Nitroarene concentrations and direct-acting mutagenicity of diesel exhaust particulates fractionated by silica-gel column chromatography

Diesel exhaust particulates were extracted with benzene-ethanol (3:1, v/v) and separated into five fractions by silica-gel column chromatography. Direct-acting mutagenic activity was assayed by the Ames test using the Salmonella typhimurium YG1024 strain. The total activity of five fractions was about four times greater than that of the crude extract, suggesting that the activities in the fractions were suppressed in the crude extract. Strong activity was observed in fraction 4 which was eluted with dichloromethane (61.5% of the total activity) and fraction 5 which was eluted with ethanol (35.3%). Nitropolycyclic aromatic hydrocarbons (NPAHs) were determined by high-performance liquid chromatography with chemiluminescence detection. They were found mainly in fraction 4, although one NPAH was in fraction 3 which was eluted with n-hexane-dichloromethane (3:1, v/v). Based on these results, 53.1% of the activity in fraction 4 was attributed to NPAHs. The contribution of 1-nitropyrene and 1,3-, 1,6- and 1,8-dinitropyrenes was great and that of the other NPAHs was small. The mutagenic compound in fraction 5 was not identified. Fractions 1 and 2, which were eluted with n-hexane, and fraction 3 suppressed the activity of fraction 4. Polycyclic aromatic hydrocarbons in fractions 2 and 3 were considered as possible suppressors of NPAHs.     

BLM (the causative gene of Bloom syndrome) protein translocation into the nucleus by a nuclear localization signal

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, sun sensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS, and BLM protein is homologous to DNA helicase. There are two putative nuclear localization signals (NLSs) within amino acid residues 1334-1349 in the C-terminus of the BLM protein, which has the distinctive structure of two basic residue arms separated by a spacer. The entire coding or deleted BLM sequences of various sizes were ligated into an enhanced green fluorescent protein (EGFP) vector and transfected into HeLa cells. The EGFP vector harboring the entire BLM coding sequence was transported to the nucleus. The BLM protein truncated at 1341 amino acid, containing an intact helicase domain and only one proximal arm, was not transported to the nucleus. The BLM protein truncated at 1357 amino acid, containing an intact helicase domain and two arms, was transported to the nucleus. The EGFP vector harboring DNA fragments encoding a protein having only the distal arms of basic amino acids in the C-terminus was also transported to the nucleus. The truncated BLM proteins corresponding to previously reported mutated BLM proteins were retained in the cytoplasm or both the cytoplasm and the nucleus as was the EGFP vector with no insert. These results show that the BLM protein translocates into the nucleus and that the distal arm of the bipartite basic residues in the C-terminus of the BLM protein is essential for targeting the nucleus.     

In-situ observation of faceted growth of benzophenone single crystals

Melt growth of benzophenone was in-situ observed under various cooling rate and temperature gradient. The melt growth experiments were performed for various cooling rate of melt and various temperature gradients. The movement of growth interface with time was measured for various growth faces such as (001), (101) and (011) and the growth rates of the respective faces were calculated. It was found that the growth rate of all the faces increased with cooling rate of the melt. Steady state growth was observed in the experiments with low temperature gradient while, the steep gradient leads to unstable growth of crystal. The observed growth rate variation was explained using the attachment energy model.     

Induced albumin secretion from HepG2 spheroids prepared using poly(ethylene glycol) derivative with oleyl groups

We developed a poly(ethylene glycol) (PEG) derivative with oleyl groups, so-called “cell adhesive”, for the promotion of human hepatocellular carcinoma HepG2 cell spheroids. Our approach was based on crosslinking of the cell membrane with a cell adhesive via a hydrophobic interaction. A cell adhesive, PEG derivative with hydrophobic oleyl groups at both ends was synthesized and characterized. HepG2 spheroids formed when the adhesive was added to cell suspensions. The size of the spheroids increased with time in culture. In addition, Ammonia elimination of HepG2 spheroid with cell adhesive was 3.4 times higher than that without cell adhesive. Furthermore, albumin secretion from HeG2 spheroids grown with the cell adhesive for 7 days was 3.3 times that from HepG2 spheroids grown without cell adhesive. Fluorescence microscopy showed greater albumin staining in spheroids grown with cell adhesive compared with spheroids grown without adhesive. This cell adhesive may be useful not only for single type of cells but also for multi types of cells to form artificial organs. This cell adhesive will be a key material for liver tissue engineering when it will apply to primary hepatocytes.     

Microfluidic device with chemical gradient for single-cell cytotoxicity assays

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.     

The effect of pituitary adenylate cyclase activating polypeptide (PACAP) on amylase secretion from guinea pig pancreatic acini

Beta-lactamase production is one of the major mechanisms of resistance amongst bacteria especially the enteric bacilli. The purpose of this study is to assess the in-vitro activity of Sulperazon, a combination of cefoperazone and an irreversible beta-lactamase inhibitor, sulbactam, against the cefoperazone resistant isolates of aerobic gram-negative bacilli. A total of 92 such strains were tested. It was found that at a concentration of < or = 8 mg/l of sulbactam added to cefoperazone 82% of Klebsiella spp, 100% of E. coli, 100% of Enterobacter spp, 33% of Pseudomonas aeruginosa, 67% of Pseudomonas spp and 62% of Acinetobacter spp that were resistant to cefoperazone alone were susceptible to the combination. Hence it is concluded that the addition of sulbactam to cefoperazone does expand the spectrum of the in-vitro activity of cefoperazone.     

Tumor necrosis factor alpha-mediated tumor regression by the in vivo transfer of genes into the artery that leads to tumors

We report that tumor necrosis factor (TNF) alpha induced a strong antitumor immune reaction when it was produced in arteries leading to tumors by gene transfer in vivo. We used a mouse model carrying a sarcoma-180 tumor in the right footpad and injected the fusogenic liposomes encapsulating the human TNF-alpha gene into the right femoral artery. Under this condition, human TNF-alpha was detected only in the artery leading to the tumor and in the tumor. There was a significant regression in tumor growth when the TNF-alpha gene was delivered into the right femoral artery, with 4 of 11 mice completely cured. No regression was observed when the TNF-alpha gene was delivered into the left femoral artery or into the tumor or when the luciferase gene was administered. Tumor regression was inhibited by the injection of anti-TNF-alpha, anti-CD4, or anti-CD8 monoclonal antibody, and CD8+ T cells accumulated in the tumors of TNF-alpha-treated mice. These results suggest that TNF-alpha expressed locally in the arteries leading to tumors efficiently suppresses tumor growth through reinforcement of an antitumor immune reaction. The significance of this phenomenon for cancer gene therapy was discussed.     

Discrepancy between surgeon''s binocular parallax perception and manipulation in the neurosurgical operation

The present study was undertaken after the detection of one case of cutaneous leishmaniasis with presumed infection in one of the three remaining wooded areas in the urban area of the city of Maringá, Southern Brazil; also in view of the lack of knowledge about sand flies and their behavior. From June to September, 1995, sand flies were caught with Falc?o traps during the night in the remaining wooded areas (Parque do Ingá, Bosque Dois and Horto Florestal). A total of 2,907 sand flies were caught in Parque do Ingá; 1,723 of them were aught in forest traps and 1,184 in wild animal shelter traps at the zoo. The results show that Lutzomyia whitmani is better adapted to the three areas under study and that it frequently occurs in wild animal shelters within the urban perimeter of Maringá.