Alpha-tocopherol protects against monocyte Mac-1 (CD11b/CD18) expression and Mac-1-dependent adhesion to endothelial cells induced by oxidized low-density lipoprotein

Alpha-tocopherol supplementation is reported to protect against cardiovascular disease and to influence cells involved in atherogenesis, such as monocytes. Interactions between monocytes and vascular endothelial cells occur early in atherogenesis, and adhesion is mediated by integrins. We evaluated the effects of alpha-tocopherol on expression of Mac-1 (CD11b/CD18) by monocytes after stimulation with oxidized low-density lipoprotein (LDL), which is implicated as a potent chemotactic agent in atherogenesis. Incubation of whole blood with oxidized LDL (100 microg/ml) increased Mac-1 expression on monocytes, and preincubation with alpha-tocopherol reduced this upregulation in a concentration dependent manner. In another experiment, whole blood was obtained from healthy adult volunteers after 10 days of alpha-tocopherol administration (600 mg/day) and was incubated with oxidized LDL (100 microg/ml). There was a decrease in the upregulation of Mac-1 compared with that measured before administration. Adherence of oxidized LDL-stimulated monocytes to human umbilical vein endothelial cells was reduced by pretreatment with alpha-tocopherol, and was also inhibited by an anti-CD18 monoclonal antibody. Experiments with protein kinase C inhibitors suggested that reduction of Mac-1 upregulation by alpha-tocopherol was secondary to a decrease of protein kinase C activity. In conclusion, alpha-tocopherol suppressed the upregulation of Mac-1 expression on monocytes by oxidized LDL.     

Plasma cortisol in cattle: Circadian rhythm and exposure to a simulated high altitude of 5,000 m

After a 2 week control period at 400 m, cattle were exposed to 5,000 m simulated altitude for 2 weeks, which was followed by a 2-week post-altitude control period. Plasma cortisol values from blood samples taken every 30 min for a total of 24 h indicated that cortisol was secreted episodically and that a circadian rhythm existed. When cortisol values were grouped into 4, 6-h periods, plasma cortisol was most abundant from 06:00 to 12:00 h with an average of 0.96µ g/100 ml and least abundant from 00:30 to 06:00 h with an average of 0.55µ g/100 ml. Plasma cortisol increased from 0.42 to 3.08µ g/100 ml during the 4 h ascent to 5,000 m and decreased to near normal levels the following day. A rhythmic plasma cortisol pattern was maintained after one day at simulated high altitude.     

Preparation of lipid-free human hemoglobin by dialysis and ultrafiltration

Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid-free hemoglobin. High pressure liquid chromatography analysis of lipid-free hemoglobin (LFHB) indicates 99-100% protein purity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119-139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one-half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.     

Effect of the dithiocarbamate pesticide zineb and its commercial formulation,the azzurro. V. Abnormalities induced in the spindle apparatus of transformed and non-transformed mammalian cell lines

Abnormalities induced in the mitotic spindle by zineb and azzurro (1.0-25.0 micro g/ml, 24h) were evaluated in Chinese hamster ovary (CHO) and HeLa cells, and in non-transformed human fibroblasts (NTHF). Spindles were stained with FITC-conjugated anti-beta tubulin. Treatment with 10.0 micro g/ml of zineb induced complete inhibition of cell viability in NTHF cells while 10.0 micro g/ml of azzurro decreased cell growth down to 62%. Higher doses of both compounds induced cell death. In HeLa and CHO cells, 15.0 micro g/ml of zineb and 10.0-15.0 micro g/ml of azzurro decreased viability, whereas 25.0 micro g/ml of both compounds was cytotoxic. A significantly decreased mitotic index (MI) was observed in NTHF treated with 5.0 micro g/ml zineb or azzurro, whereas 10.0 micro g/ml of both chemicals were necessary to induce the same phenomenon in HeLa and CHO cells. Treatment with 1.0-5.0 micro g/ml of zineb or azzurro induced a dose-dependent increase of degenerated spindles in NTHF and the number of degenerated or multipolar spindles in HeLa and CHO cells increased in a dose-dependent manner with 1.0-10.0 micro g/ml zineb and azzurro. Although zineb and azzurro were able to induce mitotic spindle abnormalities in all cell types, non-transformed cells were less resistant than immortalized cells.     

