Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination

The effects of thermal treatment on Limulus amebocyte lysate (LAL) reagent were studied. Thermal resistances of enzymes and coagulogen in LAL reagent were evaluated by aggregometry and SDS-PAGE. Although enzyme activities of LAL reagent were completely lost after heating at temperatures above 60 °C for 10 min, gelating activities of coagulogen were retained even over 80 °C. Phenylmethanesulfonyl fluoride (PMSF; 1 mmol/mL), a strong non-specific serine-protease inhibitor, did not completely inactivate serine-protease activities of LAL. As a result, complete hydrolysis of coagulogen to coagulin was unexpectedly obtained. Solvent treatment of LAL was similar in effect to thermal treatment of LAL, but there were 2 problems: complete removal of solvent from samples and increased solution turbidity during preparation. To study the application of thermal-treated LAL, we conjugated it with titania particles. LAL-conjugated titania particles were obtained as small aggregates between titania nanoparticles and thermal-treated LAL (LAL-conjugated microbeads; LCM). When the mixture of LCMs and fresh LAL reagent was reacted with endotoxin an acute aggregation of LCMs was induced prior to the aggregate formation of LAL as monitored by stirring turbidimetry. This method, endotoxin microbeads aggregometry (EMA) may provide a rapid and sensitive method for endotoxin determination.     

Soft Nanotubes with a Hydrophobic Channel Hybridized with Au Nanoparticles: Photothermal Dispersion/Aggregation Control of C60 in Water

Simple glycolipid N‐alkaroyl‐β‐D‐glucopyranosylamine 1(n) selectively self‐assembles into sheets in water, nanotubes in alcohols, and helical nanocoils in toluene. All self‐assemblies consist of bilayer membranes in which 1(n) packed in an interdigitated fashion. The outer surface of the sheet is covered with the hydrophilic glucose headgroup of 1(n), whereas those of the nanotube and helical nanocoil are covered with the hydrophobic alkyl‐chain tail of 1(n). Heat treatment of the nanotube in the presence of water induces a rearrangement of the molecular packing of the outermost surface that allows the nanotube to become an effective nanocontainer for the dispersion of fullerene (C60) in water, a result of the ability of the hydrophilic outer surface of the nanotube and the hydrophobic nanochannel to encapsulate C60. The nanotube also exhibits photothermal characteristics after being hybridized with Au nanoparticles (AuNPs). The photothermal effect of the AuNPs allows the nanotube to unfold its tubular morphology and leads to compulsive release of the encapsulated C60 to the bulk water. Application of other nanotubes with similar photostimulated transformation ability should facilitate control of the dispersion/aggregation of other carbon nanomaterials, functional aromatic compounds, and drugs with low solubility in water.     

DC dielectric characteristics and conception of insulation designfor DC GIS

In order to discuss the dielectric performance of DC GIS, the flashover characteristics of gas-insulated bus were studied under conical- and disk-type spacers. The test involves the investigation on effect of electrification for charge to be accumulated in spacers and the effect of metallic particles. As the result, it was found that the dielectric characteristics of conical-type spacer are better than disk type. Moreover, the dielectric performance of DC GIS is more affected by the characteristics of coaxial electrode system than spacer surface with regard to metallic particle. On the basis of the results of these tests, this report presents the conception of insulation design for DC GIS     

Clinical evaluation of a reagent for detection of DNA of Mycobacterium tuberculosis complex using the ligase chain reaction (LCR) method

We evaluated the clinical efficacy of LCR MTB, a reagent developed by Abbott in the USA, in the full automatic ligase chain reaction (LCR) for detection of DNA of M. tuberculosis complex using a thermostable ligase. Using 458 samples isolated from patients with tuberculosis, LCR was compared with a smear method and with a culture method, and was also compared with two other methods of gene amplification, MTD and Amplicor, using 340 and 200 of the 458 samples, respectively. The LCR method detected M. tuberculosis in 49.8% (228/458) of the samples, and was superior to the smear method (31.9%, 146/458) and the culture method (39.1%, 179/458) in sensitivity. The LCR method was also superior to the MTD and Amplicor methods; sensitivity were 37.9% (129/340) for MTD vs. 47.6% (162/340) for LCR, and 56.5% (113/200) for Amplicor vs. 59.5% (119/200) for LCR. These favorable results and the convenience of the LCR method, which enables rapid detection of target genes with a high degree of sensitivity, strongly suggest that LCR MTB is useful as a reagent for detection of M. tuberculosis using nucleic acid amplification.     

