Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a DNA repair domain containing the dihydrofolate reductase gene in Chinese hamster ovary cells

The formation and removal of UV-induced pyrimidine dimers were measured in restriction fragments near and within the essential dihydrofolate reductase (DHFR) gene in Chinese hamster ovary cells in order to map the genomic fine structure of DNA repair. Dimer frequencies were determined at 0, 8, and 24 h after irradiating the cells with 20 J/m2 UV light (254 nm). Within 8 h, the cells had removed more than 40% of the dimers from sequences near the 5' end of the gene, somewhat fewer from the 3' end, but only 2% from the 3' flanking region and 10% from a region upstream from the gene. The corresponding extent of repair in the genome as a whole is 5-10% in the 8-h period. Isoschizomeric restriction enzyme analysis was used to detect the level of methylation in the fragments in which repair was measured. We found that the only hypomethylated sites in and around the DHFR gene were in the fragment near its 5' end, which displayed maximal DNA repair efficiency. The size of the region of preferential DNA repair at the DHFR locus appears to be in the range of 50-80 kilobases, and this finding is discussed in relation to genomic domains and the structure of mammalian chromatin.     

Pathogenicity of Fusarium oxysporum Isolates from Malaysia and F. oxysporuin f. sp. elaeidis from Africa to Seedlings of Oil Palms (Elaeis guineensis)

Pathogenicity tests with Fusarium oxysporum isolated form Malaysian oil palm were made with oil palms seedlings raised form Malaysian seed as well s with wilt-susceptible seedlings gown from African seed. Oil palm seedlings grown form Malaysian seed were also inoculated with African isolates of F. oxysporum f. sp. elaeidis and F. oxysporum var. redolens. The experiments were made under normal soil moisture conditions and under water stress. F. oxysporum f. sp. elaeidis isolates form Africa were pathogenic to oil palm seedlings from Malaysian seeds but the Malaysian F oxysporum isolates were non-pathogenic to plams grown from Malaysian seed or the wilt-susceptible palms from African seed. Seedlings from Malaysian seed proved to be highly susceptible to the vascular wilt disease caused by F. oxysporum f. sp. elaeidis as 75–90% of the palms were infected. The susceptibility of the palms from Malaysian seed varied with different African isolates tested. The Yaligimba isolate from Zaire which was found to be F. oxysporum var. redolens was the most virulent. Disease was more severe when oil palm seedlings were subjected to a period of water stress. The incidence of death in the seedlings under stress conditions was 45% as compared with only 15% for palms grown under normal conditions.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Modification of glutathione levels in C3H/10T1/2 cells and its relationship to benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide-induced cytotoxicity

Levels of reduced glutathione (GSH) in C3H/10T1/2 cells were selectively altered to determine what quantitative role GSH transferase-catalyzed conjugation plays in regulating the cytotoxic effects of benzo(a)pyrene anti-7,8-dihydrodiol 9,10-epoxide (r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene, anti-diol epoxide). A 65% decrease in 10T1/2 cell GSH content from 0.16 mM (control cell GSH concentration) to 0.06 mM was accompanied by a 46% decrease in the anti-diol epoxide LD80; a 98% increase in GSH content resulted in a 44% increase in anti-diol epoxide LD80. This nonlinear relationship between changes in cellular GSH concentration and anti-diol epoxide LD80 was directly relatable to the nonlinear change in the rate of anti-diol epoxide conjugation which was catalyzed by 10T1/2 cell GSH transferases. Purified 10T1/2 cell cytosol catalyzed the GSH conjugation of anti-diol epoxide to yield a GSH conjugation product with a distinct UV absorbance spectrum; the apparent GSH Km for this cell cytosol-catalyzed reaction was 0.20 mM. Variations in the cellular GSH concentration around the GSH Km resulted in a nonlinear change in the amount of anti-diol epoxide-GSH conjugate formed, and a reciprocal change in the amount of free anti-diol epoxide available for cytotoxic alkylation events. These results clarify in quantitative, biochemical terms how GSH transferase-catalyzed conjugation can regulate the level of an electrophilic carcinogen metabolite in a biological system.     

