Specificity of Tunel Method Depends on Duration of Fixation

Recent evidence has suggested that apoptosis plays an important role in various diseases, but concerns about the specificity of the TUNEL method for detecting apoptosis have been raised. The purpose of the present study was to investigate the specificity of the TUNEL method by using immersion and perfusion fixed tissues from both normal rats and rats with heart failure. Although a few positive cells were observed in perfusion fixed tissues, a significant number of positive cells were observed in immersion fixed tissues, especially when fixed tissues were kept for an extended time before the TUNEL assay was applied. The results of TUNEL staining should be interpreted with caution. When immersion fixation is used, fixed tissues should be assayed by the TUNEL method before the DNA degenerates.     

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone alpha-subunit and placental alkaline phosphatase in HeLa cells

Production of the glycoprotein hormone common alpha-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was alpha-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and alpha-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the butyrate-mediated induction of alpha-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6-24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-alpha. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2',5'-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of alpha-subunit mRNA. There was a good correlation between alpha-subunit accumulation and corresponding levels of alpha-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.     

Regulatory mechanism of delayed-type hypersensitivity in mice. V. Augmentor cells for DTH responses

Mice primed with 1 microgram of reduced and alkylated ovalbumin (RA-OA) developed not only long-lived memory cells for delayed-type hypersensitivity (DTH), capable of differentiating into DTH-effector T cells (DTH-Te) against ovalbumin (OA) when restimulated in vitro with OA, but also spleen cells capable of augmenting recipients' DTH responses to OA when transferred into cyclophosphamide (CY)-pretreated mice. The augmenting activity in spleen cells, upon transfer, was found 7 days, but not 21 or 91 days, after priming with RA-OA, although memory DTH-Te were present throughout the period of observation. The loss of augmenting activity after day 7 of priming was not due to the presence of suppressor cells; spleen cells taken 21 days after priming failed to suppress, upon transfer, the augmenting activity in 7-day-primed spleen cells as well as induction and expression of DTH responses to OA. When 7-day-primed spleen cells were fractionated on a discontinuous bovine serum albumin density gradient, the augmenting activity was found only in the medium-density-cell layer, although memory DTH-Te were separated in the high-density layer. Augmentation of DTH-Te generation could also be demonstrated in vitro when 7-day-primed spleen cells, but not 21-day-primed spleen cells, were added to cultures of spleen cells from CY-pretreated mice. These results indicate that, in the 7-day-primed spleen, there is an augmentor cell population which is different from memory DTH-Te and interacts with CY-resistant unprimed cells to facilitate DTH-Te generation.     

Action of Protoplast-bursting Factor upon Microorganism

Protoplast-bursting factor (P. B. factor) has a little antibacterial activity and is capable of inhibiting the growth of Bacillus megaterium.

The cell suspensions required P. B. factor and Mg⁺⁺ for the oxidation of glucose-6- phosphate but did not require them for that of glucose.

Leakage of various cellular components into the surrounding menstruum occured when the cell suspension was subjected to treatment with P. B. factor. These materials were identified as protein, deoxyribonucleic acid, ribonucleic acid, and amino acids.

Under an electron microscope, the cytoplasm of the cells treated with P. B. factor was apparently less dense than the control, which seemed to suggest that the cytoplasm had leaked out of the inside of the cell through the membrane by the treatment of P. B. factor.     

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.     

Algorithms and Complexity Analyses for Control of Singleton Attractors in Boolean Networks

A Boolean network (BN) is a mathematical model of genetic networks. We propose several algorithms for control of singleton attractors in BN. We theoretically estimate the average-case time complexities of the proposed algorithms, and confirm them by computer experiments. The results suggest the importance of gene ordering. Especially, setting internal nodes ahead yields shorter computational time than setting external nodes ahead in various types of algorithms. We also present a heuristic algorithm which does not look for the optimal solution but for the solution whose computational time is shorter than that of the exact algorithms.     

Possible mechanism of nuclear translocation of proteasomes

Proteasomes (multicatalytic proteinase complexes), which are identical to the ubiquitous eukaryotic 20S particles, are localized in both the cytoplasm and the nucleus, but the mechanism of their co-localization in the two compartments is unknown. On examination of the primary structures of subunits of proteasomes, a consensus sequence for nuclear translocation of proteins, X-X-K-K(R)-X-K(R) (where X is any residue), was found to be present in some subunits and to be highly conserved in the subunits of a wide range of eukaryotes. In addition, proteasomal subunits were found to bear a cluster of acidic amino acid residues and also a potential tyrosine phosphorylation site that was located in the same polypeptide chain as the nuclear location signal. These structural properties suggest that two sets of clusters with positive and negative charges serve to regulate the translocation of proteasomes from the cytoplasm to the nucleus, and that phosphorylation of tyrosine in certain subunits may play an additional role in transfer of proteasomes into the nucleus.     

Quantitative GSL-glycome analysis of human whole serum based on an EGCase digestion and glycoblotting method

Glycosphingolipids (GSLs) are lipid molecules linked to carbohydrate units that form the plasma membrane lipid raft, which is clustered with sphingolipids, sterols, and specific proteins, and thereby contributes to membrane physical properties and specific recognition sites for various biological events. These bioactive GSL molecules consequently affect the pathophysiology and pathogenesis of various diseases. Thus, altered expression of GSLs in various diseases may be of importance for disease-related biomarker discovery. However, analysis of GSLs in blood is particularly challenging because GSLs are present at extremely low concentrations in serum/plasma. In this study, we established absolute GSL-glycan analysis of human serum based on endoglycoceramidase digestion and glycoblotting purification. We established two sample preparation protocols, one with and the other without GSL extraction using chloroform/methanol. Similar amounts of GSL-glycans were recovered with the two protocols. Both protocols permitted absolute quantitation of GSL-glycans using as little as 20 μl of serum. Using 10 healthy human serum samples, up to 42 signals corresponding to GSL-glycan compositions could be quantitatively detected, and the total serum GSL-glycan concentration was calculated to be 12.1–21.4 μM. We further applied this method to TLC-prefractionated serum samples. These findings will assist the discovery of disease-related biomarkers by serum GSL-glycomics.     

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid—locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.