A direct measurement method for the enzymatic determination of cholesteryl esters (CEs) without measuring total cholesterol (TC) and free cholesterol (FC) is described. In the first step, hydrogen peroxide generated by cholesterol oxidase from FC was decomposed by catalase. In the second step, CE was measured by enzymatic determination using a colorimetric method or a fluorometric method. The measurement sensitivity of the fluorometric method was more than 20 times that of the colorimetric method. Optimal conditions of the assay were determined, and examples of measured CE in human plasma, rat liver, and cultured cells are indicated. The method of directly measuring CE was simple and has exceptional reproducibility compared with the technique of subtracting FC from TC using each measured TC and FC. Free cholesterol (FC) has an important role as a component of cell membranes and a starting material for bile acid synthesis. However, cholesteryl ester (CE) is inactive when it is stored. In the progression of arteriosclerosis, CE accumulates in macrophages and smooth muscle cells and leads to the formation of foam cells. Determination of CE in cells or various tissues is of great importance in the fundamental research into atherosclerosis and the development of anti-atherosclerotic drugs.Several methods of enzymatic determination for FC and total cholesterol (TC) have been published (1-5). Measurement of TC has measured FC resulting from the decomposition of CE by cholesterol esterase, and FC was contained in the native sample. To measure CE, individual TC and FC are measured, and FC is then subtracted from TC (indirect assay). Measuring CE by indirect assay is difficult in a sample solution with a low ratio of CE to FC.This report describes a direct assay of CE by enzymatic determination without measuring TC and FC. In the first step, FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Hydrogen peroxide is decomposed into water and oxygen by catalase. At the second step, CE is measured by enzymatic determination using a colorimetric method or a fluorometric method. CE is hydrolyzed to FC by cholesterol esterase. FC is oxidized by cholesterol oxidase to yield the corresponding cholest-4-en-3-one and hydrogen peroxide. Thus, the enzymatic method for assaying FC is based on the measurement of hydrogen peroxide by way of peroxidase-coupled oxidation of hydrogen-sensitive probes. The hydrogen peroxide reacts with 4-aminoantipyrine or Amplex Red in the presence of peroxide to form a pigment or fluorescent products. The enzymatic reactions involved in the assay are as follows:CE content in human plasma, rat liver, and cultured cells was measured using this method, and its usefulness was evaluated.
A single intraperitoneal injection of the phenol-treated cells of Propionibacterium acnes into mice showed nonspecific resistance against subsequent lethal doses of an intraperitoneal challenge of Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes. The protection showed a biphasic pattern. The maximum protection, designated as the early phase protection, was seen in mice injected with P. acnes vaccine 1 to 2 days before the challenge, whereas the late phase protection was seen in mice vaccinated 16 to 22 days before the challenge. The activity of the reticuloendothelial system in mice after vaccination also showed a biphasic pattern with the peak on days 4 and 12. The delayed activation of the reticuloendothelial system lasted up to 3 weeks and coincided with the period of the late phase protection. The early phase resistance was markedly impaired by the treatment with hydrocortisone and carrageenan, but not by the treatment with anti-thymocyte serum, actinomycin D, or cyclophosphamide. The number of peritoneal polymorphonuclear leukocytes in vaccinated mice increased on days 1 to 2. The number of macrophages also increased at 2 to 21 days after vaccination and reached its maximum on day 14. Total activities of acid phosphatase, Nitro Blue Tetrazolium reduction, and the phagocytic activities of peritoneal exudate cells were also enhanced on and after day 1 after the injection of P. acnes vaccine.