Acute aortic dissection complicated with acute myocardial infarction--a case report of successful operation for the case of DOA

A 61-year-old woman who suddenly manifested chest and back pain was admitted after the diagnosis of acute type A dissection complicated with acute inferior myocardial infarction at another hospital. While being transported to our hospital by ambulance, her ECG repeatedly demonstrated ventricular fibrillation of cardiac arrest. She was in the state of dead on arrival (DOA) when arrived at our hospital. ECG demonstrated complete A-V block and cardiac arrest alternately. However, soon after right ventricular pacing was done, her blood pressure increased and she recovered consciousness. We therefore performed an emergency operation. We performed reconstruction of the ascending aorta and right coronary bypass grafting, since she had aortic dissection and conus brach avulsion. The postoperative course was uneventful.     

Membrane interaction of synthetic peptides related to the putative fusogenic region of PH-30 alpha, a protein in sperm-egg fusion

In order to investigate the relationship between structure and function of a putative fusogenic region of PH-30a, a protein active in sperm-egg fusion, two peptides, SFP22 and SFP23, whose sequences correspond to the residues 90-111 and 89-111 of PH-30 alpha, respectively, were chemically synthesized. An analog of SFP23, SFP23AA, which has an Ala-Ala sequence instead of the Pro-Pro sequence in SFP23, was also prepared. The CD study indicated that SFP22 and SFP23 mainly took a beta-structure in the presence of DPPC and DPPC/DPPG (3/1) vesicles, while SFP23AA showed an alpha-helical pattern though the alpha-helical content calculated was low (25-30%). alpha-Helical CD curve was observed for these peptides in trifluoroethanol. The membrane-perturbing activity of SFP22 and SFP23 was weaker than that of SFP23AA. On the other hand, the membrane-fusogenic activity of SFP22 and SFP23 to acidic phospholipid bilayers was much stronger than that of SFP23AA. All the peptides caused very weak cell lysis. These results are consistent with the reported speculation [Blobel, C. P. et al. (1992), Nature (London) 356, 248-252] that residues 90-111 of PH-30 alpha may be the fusogenic region and suggest that the Pro-Pro sequence is one of the important factors for holding the active secondary structure of the fusogenic region of PH-30 alpha in membranes.     

Modulation of motility and proliferation of glioma cells by hepatocyte growth factor

Invasive proliferation is a critical biological characteristic of gliomas. We evaluated the activities of hepatocyte growth factor (HGF) on proliferation and motility of glioma cells, comparing them with the effects of other growth factors (EGF, bFGF, PDGF-BB, TGF-beta 1). Seven primary culture lines all expressed c-met and HGF mRNA, and secreted HGF. HGF stimulated 3H-thymidine uptake of every glioma cell line (30 to 70% upregulation). Boyden chamber assay and scattering assay revealed that HGF promoted cell motility with chemokinetic and strong chemotactic activities. Concentric circle assay showed that HGF promoted two-dimensional expansion (proliferation and motility) most strongly among the growth factors studied. Further, we analyzed 23 paraffin-embedded sections of surgically resected gliomas (7 grade II, 8 grade III, and 8 grade IV) by immunohistochemistry. Expression of HGF and Met increased with malignant progression of gliomas, suggesting that gliomas stimulated their invasive proliferation by autocrine HGF production. Neurons and vasculature were HGF-positive, and Met-positive glioma cells gathered around them. The data indicate that neurons and vasculature, which are the main tracks of glioma invasion, augment chemotactic invasion and proliferation of gliomas by paracrine HGF secretion. Clearly HGF plays a critical role in invasive proliferation of glioma cells and it is therefore a candidate target of therapeutic intervention.