Influence of the mycotoxins aflatoxin B1, rubratoxin B,patulin and diacetoxyscirpenol on the fermentation activity of baker's yeast

The fermentation activity of baker's yeast (measured by the amount of produced CO₂) is inhibited by 100µg/ml and 10µg/ml aflatoxin B₁, and by 100µg/ml and 10µg/ml diacetoxyscirpenol. Lower concentrations of these mycotoxins as well as of rubratoxin B enhance the fermentation. Only 0.001µg/ml aflatoxin B₁, 0.00001µg/ml diacetoxyscirpenol and 0.01µg/ml rubratoxin B are without effect or slightly inhibitory. Patulin in all concentrations tested does not influence the CO₂ production significantly. Cytochemical studies show that the enzyme alcohol dehydrogenase is inhibited by 100µg/ml and enhanced by 1µg/ml and 0.1µg/ml aflatoxin B₁. It is suggested that the influence of at least aflatoxin B₁ on the fermentation activity of the yeast cells is due to an interaction with alcohol dehydrogenase. It is possible that the activity of other enzymes of yeast is also influenced by mycotoxins.     

Mutagenic and genotoxic evaluation of bisphenol F diglycidyl ether (BFDGE) in prokaryotic and eukaryotic systems

The epoxy resin bisphenol F diglycidyl ether (BFDGE), was examined for its mutagenicity in prokaryotic assays (Salmonella typhimurium His(-) and Escherichia coli Trp(-) tests) and its genotoxicity in eukaryotic systems (sister chromatid exchange (SCE) and micronucleus tests in human lymphocytes), in the presence or absence of an exogenous metabolizing system (S9 from rat liver). In the prokaryotic tests, the concentrations of BFDGE ranged between 100 and 5000 micro g per plate, and in the eukaryotic assays from 12.5 to 62.5 micro g/ml. The compound is able to induce mutagenic effects in bacterial strains TA100, TA1535, WP2uvrA and IC3327, as revealed by the increase observed in the number of induced revertants. With respect to the genotoxicity assays, BFDGE induces an increase in the frequency of sister chromatid exchanges and micronuclei in human peripheral blood lymphocytes.     

Sensitive fluorimetric method for the determination of putrescine,spermidine and spermine by high-performance liquid chromatography and its application to human blood

A fast and sensitive method for the determination of putrescine, spermidine and spermine by high-performance liquid chromatography is described. These compounds are converted to their fluorescent dansyl derivatives and are separated by a reversed-phase chromatographic system (Micropak CH-10) with water and acetonitrile as mobile phase. The sensitivity of the method is 30 pmoles.The application of the method to the determination of polyamines in blood is described. It was found that most of the polyamines circulating in blood are localized in the erythrocytes, their content in normal human blood being spermidine 14.1 ± 3.1, and spermine 8.4 ± 2.8 nmoles/ml packed erythrocytes. Putrescine is not present in normal human erythrocytes. The polyamine level in serum is less than 0.1 nmole/ml.The polyamine content of the erythrocytes from patients with malignant neoplasm was significantly elevated.     

Gangliosides of human, bovine, and rabbit plasma

Gangliosides were isolated from human, bovine, and rabbit plasma and were quantified by gas-liquid chromatography. Purification was achieved by sequential use of partitioning in solvents, DEAE-Sephadex chromatography, base treatment, and silicic acid chromatography. Human and bovine plasma yielded slightly more than 1 micro mole of lipid-bound sialic acid/100 ml; for rabbit plasma the value was 0.28 micro mole/100 ml. The total bovine plasma ganglioside fraction contained equal amounts of N-acetylneuraminic and N-glycolylneuraminic acids, rabbit plasma gangliosides had about 1% of the latter, and the human plasma sample contained only the former. Thin-layer chromatography revealed important differences among the plasmas from the three species, but all possessed hematosides and hexosamine-containing gangliosides. The approximate ratios of these two categories, based on sialic acid content, were (hematosides: hexosamine-type): human, 2:1; rabbit, 3:2; and bovine, 2:3. The fatty acid compositions of both categories were characteristic of extraneural gangliosides and included six major acids: palmitic, stearic, behenic, tricosanoic, lignoceric, and nervonic. The major long-chain base in each sample was sphingosine, while only a trace of the C(20) isomer was detected.     

The Large Scale Purification of Ubiquitin from Human Erythrocytes

A simple, reproducible method for the large-scale purification of active ubiquitin from human erythrocytes is described. Erythrocytes contain 100 μg free ubiquitin per cc of packed cells, of which 44% can be recovered in homogeneous form by a combination of heat treatment, ammonium sulfate fractionation, and ion exchange chromatography.     