Staphylococcal enterotoxin B (SEB), a toxin produced by Staphylococcus aureus, causes food poisoning and other fatal diseases by inducing high levels of pro-inflammatory cytokines. These cytokines are released from CD4+ T cells and major histocompatibility complex (MHC) class II antigen-presenting cells, which are activated through binding of wild-type (WT) SEB to both the MHC class II molecule and specific T-cell receptor Vbeta chains. Here, we focused on a trypsin/cathepsin cleavage site of WT SEB, which is known to be cleaved in vivo between Lys97 and Lys98, located within the loop region. To know the function of the cleavage, an SEB mutant, in which both of these Lys residues have been changed to Ser, was examined. This mutant showed prolonged tolerance to protease cleavage at a different site between Thr107 and Asp108, and structural analyses revealed no major conformational differences between WT SEB and the mutant protein. However, differential scanning calorimetric analysis showed an increase in enthalpy upon thermal denaturation of the mutant protein, which correlated with the speed of cleavage between Thr107 and Asp108. The mutant protein also had slightly increased affinity for MHC. In the in vivo experiment, the SEB mutant showed lower proliferative response in peripheral blood mononuclear cells and had lower cytokine-induction activity, compared with WT SEB. These results highlight the importance of the flexible loop region for the functional, physical and chemical properties of WT SEB, thus providing insight into the nature of WT SEB that was unrevealed previously.
A lividomycin-phosphorylating enzyme from a lividomycin-resistant strain of Escherichia coli carrying an R factor was partially purified by fractionation with ammonium sulfate and Sephadex G-100 column chromatography. The enzyme inactivated, in the presence of adenosine triphosphate and Mg'+, several antibiotics having a D-ribose moiety linked to 2-deoxystreptamine, i.e., lividomycin A and B, neomycin, paromomycin, and vistamycin, but did not inactivate the kanamycins, streptomycin, or the gentamicin C components. Chemical studies of the inactivated product suggested that the phosphorylated site of the inactivated lividomycin was the hydroxyl group of the D-ribose moiety.Since Okamoto and Suzuki (18) reported the enzymatic inactivation of antibiotics by a resistant strain of Escherichia coli carrying an R factor, many papers concerning drug inactivation by resistant strains have been published (2, 5-7, 9, 10, 12, 13, 15, 19-22, 24 (24) and Pseudomonas aeruginosa (10), because both LV-A (17) and gentamicin C (3) lack the hydroxyl group at the C-3 position of the D-amino-glucose moiety linked to 2-deoxystreptamine. It was found, however, that LV-A was inactivated by cell-free extracts from lividomycin (LV)-resistant strains of E. coli (24) and P. aeruginosa (11), and that the inactivated product was a monophosphorylated derivative of the drug. This paper deals with the phosphorylated site of LV and some properties of LV-phosphorylating enzyme from E. coli carrying an R factor. Washed cells were suspended in 30 ml of the same solution and disrupted through a French press under a pressure of 600 kg/Cm2. The suspension of disrupted cells was centrifuged at 105,000 X g for 60 min, and the supernatant fluid was designated as the S-105 fraction. The inactivating enzymes in the S-105 fraction were precipitated by ammonium sulfate at 33 to 66% saturation, collected by centrifugation, and dissolved in TMK solution. The solution was passed twice through a Sephadex G-100 column and developed with TMK solution. The fractions which showed LV-inactivating activity were collected and used as an enzyme solution. The protein concentration was adjusted to 1.0 mg/ml.
Carpetimycins A and B showed widely broad spectra and potent activity against gram-positive and gram-negative bacteria, including various species of anaerobic bacteria. The antimicrobial activity of carpetimycin A was 8 to 64 times greater than that of carpetimycin B and 4 to 128 times greater than that of cefoxitin. The inhibitory concentration of carpetimycin A required to inhibit more than 90% of clinical isolates was 0.39 ,ug/ml for Escherichia coli and Klebsiella and 1.56 ,ug/ml for Proteus and Staphylococcus aureus. At a concentration of 3.13 ,ug/ml, carpetimycin A inhibited almost all clinical isolates of Enterobacter and Citrobacter, which showed resistance to many clinically used ,B-lactam antibiotics. Carpetimycins A and B furthermore were shown to have potent inhibitory activities against several kinds of 1-lactamases produced by P-lactan-resistant strains; they inhibited not only penicillinase-type P-lactamases but also cephalosporinase-type P-lactamases, which were insensitive to clavulanic acid. In combination with P-lactam antibiotics such as ampicillin, carbenicillin, and cefazolin, carpetimycins A and B showed synergistic activities against P-lactam-resistant bacteria.
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