Forms of human serum high density lipoprotein protein

Delipidation by ethanol-diethyl ether at -10 degrees C of human serum high-density lipoprotein (HDL, d 1.063-1.21) or of its subclasses HDL(2) (d 1.063-1.120) and HDL(3) (d 1.120-1.21), yielded proteins-alphaP, alphaP(2), and alphaP(3)-containing 3% phospholipid (largely lecithin) and 3.3% carbohydrate (glucosamine:L-fucose:D-galactose, D-mannose:sialic acid, 1.00:41 : 0.56:0.31). Solubility data and analytical ultracentrifugal analyses indicated that, upon lipid removal, HDL protein aggregates readily; the aggregation is dependent upon pH and ionic strength of the solvent medium. Subunits of 21,000 mol wt were obtained by acetylation or addition of sodium dodecyl sulfate (SDS). HDL and alphaP elicited in the rabbit a similar immunological response. By agar gel immunoelectrophoresis both anti-HDL and anti-alphaP sera detected a major and two minor antigenic determinants in HDL, HDL(3), alphaP, alphaP(2), and alphaP(3). HDL(2), antigenically homogeneous, gave an immunoelectrophoretic pattern of HDL(3) upon mixing with alphaP. alphaP, alphaP(2), and alphaP(3) exhibited a single antigenic determinant after treatment with SDS (0.5 M) or upon acetylation. Native or delipidated forms of HDL, HDL(2), and HDL(3) were separated by vertical starch gel electrophoresis into several components, which showed identical reactions against anti-HDL or anti-alphaP sera. The data suggest that (a) the proteins of HDL, HDL(2), and HDL(3) are made of subunits, probably identical, of an average molecular weight of 21,000; (b) the difference in antigenic behavior between HDL(2) and HDL(3) is due to the presence in the latter of a lipid-poor protein; (c) antigenic polymorphism of alphaP is probably related to the presence in solution of monomeric and polymeric forms having different reactivity against anti-HDL and anti-alphaP sera.     

In vitro interaction between ceruloplasmin and human serum transferrin

The thermodynamics of the interactions of serum apotransferrin (T) and holotransferrin (TFe(2)) with ceruloplasmin (Cp), as well as those of human lactoferrin (Lf), were assessed by fluorescence emission spectroscopy. Cp interacts with two Lf molecules. The first interaction depends on pH and μ, whereas the second does not. Dissociation constants were as follows: K(11Lf) = 1.5 ± 0.2 μM, and K(12Lf) = 11 ± 2 μM. Two slightly different interactions of T or TFe(2) with Cp are detected for the first time. They are both independent of pH and μ and occur with 1:1 stoichiometry: K(1T) = 19 ± 7 μM, and K(1TFe2) = 12 ± 4 μM. These results can improve our understanding of the probable process of the transfer of iron from Cp to T in iron and copper transport and homeostasis.     

In vitro effect of air pollutants on human bronchi

A useful approach for constructing dose–response relationships and for studying the underlying mechanisms by which a xenobiotic agent enhances airway reactivity is to measure the response of an isolated airway following ex vivo exposure to a pollutant. We have in this way determined the dose–response relationship between ex vivo exposure to pollutants such as nitrogen dioxide (NO₂), the aldehyde acrolein, and ozone (O₃) and the reactivity to agonists in human isolated bronchial smooth muscle. We have also investigated the underlying alteration in the cellular mechanisms of airway smooth-muscle contraction induced by such exposure and found that it is related to alteration in calcium signaling at the site of the airway smooth-muscle cell. Finally, although there is epidemiological evidence that an increase in allergic diseases such as asthma may be linked to air pollution, there are few experimental data to address this issue. The final aim of this study was therefore to investigate the interaction between passive sensitization and exposure to pollutants in human isolated airways. We have examined (i) the effect of a pre-exposure to pollutants on the contraction of sensitized bronchi in response to a specific antigen and (ii) the effect of passive sensitization on the contraction in response to nonspecific agonists in bronchi pre-exposed to pollutants. The results indicate a combined effect of immunological sensitization and exposure to pollutants; that is, passive sensitization and exposure to pollutants act in a synergistic manner on human bronchial smooth-muscle reactivity in response to both specific antigens and nonspecific agonists. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of surface active agents on microbial culture

Summary The concentration of several surface active agents required to inhibit the initiation of growth ofAspergillus foetidus in both solid and liquid media was estimated. The culturing in liquid media involved both shake flask and fermenter cultures.     