Quantitative evaluation of the effects of human carcinogens and related chemicals on human foreskin fibroblasts

Ten compounds representative of diverse classes of chemicals were evaluated for their cytotoxicity and transforming ability to human skin fibroblasts in vitro. Only five of the ten compounds were highly cytotoxic in the 0-100 µg/ml range and their order of cytotoxicity was: 2,5-bis(1-aziridinyl)-3,6-bis(carboethoxyamino)-1,4-benzoquinone (AZQ) > cis platin > bis(chloromethyl)ether (BCME) > acrylonitrile > afatoxin BI (AFBI). The other five compounds, afatoxin B2 (AFB2), methylmethacrylate, 1-naphthylamine (1-NA), 2-naphthylamine (2-NA), and cyclophosphamide, exhibited less than 40% inhibition of colony formation even at 100 µg/ml of the compound (the maximum concentration of AFB2 used was 50 µg/ml due to its low solubility). Anchorage-independent growth of exposed cells in soft agar was used as a biological endpoint for the expression of chemical transformation. AFB₁ had strong transforming ability, whereas AFB₂ was a weak transforming agent. The transforming abilities of acrylonitrile, AZQ, BCME, cis-platin, methylmethacrylate and 2-NA ranged between those of AFBI and AFB2. 1-NA also induced the soft agar growth property in the treated cells even though this compound has not been shown to be carcinogenic. AFB₁, AZQ, cisplatin, cyclophosphamide and 1-NA exhibited a dose dependent increase in soft agar growth frequency for at least three consecutive concentrations. The data suggest that anchorage-independent colony forming ability of exposed cells is a reliable marker to measure the carcinogenic potential of various hazardous chemicals.Abbreviations AZQ 2,5-bis(1-aziridinyl)-3,6-bis(carboethoxyamino)-1,4-benzoquinone - AFB aflatoxin B₁ - AFB2 aflatoxin 132 - AI anchorage independent - B[a]P benzo[a]pyrene - BCME bis(chloromethyl)ether; cis-platin, cis-diammine-dichloroplatinum - CM complete medium - E.D.50 effective dose which produced 50% cytotoxicity - CP cyclophosphamide - HNF human neonatal foreskin - HPLC high pressure liquid chromatography - 1-NA 1-naphthylamine - 2-NA 2-naphthylamine - PDL population doubling     

Effect of ibuprofen on monocyte activation by liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (CGP 19835A): Can ibuprofen reduce fever and chills without compromising immune stimulation?

The purpose of this study was to determine the effects of ibuprofen on the ability of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) to activate human blood monocytes in vitro. We undertook these experiments because the major toxic side-effects following L-MTP-PE infusion, fever and chills, could be prevented when ibuprofen was given orally immediately before L-MTP-PE infusion. It was therefore important to determine whether ibuprofen interfered with the macrophage-activation properties of L-MTP-PE. Peripheral blood monocytes were isolated from normal donors, then incubated with L-MTP-PE in the presence or absence of ibuprofen. The cytotoxic properties of the monocytes were assessed by a radioisotope-release assay against A375 cells. Ibuprofen at dose levels of 40 µg/ml suppressed the generation of the cytotoxic phenotype but did not interfere with the killing process once the cells were activated. Interleukin-1 (IL-1) and tumor necrosis factor (TNF) production, as well as the mRNA expression of these cytokines, was suppressed by 40 µg/ml ibuprofen. Since IL-1 and TNF play a crucial role in the cytotoxic function of monocytes, these findings may explain the mechanism by which ibuprofen inhibited the generation of the cytotoxic phenotype by L-MTP-PE. By contrast, ibuprofen dose levels up to 10 µg/ml had no effect on the generation of monocyte-mediated cytotoxicity by L-MTP-PE and no effect on the production, secretion, or mRNA expression of TNF and IL-1. Therefore, we concluded that if ibuprofen is to be used to control the side-effects of L-MTP-PE, blood levels of up to 10 µg/ml are desirable. In two of three patients, we determined that an oral dose of 200 mg given immediately before L-MTP-PE infusion could achieve these desired blood levels.     