The ultrastructural localization of two basement membrane components: entactin and laminin in rat tissues

The localization of two noncollagenous components of basement membranes, laminin and entactin, was determined in rat kidney, muscle, and small intestine using electron immunohistochemistry. In the renal glomerulus anti-laminin antibodies reacted with the basement membrane of peripheral capillary loops and with mesangial matrix. In the peripheral capillary loop laminin was preferentially distributed in both laminae rarae. This was in contrast to anti-entactin that localized in peripheral capillary loops but not in mesangial matrix. Even in the peripheral capillary loops it had a different distribution than laminin. Entactin was found predominantly in the lamina rara interna. In renal tubular basement membranes both antibodies localized throughout the full thickness of the basement membranes, with laminin having a preferential distribution in the lamina rara, whereas entactin was more evenly distributed. In the basement membrane of the duodenal mucosa entactin localized in the lamina densa, whereas laminin was present in both laminae. In skeletal muscle both antibodies had similar localization in all basement membranes. These results demonstrate that entactin is an intrinsic component of basement membranes. They also demonstrate that basement membranes from different tissues have subtle variations in content and/or assembly of the different components. It is likely that these variations may be reflected in different functional properties.     

Effect of platelet-activating factor on airway vascular permeability: possible mechanisms

We studied the effects of the potent inflammatory mediator, platelet-activating factor (PAF), on vascular permeability in airways (and other tissues) of guinea pigs by measuring extravasation of circulating Evans blue dye. PAF caused a dose-dependent increase in vascular permeability. At 1 ng/kg iv, PAF caused an increase in Evans blue extravasation of 220% (P less than 0.05) in the trachea, with the greatest effect at a dose of 100 ng/kg (858%; P less than 0.01). Histamine (150 micrograms/kg iv) caused a 320% increase over base line in the trachea and 200% in main bronchi; this effect was equivalent to that induced by 10 ng/kg PAF in the trachea and 1 ng/kg in main bronchi. The duration of effect of PAF was greatest in main bronchi (less than 10 min). Platelet depletion with a cytotoxic antibody, or the cyclooxygenase inhibitor, indomethacin, or the cyclooxygenase-lipoxygenase inhibitor, BW 7556, did not affect the vascular permeability response to PAF. The PAF-receptor antagonist, BN 52063, inhibited Evans blue extravasation in the airways in a dose-dependent manner, with complete inhibition at 5 mg/kg. Thus PAF-induced airway vascular leakage is mediated by specific receptors but not by products of arachidonic acid metabolism or by platelets. Increased airway microvascular leakage induced by PAF may lead to plasma extravasation and airway edema, factors that may contribute to the airway narrowing and hyperresponsiveness induced by PAF.     

A simple purification of calmodulin by reversed phase chromatography with hydrophobic polymer 3520

A new adsorption chromatography procedure for the purification of calmodulin from bovine brain was developed using polymeric adsorbent 3520. Calmodulin was first isolated by DEAE-Cellulose column chromatography and further purified to apparent homogeneity following elution with 50% ethanol from the adsorbent column. Polyacrylamide gel electrophoresis showed one band either in the presence of Ca2+ or EGTA. The polymeric adsorbent 3520 is a non-polar polymer lacking exchangeable groups. The selective adsorption of calmodulin is based on hydrophobic interaction within the matrix, and is Ca2+ independent. Neither high salt (0.5 M NaC1) nor EGTA (5 mM) was able to elute the CaM from the adsorption column whereas ethanol (50%) eluted it completely. This method is simple to use and it provides highly purified calmodulin with high yield.     

The effect of trifluoroperazine on the sarcoplasmic reticulum membrane

The inhibitory effect of trifluoroperazine (25-200 microM) on the sarcoplasmic reticulum calcium pump was studied in sarcoplasmic reticulum vesicles isolated from skeletal muscle. It was found that the lowest effective concentrations of trifluoroperazine (10 microM) displaces the Ca2+ dependence of sarcoplasmic reticulum ATPase to higher Ca2+ concentrations. Higher trifluoroperazine concentrations (100 microM) inhibit the enzyme even at saturating Ca2+. If trifluoroperazine is added to vesicles filled with calcium in the presence of ATP, inhibition of the catalytic cycle is accompanied by rapid release of accumulated calcium. ATPase inhibition and calcium release are produced by identical concentrations of trifluoroperazine and, most likely, by the same enzyme perturbation. These effects are related to partition of trifluoroperazine ino the sarcoplasmic reticulum membrane, and consequent alteration of the enzyme assembly within the membrane structure, and of the bilayer surface properties. The effect of trifluoroperazine was also studied on dissociated ('chemically skinned') cardiac cells undergoing phasic contractile activity which is totally dependent on calcium uptake and release by sarcoplasmic reticulum, and is not influenced by inhibitors of slow calcium channels. It was found that trifluoroperazine interferes with calcium transport by sarcoplasmic reticulum in situ, as well as with the role of sarcoplasmic reticulum in contractile activation.