In vitro alteration of association patterns of human acrocentric chromosomes

Summary Association patterns of the human acrocentric chromosomes are supposed to reflect the metabolic and structural behavior of interphase nucleoli. Attempts were made to alter the association patterns by the action of chemical and physical agents (glucose, actinomycin D, temperature), which presumably influence nucleolar ultrastructur and function.A decrease in association frequency was noted after doubling the concentration of glucose in the culture medium. This effect might be due to elevated synthetic activity and growth rate of PHA-stimulated lymphocytes in the presence of excess glucose. More pronounced changes were obtained by increasing the temperature to 41.5°C 6 hrs prior to harvesting. This finding probably reflects the well known ultrastructural and biochemical lesions caused by supranormal temperatures in the nucleoli of mammalian cells. Addition of actinomycin D did not yield any significant response. Due to the mechanism of action of this inhibitor, its application is restricted to a time interval starting late in G₂.

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Zusammenfassung Die Assoziationsmuster der menschlichen akrozentrischen Chromosomen werden als Ausdruck des metabolischen und strukturellen Verhaltens der Interphasennucleoli angesehen. Es wurde versucht, diese Muster durch Einwirkung chemischer und physikalischer Agentien (Glucose, Actinomycin D, Temperatur) zu verändern, von denen angenommen werden kann, daß sie die Funktion und die Ultrastruktur des Nucleolus beeinflussen.Verdoppelung des Glucosegehaltes im Kulturmedium bewirkte eine Verringerung der Assoziationshäufigkeit. Dieser Effekt beruht vermutlich auf einer vermehrten synthetischen Aktivität und erhöhter Wachstumsgeschwindigkeit bei hohen Glucosekonzentrationen. Deutlichere Veränderungen wurden durch Erhöhung der Temperatur, 6 Std vor dem Abbrechen der Kulturen, auf 41,5°C erzielt. Dieses Ergebnis steht wahrscheinlich mit den bekannten ultrastrukturellen und biochemischen Störungen in Zusammenhang, die man durch erhöhte Temperaturen im Nucleolus von Säugerzellen erzeugen kann. Mit Actinomycin D ergaben sich keine signifikanten Veränderungen der Assoziationsmuster. Auf Grund seines Wirkungsmechanismus ist die Anwendung dieses Inhibitors auf das Zeitintervall zwischen der späten G₂-Phase und der Mitose beschränkt.

     

In vitro selective effect of melphalan on human T-cell populations

Summary In vitro treatment with 2 g/2×10⁶ cells melphalan (l-PAM: l-phenylalanine mustard) significantly decreased the total number of T lymphocytes from peripheral blood (PBL) of healthy human donors and of the OKT₄ population (precursor suppressor/helper/inducer) T cells as defined by monoclonal antibodies OKT₃ and OKT₄, respectively. No changes in the OKT ₈ ⁺ lymphocyte population (cytotoxic/mature suppressor cells) were observed following the same treatment. Preincubation of PBL with l-PAM at concentrations that do not affect the rate of DNA synthesis in PHA-stimulated lymphocytes inhibited the generation of T suppressor lymphocytes by ConA, as shown by their effect on PHA stimulation. Treatment of allogeneic PBL with l-PAM had no effect on mature suppressor T cells already induced by Con A, as shown by incubation of PBL with l-PAM after incubation with ConA.     