Direct sensitivity test of the MB/BacT system

In order to evaluate the direct-method test of sensitivity to drugs used in the principal tuberculosis treatment regimes, in the Organon Teknika MB/BacT system, we tested 50 sputum samples positive to microscopy taken from patients with pulmonary tuberculosis and with clinical indications for an antibiogram, admitted sequentially for examination during the routine of the reference laboratory. The material was treated v/v with 23% trisodium phosphate solution, incubated for 24 h at 35 degrees C, and neutralized v/v with 20% monosodium phosphate solution. The material was then centrifuged and the sediment inoculated into flasks containing Rifampin - 2 micro g/ml, Isoniazid - 0.2 micro g/ml, Pyrazinamide - 100 micro g/ml, Ethambutol - 2.5 micro g/ml, Ethionamide - 1.25 micro g/ml, and Streptomycin - 2 micro g/ml. The tests were evaluated using the indirect method in the BACTEC 460 TB (Becton Dickinson) system as the gold standard. The results showed that the Rifampin test performed best, i.e., 100% sensitivity at 95% Confidence Interval (82.2-100) and 100% specificity at 95% Confidence Interval (84.5-100), followed by Isoniazid and Pyrazinamide. In this experiment, 92% of the materials showed a final reading in 30 days; this period represents the time for primary isolation as well as the results of the sensitivity profile, and is within Centers for Disease Control and Prevention recommendations regarding time for performance of the antibiogram. The inoculated flasks showed no contamination during the experiment. The MB/BacT is shown to be a reliable, rapid, fully automated nonradiometric system for the tuberculosis antibiogram.     

A sensitive cyclic nucleotide phosphodiesterase assay for transient enzyme kinetics

A rapid and sensitive method was developed for the quantitative determination of alpha-tocopherol in tissues and plasma of rats and mice. Tissue and plasma were extracted in acetone and chromatographed on a reverse-phase C18 column with 2% water in methanol. Fluorescence and ultraviolet detection were used for tissue and plasma alpha-tocopherol levels, respectively. Extraction of tissues and plasma was found to be more complete in acetone than in other solvent systems analyzed. The average recovery of alpha-tocopherol added to tissue samples was 97%. As little as 0.1 g of tissue or 0.1 ml plasma can be accurately used for analysis. The method is sensitive to 0.05 micrograms alpha-tocopherol/g tissue.     

Measurement of protein concentration by quantitative electron microscopy

The method of quantitative electron microscopy was applied to the measurement of protein concentration in thin sections. The human erythrocyte was selected as a model because of its apparently uniform protein concentration. Phosphotungstic acid (PTA) in aqueous solution was used as a reversible stain for protein, and PTA-stained Dowex resin spheres were embedded along with the red cells as standards for measurement of section thickness. The mass of stain removed from a given area of sectioned red cell by buffer (pH 7.4) was measured by quantitative electron microscopy. From the stoichiometry of the reaction between PTA and red cell protein established in this study, the amount of protein present in the measured area was calculated. From this amount of protein and the measured thickness, the concentration of protein was calculated and expressed as g/100 ml, for comparison with the clinical laboratory value for hemoglobin. Groups of red cells from the same sample were measured on 3 different days and their mean values (g/100 ml ± SD) were 29 ± 3.9, 30 ± 2.7, and 33 ± 4.6, compared to the clinical laboratory value of 32.1 g/100 ml packed cells, after correction for volume change and protein loss during fixation.     

Effects of insulin on glucose metabolism in isolated human fat cells

Isolated fat cells were used for the study of in vitro effects of insulin on glucose metabolism in human and rat adipose tissue. In human subcutaneous fat cells, effects of insulin could be detected at concentrations of glucose in the medium from 1 to 10 micro moles/ml. Cellular responsiveness was inversely proportional to the glucose level. At a constant concentration of 6 micro moles of glucose per ml, the effects of insulin at various concentrations up to 500 micro U/ml were investigated. At the highest concentration, which gave the maximal response, there was a 100% increase in the conversion of glucose-U-(14)C to glyceride-glycerol and a 40% increase in glucose oxidation. The dose-response curve was steepest between 2 and 20 micro U/ml. Rat epididymal fat cells were much more responsive to insulin. Glucose lipogenesis and pentose cycle activity could also be demonstrated in rat cells, whereas these activities could not be shown in fat cells from human omental and subcutaneous tissue. The findings for human cells are attributed to changes in cellular activity during preparation.     

