Biomedical science最新文献

A scheme for the partial purification of a Legionella pneumophila product possessing ADP-ribosyl-transferase and NAD-glycohydrolase activities is presented. The purification steps consisted of gel chromatography, ion-exchange, hydrophobic interaction chromatography, and chromatofocusing. The partially purified preparation modified eukaryotic components of molecular mass 20-25 kDa, which it is proposed are GTP-binding proteins. Addition of bivalent cations as well as ATP to the reaction buffer was necessary for ADP-ribosylation. NAD (50 microM) and nicotinamide (16 mM) greatly inhibited incorporation of ADP-ribose into acceptor proteins.

A spin-labelled fatty acid with an epr spectrum that is sensitive to the localization of the probe was used to show that albumin binds free fatty acids present in solution and also free fatty acids present in low density lipoproteins (LDL). Furthermore, albumin binds the thiobarbituric acid-reactive (TBA-reactive) products formed during the oxidation of linolenic acid, whereas the TBA-reactive substances formed during the oxidation of LDL are not bound by albumin. Linolenic acid bound to albumin essentially does not undergo peroxidation in the presence of ferrous ions, in contrast to a suspension of linolenic acid and LDL in which peroxidation occurs quite readily in the presence of ferrous ions. The highest rate of oxidation was found for linolenic acid alone. Albumin-bound spin-labelled fatty acid was essentially not reduced by ferrous ions, whereas free fatty acid or fatty acid incorporated into LDL was reduced quite rapidly, the highest rate of reduction being for free fatty acids. Thus the ability of fatty acids to undergo oxidation correlates with their accessibility to ferrous ions. The data obtained indicate that serum albumin is a relatively effective antioxidant in the blood and its mode of action is based on the immobilization of free fatty acids.

Lymphoid cell phenotype was investigated in the peripheral blood, synovial fluid, and synovial tissue of sixteen patients with rheumatoid arthritis (RA). In the peripheral blood of RA patients the proportion of cells expressing HLA-DR and beta 2-microglobulin receptors was higher than in normal controls, whereas the proportion of cells that were CD5+ (i.e. were T lymphocytes) was lower. Expression of the other cell surface antigens studied remained in the normal range. In the synovial tissue and synovial fluid of RA patients there was an increased percentage of cells expressing HLA-DR, beta 2-microglobulin receptors, CD25, CD5, CD4, and Thy-1, but the proportion of CD8+ cells was significantly decreased compared with that seen in peripheral blood. The CD4+/CD8+ ratio in RA joints was therefore significantly higher than that in peripheral blood. The proportion of cells expressing HLA-DR correlated with disease activity.

Water-soluble polymeric amide and azo derivatives of benzidine, based on the terpolymer N-vinylpyrrolidone-crotonic acid-p-crotonoylaminophenol, which possesses adjuvant activity, were synthesized. By means of a double diffusion reaction in agar and a complement-binding reaction it was found that immunization of rabbits with the benzidine-polymer antigens (with or without complete Freund's adjuvant) led to the formation of highly specific antibodies against the hapten benzidine. Sera from rabbits immunized with these conjugates of benzidine may thus be used for the detection of benzidine in the sera of workers exposed to this chemical.

Irradiation of human peripheral blood lymphocytes with an He-Ne laser (at 632.8 nm) at doses between 28 and 112 J m-2 caused changes in the chromatin during the first 6 h after exposure that were similar to those found after stimulation of the lymphocytes by phytohaemagglutinin (PHA), i.e. it increased chromatin accessibility to the low-molecular-mass ligand acridine orange (AO) and increased incorporation of the labelled RNA precursor [14C]uridine into the cells. The curves of AO-chromatin binding and RNA synthesis after either He-Ne irradiation with an He-Ne laser (56 J m-2) or PHA treatment were multipeak in nature. For the first 6 h after stimulation the curves for the two treatments were similar. After 7 h, the rate of RNA synthesis in laser-irradiated lymphocytes dropped to the control level, whereas in the PHA-stimulated cells [14C]uridine incorporation increased substantially. Unlike the case with PHA, treatment with an He-Ne laser did not induce resumption of DNA synthesis in lymphocytes. Lymphocytes irradiated by laser in the presence of interleukin-2 (IL-2) retained the level of labelled-thymidine incorporation characteristic of intact cells cultivated in the presence of IL-2. On the other hand, irradiation by an He-Ne laser produced a potentiating action on the response of peripheral blood lymphocytes to PHA, with thymidine incorporation being stimulated. This effect may explain the mechanism of wound healing by an He-Ne laser radiation: chromatin activation in the cells of wounds and ulcers makes these cells more responsive to the natural stimulators present in tissues.

