate dehydrogenase, which catalyzes the first step, the hydroxylation of quinolinic acid, was solubilized from a membrane fraction, partially purified, and characterized. The enzyme catalyzed the incorporation of oxygen atoms of H₂O into the hydroxyl group. The dehydrogenase hydroxylated quinolinic acid and pyrazine-2,3-dicarboxylic acid to form 6-hydroxyquinolinic acid and 5-hydroxypyrazine-2,3-dicarboxylic acid, respectively. Phenazine methosulfate was the preferred electron acceptor for quinolinate dehydrogenase. 6-Hydroxyquinolinate decarboxylase, catalyzing the nonoxidative decarboxylation of 6-hydroxyquinolinic acid, was purified to homogeneity and characterized. The purified enzyme had a molecular mass of approximately 221 kDa and consisted of six identical subunits. The decarboxylase specifically catalyzed the decarboxylation of 6-hydroxyquinolinic acid to 6-hydroxypicolinic acid, without any co-factors. The N-terminal amino acid sequence was homologous with those of bacterial 4,5-dihydroxyphthalate decarboxylases.     

S-Nitrosated human serum albumin dimer is not only a novel anti-tumor drug but also a potentiator for anti-tumor drugs with augmented EPR effects

Macromolecules have been developed as carriers of low-molecular-weight drugs in drug delivery systems (DDS) to improve their pharmacokinetic profile or to promote their uptake in tumor tissue via enhanced permeability and retention (EPR) effects. In the present study, recombinant human serum albumin dimer (AL-Dimer), which was designed by linking two human serum albumin (HSA) molecules with the amino acid linker (GGGGS)(2), significantly accumulated in tumor tissue even more than HSA Monomer (AL-Monomer) and appearing to have good retention in circulating blood in murine colon 26 (C26) tumor-bearing mice. Moreover, we developed S-nitrosated AL-Dimer (SNO-AL-Dimer) as a novel DDS compound containing AL-Dimer as a carrier, and nitric oxide (NO) as (i) an anticancer therapeutic drug/cell death inducer and (ii) an enhancer of the EPR effect. We observed that SNO-AL-Dimer treatment induced apoptosis of C26 tumor cells in vitro, depending on the concentration of NO. In in vivo experiments, SNO-AL-Dimer was found to specifically deliver large amounts of cytotoxic NO into tumor tissue but not into normal organs in C26 tumor-bearing mice as compared with control (untreated tumor-bearing mice) and SNO-AL-Monomer-treated mice. Intriguingly, S-nitrosation improved the uptake of AL-Dimer in tumor tissue through augmenting the EPR effect. These data suggest that SNO-AL-Dimer behaves not only as an anticancer therapeutic drug, but also as a potentiator of the EPR effect. Therefore, SNO-AL-Dimer would be a very appealing carrier for utilization of the EPR effect in future development of cancer therapeutics.     

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.