Improving cell-free protein synthesis for stable-isotope labeling

Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium l-glutamate (l-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium d-glutamate (d-Glu system). The productivity of the d-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the ¹H–¹⁵N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the d-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the d-Glu system. These results indicate that the d-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Distinct cell surface proteome profiling by biotin labeling and glycoprotein capturing

We performed here MS-based cell surface proteome profiling of HCT-116 cells by two distinct methods based on biotin labeling and glycoprotein capturing. In total, 742 biotinylated and 219 glycosylated proteins were identified by the biotin labeling and glycoprotein capturing, of which 224 and 138 proteins known to be located on plasma membrane were included, respectively, according to ingenuity pathway analysis. Although 104 plasma membrane proteins were identified by both methods, the rest of 154 were identified only by one. Almost all the identified plasma membrane proteins possessed consensus N-glycosylation sites, and proteins having various numbers of glycosylation sites were identified by both methods. Thus, the discrepancies of the identified proteins obtained from those two methods might not be only due to the number of glycosylation sites, but also to the expression and/or glycosylation level of the cell surface proteins. We also identified 312 N-glycosylated proteins from xenograft samples by glycoprotein capturing of which 135 were known as plasma membrane proteins. Although a number of highly-expressed plasma membrane proteins were common between culture and xenograft cells, some proteins showed culture- or xenograft-specific expression, suggesting that those proteins might contribute to grow in different environment.     

Phylogeny and Reclassification of Hemistasia phaeocysticola (Scherffel) Elbrächter & Schnepf, 1996

Hemistasia phaeocysticola is a marine flagellate that preys on diatoms and dinoflagellates among others. Although its morphology and ultrastructure were previously observed and characterized, its phylogenetic position has not been analyzed using molecular sequence data. This flagellate was classified as a kinetoplastid on the basis of the presence of a kinetoplast in the mitochondrion. However, several morphological characteristics similar to those of diplonemids, a sister group of kinetoplastids, have also been noted. Herein, we report that H. phaeocysticola branches within the diplonemid clade in the phylogenetic tree reconstructed by analyzing 18S rRNA gene sequences. Its systematic placement based on this finding is also discussed.     

Rapid induction of alpha-amylase by nongrowing mycelia of Aspergillus oryzae.

A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.     

Characteristics and gender differences concerning pulmonary hemodynamics in Clawn miniature pigs.

The characteristics and gender differences of the pulmonary hemodynamic parameters of 16 Clawn miniature pigs were examined and the data were compared with reports concerning dogs and other pig species. The pulmonary systolic, diastolic and mean arterial blood pressures of the mini-pig were slightly higher than those of the dog, respectively, but both the right atrial pressure and pulmonary capillary wedge pressure were within the normal physiological ranges of the dog. Concerning gender differences in hemodynamic parameters of the mini-pig, the female values, except the right atrial pressure, were slightly higher than those of the male, but no significant differences were recognized. The present study results will help pulmonary researchers understand the differences between Clawn miniature pigs and dogs for accurate analysis of experimental results.     

Sequence Analysis of 18S-28S rRNA Spacer Regions from Saccharomyces kunashirensis, S. martiniae, S. rosinii, and S. transvaalensis

Sequences of two internal transcribed spacer regions between 18S and 28S rRNA for recently described yeasts species, Saccharomyces kunashirensis, S. martiniae, S. rosinii, and S. transvaalensis, were determined to assess their phylogenetic relationship to the other Saccharomyces species. In the two phylogenetic trees constructed by the neighbor-joining method, independent branches reflected that delimitation of the four new species was valid. Received: 17 June 1998 / Accepted: 7 August 1998     

MIC-A allele profiles and HLA class I associations in Behçet's disease

Recently a new family of non-classical MHC molecules, the MHC class I chain-related protein (MIC), encoded by genes located in the major histocompatability complex have been identified. On the basis of the location of MIC genes and the structure and expression of MIC molecules it has been postulated that MIC may be a susceptibility factor in Behçet's disease (BD). We investigated the association of the 16 described external domain alleles and the transmembrane triplet repeats of MIC-A with BD in a Middle Eastern population. DNA from ninety-five patients and 102 age- and sex-matched controls were analyzed by polymerase chain reaction using allele specific primers. Our results show an increase of MIC-A*009 in the BD patient group 44/95 (46%) compared with controls 24/102 (24%) (χ²=11.3, OR=2.8, P=0.00078). MIC-A*009 was also found to be strongly associated with HLA-B51 in the patients 39/44 (88%) when compared with controls 10/24 (42%) (χ²=4, P=0.04). MIC-A*009 was also found in linkage disequilibrium with HLA-B52, but only in controls. The A6 form of a MIC-A transmembrane triplet repeat was found to be significantly raised in the patients (80/95; 84%;) compared with controls (58/102, 57%) (χ²=17.5, OR=4, P=0.000028). Although the MIC-A associations described are highly significant, the association with HLA-B51 independently remains the most significant factor (χ²=56.8, P<10^–6). The data suggests that as both MIC-A*009 and A6 are in strong linkage disequilibrium with HLA-B51, they are unlikely to be the susceptibility gene for BD but may be markers for additional risk factors.     

Geranylgeranylacetone induces apoptosis in HL-60 cells.

Geranylgeranylacetone (GGA) induces apoptosis in human leukemia HL-60 cells in a dose- and time-dependent manner. This effect was completely prevented by the pan-caspase inhibitor z-Val-Ala-Asp(OMe) fluoromethylketone, thereby implicating the caspase cascade in the process. Prior to DNA fragmentation, GGA treatment markedly activated caspase-3(-like) proteases, which might be responsible for the observed apoptosis. In addition, GGA treatment interfered with the processing and membrane localization of Rap1 and Ras, and these changes may be a result of apoptosis. Moreover, nitric oxide donors significantly accentuated the GGA-induced apoptosis, suggesting that the apoptotic pathway induced by GGA might be regulated by a redox-sensitive mechanism. Taken together, these data suggest that the isoprenoid, GGA, is an effective inducer of apoptotic cell death in HL-60 cells.