Detection and measurement of opium alkaloids and metabolites in urine of opium eaters by methane chemical ionization mass fragmentography

A gas chromatographic—mass spectrometric assay for eight opium alkaloids in human urine following opium ingestion is described. The compounds were extracted from urine with methylene chloride—isopropanol (7:3, v/v) at pH 9.5, evaporated, derivatized with Tri-Sil Z and analyzed by methane chemical ionization mass fragmentography. The method is sensitive to ca. 0.01 μg/ml for morphine and codeine and ca. 0.05 μg/ml for the other compounds. Adsorption problems on the gas chromatography column prevented obtaining reproducible results for the measurement of noscapine. Extraction efficiencies over the pH range of 8–11 for the eight compounds are reported. Retention times of the opium alkaloids were determined using five different liquid phases (3%) on Gas-Chrom Q (100–120 mesh) and two column lengths (36 cm and 183 cm). The 36-cm column packed with OV-210 was selected for use in the assay. Ions were selected for monitoring for each component from their methane chemical ionization spectrum to provide the needed sensitivity and specificity for analysis of a multi-component mixture. The assay was used for the analysis of an “opium eater's” urine. Morphine, codeine, nomorphine, norcodeine and noscapine were detected; however, no evidence was obtained for thebaine, papaverine or oripavine. Unconjugated morphine (0.64 μg/ml) was present at nearly twice the concentration of codeine (0.37 μg/ml) and normorphine and norcodeine were present in equal amounts (ca. 0.15 μg/ml).     

Pectins in the fruits of Japanese quince (Chaenomeles japonica)

The pectin content, composition and physico-chemical properties were studied in the fruits of two genotypes of Japanese quince. On average, the fruits contained 11 g pectins/100 g dry fruit and 1.4 g pectins/100 g fresh fruit. A sequential extraction was used to isolate the pectins from the fruits. The cells from the flesh were examined using a confocal laser scan microscope, fresh and after each extraction step of the sequence. The dilute acid conditions were the most efficient for pectin extraction. Pectins extracted by water or potassium oxalate had higher (>600 ml/g) intrinsic viscosities than the pectins extracted by dilute acid (<400 ml/g). Anionic exchange chromatography was performed on the acid-extracted pectins. They were constituted of four populations, the first one being mainly composed of arabinans, the second one of homogalacturonans, the third one of rhamnogalacturonans. The composition of the last one varied with the genotype studied.     

Inter- and intra-individual variation in plasma and red blood cell vitamin E after supplementation

To establish the range of individual blood responses to supplemental vitamin E, 30 healthy subjects ingested 75 mg of deuterium-labelled alpha-tocopherol with a standard breakfast. Blood was collected at 6, 9, 12, 27 and 51 h post ingestion and deuterated (d6) and non-deuterated (do) alpha-tocopherol concentrations were determined in plasma and red blood cells (RBC) by GC-MS. To examine intra-individual responses, 6 of these subjects were re-examined at 6-month intervals over a 30-month period. Post ingestion, the amount of d6-alpha-tocopherol in blood increased rapidly with time with maximal concentrations seen at 12 h (plasma) and 27 h (RBC) in most subjects. At these times, d6-alpha-tocopherol concentration ranged from 0.3-12.4 micromol/l in plasma and 0.6-4.09 micromol/l packed cell in RBC. Area under the curve calculations indicated inter-individual differences of alpha-tocopherol uptake to be 40-fold for plasma (12.9-493.3 micromol h/l) and 6-fold for RBC (24.4-146.1 micromol h/l packed RBC). Intra-individual variation in alpha-tocopherol uptake was small in comparison and remained relatively constant over the 30-month period. We conclude that vitamin E uptake varies widely in the normal population, although it is comparatively stable for an individual over time. These differences likely arise from variations in the regulation of vitamin E uptake and metabolism between subjects. Factors regulating this process must be better understood before the optimal intake of vitamin E can be ascertained.     

Oxidative modulation of NF-kappaB signaling by oxidized low-density lipoprotein

Oxidized low-density lipoprotein (oxLDL) modifies macrophage inflammatory responses in the pathogenesis of atherosclerosis. In the present study, we focused on gamma-glutamylcysteine synthetase (gamma-GCS), a rate limiting enzyme of glutathione synthesis, and examined whether inflammatory stimulation of gamma-GCS gene in macrophages by lipopolysaccharide (LPS) is modified when the cells were exposed to oxLDL. We found that the nuclear factor-kappaB (NF-kappaB)-mediated induction of gamma-GCS by LPS (100 ng/ml) was suppressed by a 48-h pre-treatment with oxLDL (50 micro/ml), and this was due to a decrease in the DNA-binding activity of NF-kappaB. Furthermore, pre-treatment with oxLDL caused a carbonylation of NF-kappaB subunit p65. With alpha-tocopherol, the oxLDL-induced carbonylation of proteins decreased with a restoration of DNA-binding activity of NF-kappaB. Together, these indicate that oxidative modification of NF-kappaB suppresses LPS-induced expression of gamma-GCS gene in ox-LDL-treated cells, suggesting an implication of oxLDL-induced modulation of NF-kappaB signaling with atherosclerosis.