The cryoablation of lung tissue using liquid nitrogen in gel and in the ex vivo pig lung.

PURPOSES: To examine the efficiency of cryoablation using liquid nitrogen in lung tissue, we measured the size and temperature distribution of the frozen area (iceball) in gel and in the ex vivo pig lungs. METHODS: Cryoprobes with diameters of 2.4 and 3.4 mm (2.4D and 3.4D, respectively) were used. Three temperature sensors were positioned at the surface of the cryoprobe and at distances of 0.5 and 1.5 cm from the cryoprobe. The ex vivo pig lungs were perfused with 37 °C saline and inflated using ventilator to simulate in vivo lung conditions. RESULTS: In gel, the 2.4D and 3.4D probes made iceballs of 3.9 ± 0.1 and 4.8 ± 0.3 cm in diameter, respectively, and the temperature at 1.5 cm from those probes reached -32 ± 8 and -53 ± 5 °C, respectively. In the pig lung, the 2.4D and 3.4D probes made iceballs of 5.2 ± 0.1 and 5.5 ± 0.4 cm in diameter, respectively, and the temperature at 1.5 cm from these probes reached -49 ± 5 and -58 ± 3 °C, respectively. CONCLUSION: Liquid nitrogen cryoablation using both 2.4D and 3.4D probes made iceballs that were of sufficient size, and effective temperatures were reached in both gel and the ex vivo pig lung.

Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome.

Pokeweed antiviral protein (PAP) isolated from Phytolacca americana is a ribosome-inactivating protein (RIP) that has RNA N-glycosidase (RNG) activity towards both eukaryotic and prokaryotic ribosomes. In contrast, karasurin-A (KRN), a RIP from Trichosanthes kirilowii var. japonica, is active only on eukaryotic ribosomes. Stepwise selection of chimera proteins between PAP and KRN indicated that the C-terminal region of PAP (residues 209-225) was critical for RNG activity toward prokaryotic ribosomes. When the region of PAP (residues 209-225) was replaced with the corresponding region of KRN the PAP chimera protein, like KRN, was active only on eukaryotic ribosomes. Furthermore, insertion of the region of PAP (residues 209-225) into the KRN chimera protein resulted not only in the detectable RNG activity toward prokaryotic ribosome, but also activity toward the eukaryotic ribosomes as well that was seven-fold higher than for the original KRN. In this study, the possibility of genetic manipulation of the activity and substrate specificity of RIPs is demonstrated.

Purification and characterization of UDP-glucose : curcumin glucoside 1,6-glucosyltransferase from Catharanthus roseus cell suspension cultures.

Catharanthus roseus cell suspension cultures converted exogenously added curcumin to a series of curcumin glucosides that possessed drastically enhanced water solubility. A cDNA clone encoding a glucosyltransferase responsible for glucosylation of curcumin to form curcumin 4'-O-glucoside was previously isolated, and in the present study a novel sugar-sugar glycosyltransferase, UDP-glucose:curcumin glucoside glucosyltransferase (UCGGT), was purified approximately 900-fold to apparent homogeneity from cultured cells of C. roseus. The purified enzyme (0.2% activity yield) catalyzed 1,6-glucosylation of curcumin 4'-O-glucoside to yield curcumin 4'-O-gentiobioside. The molecular weight and isoelectric point were estimated to be about 50 kDa and 5.2, respectively. The enzyme showed a pH optimum between 7.5 and 7.8. Both flavonoid 3-O- and 7-O-glucosides were also preferred acceptor substrates of the enzyme, whereas little activity was shown toward simple phenolic glucosides such as arbutin and glucovanillin, cyanogenic glucoside (prunasin) or flavonoid galactoside. These results suggest that UCGGT may also function in the biosynthesis of flavonoid glycosides in planta.

DNA authentication of Plantago Herb based on nucleotide sequences of 18S-28S rRNA internal transcribed spacer region.

Internal transcribed spacer (ITS) regions of nuclear ribosomal RNA gene were amplified from 23 plant- and herbarium specimens belonging to eight Plantago species (P. asiatica, P. depressa, P. major, P. erosa, P. hostifolia, P. camtschatica, P. virginica and P. lanceolata). Sequence comparison indicated that these Plantago species could be identified based on the sequence type of the ITS locus. Sequence analysis of the ITS regions amplified from the crude drug Plantago Herb obtained in the markets indicated that all the drugs from Japan were derived from P. asiatica whereas the samples obtained in China were originated from various Plantago species including P. asiatica, P. depressa, P. major and P. erosa.

An apoptotic inducer, aralin, is a novel type II ribosome-inactivating protein from Aralia elata.

We recently found that aralin, a novel cytotoxic protein consisting of two subunits, from Aralia elata selectively induces apoptosis in transformed cells as compared to normal cells. Here we report that aralin is a lectin specific for galactose (Gal) and its derivatives, and possesses RNA N-glycosidase activity as a new type II ribosome-inactivating protein (RIP). The RNA N-glycosidase activity of aralin was detected in cell-free and whole cell systems by the generation of an R-fragment from 28S rRNA. Coinciding with appearance of the R-fragment in aralin-treated cells, significant inhibition of protein synthesis was observed prior to the onset of apoptosis. Aralin-evoked cell death was efficiently repressed by the addition of Gal and its derivatives. Interestingly, melibiose preferentially protected normal cells from apoptosis as compared with transformed cells. Using rhodamine-coupled aralin, the aralin receptor could be clearly detected around the cell surface of transformed cells, but to a lesser extent on normal cells. Receptor binding was suppressed by Gal. These results indicate that aralin is incorporated into cells via its Gal-containing cell surface receptor and induces apoptosis through its RIP activity. Moreover, the expression level and/or structural changes of the aralin receptor may affect the sensitivity toward aralin.

Effect of N- and C-terminal deletions on the RNA N-glycosidase activity and the antigenicity of karasurin-A, a ribosome-inactivating protein from Trichosanthes kirilowii var. japonica.

Karasurin-A, from root tubers of Trichosanthes kirilowii var. japonica, is a type I ribosome-inactivating protein (RIP) that displays activity of RNA N-glycosidase to remove an adenine in the conserved sarcin/ricin loop of the largest RNA in the ribosome. We expressed recombinant proteins of karasurin-A and its various mutants with N- or C-terminal deletions in Escherichia coli as fusion proteins with maltose-binding protein (MBP), and compared their enzymatic activities and antigenicities. Muteins of karasurin-A generated by deleting either the first 100 N-terminal or the last 30 C-terminal amino acid residues lost activity of RNA N-glycosidase. The mutant proteins whose 80 N-terminal or 20 C-terminal amino acids were deleted could depurinate rRNA although the activities were decreased drastically. The antigenicities of the recombinant proteins were considerably reduced by deleting 20 amino acid residues from either N- or C-terminal regions.