Substrate specificity of ribose-5-phosphate isomerases from <Emphasis Type="Italic">Clostridium difficile</Emphasis> and <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

The activity of ribose-5-phosphate isomerases (RpiB) from Clostridium difficile for d-ribose isomerization was optimal at pH 7.5 and 40°C, while that from Thermotoga maritima for l-talose isomerization was optimal at pH 8.0 and 70°C. C. difficile RpiB exhibited activity only with aldose substrates possessing hydroxyl groups oriented in the right-handed configuration (Fischer projections) at the C2 and C3 positions, such as d-ribose, d-allose, l-talose, l-lyxose, d-gulose, and l-mannose. In contrast, T. maritima RpiB displayed activity only with aldose substrates possessing hydroxyl groups configured the same direction at the C2, C3, and C4 positions, such as the d- and l-forms of ribose, talose, and allose.     

Mannose production from fructose by free and immobilized <Emphasis Type="SmallCaps">d</Emphasis>-lyxose isomerases from <Emphasis Type="Italic">Providencia stuartii</Emphasis>

A recombinant d-lyxose isomerase from Providencia stuartii was immobilized on Duolite A568 beads which gave the highest conversion of d-fructose to d-mannose among the various immobilization beads evaluated. Maximum activities of both the free and immobilized enzymes for fructose isomerization were at pH 7.5 and 45°C in the presence of 1 mM Mn²⁺. Enzyme half-lives were 14 and 30 h at 35°C and 3.4 and 5.1 h at 45°C, respectively. The immobilized enzyme in 300 g fructose/l (replaced hourly), produced 75 g mannose/l at 35°C = 25% (w/w) yield with a productivity of 75 g mannose l⁻¹ h⁻¹ after 23 cycles.     

tsORFdb: Theoretical Small Open Reading Frames (ORFs) database and massProphet: Peptide Mass Fingerprinting (PMF) tool for unknown small functional ORFs

Peptide mass fingerprinting (PMF) has become one of the most widely used methods for rapid identification of proteins in proteomics research. Many peaks, however, remain unassigned after PMF analysis, partly because of post-translational modification and the limited scope of protein sequences. Almost all PMF tools employ only known or predicted protein sequences and do not include open reading frames (ORFs) in the genome, which eliminates the chance of finding novel functional peptides. Unlike most tools that search protein sequences from known coding sequences, the tool we developed uses a database for theoretical small ORFs (tsORFs) and a PMF application using a tsORFs database (tsORFdb). The tsORFdb is a database for ORFeome that encompasses all potential tsORFs derived from whole genome sequences as well as the predicted ones. The massProphet system tries to extend the search scope to include the ORFeome using the tsORFdb. The tsORFdb and massProphet should be useful for proteomics research to give information about unknown small ORFs as well as predicted and registered proteins.     

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.     

Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide

TLR2 in association with TLR1 or TLR6 plays an important role in the innate immune response by recognizing microbial lipoproteins and lipopeptides. Here we present the crystal structures of the human TLR1-TLR2-lipopeptide complex and of the mouse TLR2-lipopeptide complex. Binding of the tri-acylated lipopeptide, Pam(3)CSK(4), induced the formation of an "m" shaped heterodimer of the TLR1 and TLR2 ectodomains whereas binding of the di-acylated lipopeptide, Pam(2)CSK(4), did not. The three lipid chains of Pam(3)CSK(4) mediate the heterodimerization of the receptor; the two ester-bound lipid chains are inserted into a pocket in TLR2, while the amide-bound lipid chain is inserted into a hydrophobic channel in TLR1. An extensive hydrogen-bonding network, as well as hydrophobic interactions, between TLR1 and TLR2 further stabilize the heterodimer. We propose that formation of the TLR1-TLR2 heterodimer brings the intracellular TIR domains close to each other to promote dimerization and initiate signaling.     

Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544

The enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid was investigated in 53 Rhodococcus and Pseudomonas related strains. Rhodococcus erythropolis ATCC 25544 was selected as it showed the highest enantioselectivity. The enantioselectivity was due to the amidase activity in a two-step reaction involving nitrile hydratase. The enantiomeric excess of the amidase was highest at pH 7.0 and decreased significantly above 20 °C. For the enantioselective production of (S)-2,2-dimethylcyclopropane carboxylic acid, the optimum reaction conditions of the cells were determined to be pH 7.0, 20 °C, and 10% (v/v) methanol and were the same as the optimum pH and temperature for the enantioselective conversion by the amidase. Under these conditions, the R. erythropolis ATCC 25544 cells, which harbored nitrile hydratase and amidase enzymes, produced 45 mM (S)-2,2-dimethylcyclopropane carboxylic acid from racemic 100 mM 2,2-dimethylcyclopropane carbonitrile with an 81.8% enantiomeric excess after 64 h.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

G Protein betagamma subunits augment UVB-induced apoptosis by stimulating the release of soluble heparin-binding epidermal growth factor from human keratinocytes

UV radiation induces various cellular responses by regulating the activity of many UV-responsive enzymes, including MAPKs. The betagamma subunit of the heterotrimeric GTP-binding protein (Gbetagamma) was found to mediate UV-induced p38 activation via epidermal growth factor receptor (EGFR). However, it is not known how Gbetagamma mediates the UVB-induced activation of EGFR, and thus we undertook this study to elucidate the mechanism. Treatment of HaCaT-immortalized human keratinocytes with conditioned medium obtained from UVB-irradiated cells induced the phosphorylations of EGFR, p38, and ERK but not that of JNK. Blockade of heparin-binding EGF-like growth factor (HB-EGF) by neutralizing antibody or CRM197 toxin inhibited the UVB-induced activations of EGFR, p38, and ERK in normal human epidermal keratinocytes and in HaCaT cells. Treatment with HB-EGF also activated EGFR, p38, and ERK. UVB radiation stimulated the processing of pro-HB-EGF and increased the secretion of soluble HB-EGF in medium, which was quantified by immunoblotting and protein staining. In addition, treatment with CRM179 toxin blocked UV-induced apoptosis, but HB-EGF augmented this apoptosis. Moreover, UVB-induced apoptosis was reduced by inhibiting EGFR or p38. The overexpression of Gbeta(1)gamma(2) increased EGFR-activating activity and soluble HB-EGF content in conditioned medium, but the sequestration of Gbetagamma by the carboxyl terminus of G protein-coupled receptor kinase 2 (GRK2ct) produced the opposite effect. The activation of Src increased UVB-induced, Gbetagamma-mediated HB-EGF secretion, but the inhibition of Src blocked that. Overexpression of Gbetagamma increased UVB-induced apoptosis, and the overexpression of GRK2ct decreased this apoptosis. We conclude that Gbetagamma mediates UVB-induced human keratinocyte apoptosis by augmenting the ectodomain shedding of HB-EGF, which sequentially activates EGFR and p38.     

Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells

A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.