It is well known that during certain pathological processes phagocytes acquire the ability to generate activated oxygen species during phagocytosis. The priming of phagocytes by cytokines and water-soluble products of lipid peroxidation (LPO) is described. Preincubation of human polymorphonuclear leukocytes (PMNL) with the water-soluble products of LPO or oxidised liposomes for 15-20 min at 37 degrees C enhanced their functional activity when they were stimulated by opsonised zymosan or latex particles. There was a 2-3-fold increase in luminol-dependent chemiluminescence response of cells stimulated in this way, and an increase in Fc-receptor expression on the PMNL surface. An endogenous cytokine alone did not activate the phagocytes for an oxidative burst response, but preincubation of murine peritoneal macrophages (MP) and human PMNL with cytokines (molecular mass 20-30 kDa) for 3-48 h at 37 degrees C enhanced the cell chemiluminescence response to opsonised zymosan by a factor of 5-9 for MP and a factor of 2-3 for PMNL. Treatment of phagocytes with the cytokine complex also increased other effector functions of the phagocytes such as tumouricidal activity, phagocytosis, secretion of interleukin-1, and antiparasitic activity. The protein synthesis inhibitor cycloheximide abolished cytokine-induced priming of MP (but not of PMNL). The mechanisms of short-term and prolonged priming of the two types of phagocytes (MP and PMNL) are discussed.

Thermal denaturation of myosin subfragment 1 (S1) isoforms from rabbit skeletal muscle containing the different alkali light chains A1 and A2 [S1(A1) and S1(A2), respectively] were studied by various methods. Turbidity measurements showed that thermally induced (heating rate 1 degrees C min-1) aggregation of S1(A1) occurs at lower temperatures than that of S1(A2). However, the temperature dependences of the tryptophan fluorescence spectrum and that for ATPase inactivation were the same for S1(A1) and S1(A2). Thermal denaturation of the S1 isoforms was also studied by differential scanning microcalorimetry with the 'successive annealing' method. Three independently melting cooperative regions (domains) were revealed in the molecules of both isoforms. Heat sorption curves for the S1 isoforms were different only for the most thermolabile domain, which had a maximum at 36 degrees C for S1(A1) and at 40.5 degrees C for S1(A2). Two other peaks had maxima at 46-47 degrees C and 50-51 degrees C for both isoforms. It is proposed that alkali light chains A1 and A2 differently affect the conformation of the most thermolabile domain, which probably does not contain trytophan residues and does not take part directly in the formation of the active site of the S1 ATPase.

The action of a new anticancer agent, the semisynthetic alkyl-phospholipid plasmanyl-(N-acyl)-ethanolamine (sPNAE), namely 1-O-octadecyl-2-oleoyl-sn-glycero-3-phospho-(N-palmitoyl)-ethanolamine, on protein kinase C (PKC) was investigated, and it was found to inhibit in a dose-dependent manner PKC isolated from mouse brain. The inhibition was competitive with respect to phosphatidylserine (K(i) = 20 microM). Lyso-PNAE, a possible cell metabolite of sPNAE, also inhibited PKC. A two-site model was used to calculate the binding affinity and the number of binding sites for phorbol ester in a culture of human melanoma BRO cells. The values of Kd, the dissociation constant, were K'd = 0.5 nM and K"d = 72 nM, whereas the values of Bmax, the number of binding sites, were B'max = 4.6 x 10(4) sites cell-1, and B"max = 2.9 x 10(5) sites cell-1. sPNAE was able to reduce the affinity of BRO cells for phorbol ester with almost no changes in the number of binding sites: K'd = 1.6 nM, K"d = 557 nM, and B'max = 4 x 10(4), B"max = 1.9 x 10(5). These data suggest that sPNAE may inhibit PKC in intact cells. Since various inhibitors of PKC may enhance the antiproliferative activity of cis-diamminedichloroplatinum(II) (cis-DDP), we investigated the effect of the combination of sPNAE and cis-DDP on the proliferation of BRO cells. sPNAE synergistically enhanced the antiproliferative activity of cis-DDP.

Intraperitoneal administration of dermorphine induces dose-dependent changes in the temperature of the body and the tail skin of rats. The character of these changes is largely determined by the ambient temperature, i.e. it depends on the initial functional state of the thermoregulation system. Pretreatment with naloxone reduces the dermorphine-induced effects on thermoregulation but does not eliminate them completely.