In vitro effect of actinomycin D on human neutrophil function

The effect of actinomycin D (ACT-D) on human neutrophil chemotaxis, chemiluminescence (CL), superoxide (O2-) production, phagocytic uptake, and intracellular bacterial killing has been examined. The viability of the ACT-D-treated neutrophils was 98% even at a concentration of 10 micrograms/ml for 4 hr. Using fMLP as the chemotactic factor, depressed chemotaxis was demonstrated following ACT-D (1-10 micrograms/ml) pretreatment of neutrophils as compared with the non-treated controls. Similar ACT-D pretreatment produced the depressed responses in phorbol myristate acetate-induced CL and superoxide production by neutrophils. Moreover, using heat-inactivated human serum as an opsonin for Salmonella enteritidis (NCTC 6676), there was a significant difference in intracellular killing (P less than 0.01) but no difference in phagocytic uptake between ACT-D-treated and non-treated neutrophils. These studies indicate that ACT-D profoundly impairs both intracellular bacterial killing by human neutrophil through an effect on respiratory burst activity and directed cell migration of human neutrophils.     

In vitro effect of lead acetate on human erythropoietic progenitors

Lead is known to induce hematological disturbances resulting from abnormalities in cell differentiation and hemoglobin synthesis during hematopoiesis. The aim of the present work was to study human erythropoiesisin vitro in the presence of lead. Human erythroblastic progenitors, burst-forming units–erythroid (BFU-E), were exposed to lead acetate at increasing concentrations during 14 days of culture. Hematotoxicity was evaluatedin vitro according to proliferation and differentiation of cell colonies arising from BFU-E development. The ability of cells to synthesize proteins, porphyrins, and hemoglobin was measured by spectrophotometric tests and by high-pressure liquid chromatography (HPLC). Results showed that in the presence of 10^–3 mol/L lead acetate, no hemoglobinized cells were observed in culture and no fluorescent porphyrins were detected in cells. Up to 10^–3 mol/L, lead acetate is not cytotoxic, i.e., it does not induce cell destruction. The present work demonstrates that lead acetate interferes with the porphyrin synthesis of human erythroblastic progenitors in vitro. The decrease of porphyrin content with 10^–5 mol/L lead acetate suggest that δ-aminolevulinic acid dehydratase can be inhibited by lead acetate duringin vitro erythropoiesis. In vivo erythropoiesis occurs in the bone marrow. As about 95% of the body burden of lead in adults is located in the bones with a biological half-life of some years, the concentration of lead acetate found to block porphyrin synthesis in vitro has to be compared with in situ bone marrow lead concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antioxidative effect of citrus essential oil components on human low-density lipoprotein in vitro

We studied the antioxidative action to evaluate the effect of citrus essential oil components on human LDL in vitro. Among the authentic volatile compounds tested, gamma-terpinene showed the strongest antioxidative effect, and inhibited both the Cu(2+)-induced and AAPH-induced oxidation of LDL. gamma-Terpinene added after 30 min (mid-lag phase) and 60 min (propagation phase) of incubation of LDL with Cu(2+) inhibited LDL oxidation.     

In vitro protein production by the human endometrium

Protein secretion by the human endometrium was studied in vitro in medium after incubation of tissue minces (n = 10) or glands isolated by collagenase digestion (n = 4) from tissues obtained at the time of curettage from normal women. Samples were incubated in a serum-free medium for 24 h at 38 degrees C in the presence of radiolabeled methionine. Dialyzed medium from each sample was subjected to two-dimensional gel separation, and detected by protein staining. Although 5 of the 27 proteins were considered to be present in the labeling experiments by only one of the three observers, there was agreement about the presence of the 22 others. In addition, the observers categorized the proteins into three groups for purposes of analysis: a) those associated with the follicular phase of the cycle; b) those associated with the luteal phase; and c) those not cycle-related. One protein triplet, labeled #27, showed a significant relation to the luteal phase (p less than 0.01). A complete lack of similarity between the pattern of labeled proteins obtained from the medium and labeled proteins obtained from lysates of cells incubated in the same experiments makes it unlikely that cellular lysis, as opposed to secretion, contributed to the pattern of proteins studied in these experiments.     

In vitro inhibition effect of some coumarin compounds on purified human serum paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro effect of some hydroxy and dihydroxy ionic coumarin derivatives (1–20) on purified PON1 activity was investigated. Among these compounds, derivatives 11–20 are water soluble. In investigated compounds, compounds 6 and 13 were found the most active (IC₅₀?=?35 and 34?µM) for PON1, respectively. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.