Characterization of monocyte adherence to human macrovascular and microvascular endothelial cells

The interaction of monocytes with cultured large vessel venous and arterial endothelial cells (EC) and with cultured microvascular EC was studied. Analysis of time-lapse microcinematographic video recordings showed that monocytes adhere rapidly to the surface of EC and subsequently remain spherical and fixed to the initial site of adherence. Some monocytes adherent to EC stretch out within 30 to 90 min and migrate over the EC surface or become stretched for about 10 to 30 min and then detach from the EC surface and move rapidly over the EC monolayer. It was shown that the interaction of monocytes with EC is dynamic, that the morphology of monocytes adherent to EC changes constantly, and that stretching of the monocytes over the surface of the EC is not an inevitable and irreversible consequence of binding. A quantitative adherence assay was developed in which both the morphology and the number of monocytes bound to EC were determined. For each type of EC the number of monocytes bound to a single EC was found to be linearly related to the number of monocytes added and was lower for smaller EC. The adherence of monocytes to venous and arterial EC followed a different time course than the adherence to capillary EC and adherence to both types of macrovascular EC was higher than adherence to microvascular EC was higher than adherence to microvascular EC. The percentage of adherent monocytes with a stretched morphology was lower when these cells were adherent to capillary EC than to both types of macrovascular EC and increased upon addition of serum. Adherence of monocytes to venous, arterial, and capillary EC was partially inhibited by mAb directed against the alpha-chain of lymphocyte function-associated Ag-1 or C3bi receptor (with mAb LM2/1, but not with mAb OKM1) and by mAb against the common beta-chain of the three leukocyte adhesion molecules. The degree of inhibition of monocyte adherence to EC by mAb against lymphocyte function-associated Ag-1 alpha and the common beta-chain was dependent on the type of EC and was higher for venous EC (57 to 70% inhibition) than for arterial (40 to 44% inhibition) and capillary (44 to 49% inhibition) EC. Inhibition of monocyte adherence obtained with anti-C3bi receptor-alpha mAb was similar for each EC type. mAb against p150, 95 did not affect adherence. None of the mAb could block binding completely; combinations of the mAb also did not result in increased inhibition of monocyte adherence to EC.     

Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.     

Complex between single-stranded DNA and gene 5 protein of bacteriophage M13 studied with linear dichroism and ultraviolet absorption

We have studied complexes between the gene 5 protein (gp5) of bacteriophage M13 and various polynucleotides, including single-stranded DNA, using ultraviolet absorption and linear dichroism. Upon complex formation the absorption spectra of both the protein and the polynucleotides change. The protein absorption changes indicate that for at least two of the five tyrosine residues per protein monomer the environment becomes less polar upon binding to the polynucleotides but also to the oligonucleotide p(dT)8. All gp5-polynucleotide complexes give rise to intense linear dichroism spectra. These spectra are dominated by negative contributions from the bases, but also a small positive dichroism of the protein can be discerned. The spectra can be explained by polynucleotide structures, which are the same in all complexes. The base orientations are characterized by a substantial inclination and propellor twist. The number of possible combinations of inclination and propeller twist values, which are in agreement with the linear dichroism results, is rather limited. The base orientations with respect to the complex axis are essentially different from those in the complex with the single-stranded DNA-binding protein gp32 of bacteriophage T4.     

Extreme differences in charge changes during protein evolution

Summary The maintenance of a proper distribution of charged amino acid residues might be expected to be an important factor in protein evolution. We therefore compared the inferred changes in charge during the evolution of 43 protein families with the changes expected on the basis of random base substitutions. It was found that certain proteins, like the eye lens crystallins and most histones, display an extreme avoidance of changes in charge. Other proteins, like phospholipase A2 and ferredoxin, apparently have sustained more charged replacements than expected, suggesting a positive selection for changes in charge. Depending on function and structure of a protein, charged residues apparently can be important targets for selective forces in protein evolution. It appears that actual biased codon usage tends to decrease the proportion of charged amino acid replacements. The influence of nonrandomness of mutations is more equivocal. Genes that use the mitochondrial instead of the universal code lower the probability that charge changes will occur in the encoded proteins.     

Effect of glutathione depletion on the cytotoxicity of xenobiotics and induction of single-strand DNA breaks by ionizing radiation in isolated hamster round spermatids

The role of glutathione (GSH) in cellular protection mechanisms in round spermatids from hamsters was studied. Isolated spermatids were largely depleted of GSH by treating the cells for 2 h with the GSH conjugating agent diethyl maleate (DEM). This treatment resulted in a 90% decrease of the cellular GSH content, but did not affect the ATP content. Exposure of isolated spermatids to cumene hydroperoxide (CHP), a compound which is detoxicated by the GSH redox cycle, showed that the cytotoxicity of the peroxide was markedly potentiated by GSH depletion of the cells. The cytotoxicity was reflected by the cellular ATP content. A decrease of the ATP content of the GSH-depleted spermatids was observed at 5-6-fold lower CHP concentrations, as compared to control cells. An increased cytotoxicity in GSH-depleted cells was also observed using 1-chloro-2,4-dinitrobenzene (CDNB), which is a reactive compound that is detoxicated by glutathione conjugation. The induction of single-strand DNA breaks by gamma radiation was 3-5-fold higher in GSH-depleted spermatids as compared to control cells. This radiation-induced damage was estimated under hypoxic conditions (500 p.p.m. O2 in N2). GSH depletion did not affect the repair of single-strand DNA breaks following the irradiation. The present results indicate that cellular GSH has an important function in the defence mechanisms of round spermatids against peroxides, electrophilic xenobiotics and radiation-induced DNA damage.     

Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells.

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.     

Isolation and partial characterization of an unusual human immunodeficiency retrovirus from two persons of west-central African origin.

An unusual human retrovirus was isolated from two patients with persistent generalized lymphadenopathy who originate from West-Central Africa and are currently residing in Belgium. Although the virus shared a number of the same biological and morphological properties as human immunodeficiency retrovirus type 1 (HIV-1) and HIV-2, significant antigenic differences could be demonstrated. Several of the viral proteins also differed in molecular weight from the corresponding HIV-1 and HIV-2 proteins. Partial chemical cleavage of the most highly conserved viral proteins resulted in patterns which differed from those of HIV-1 and HIV-2. Furthermore, nucleic acid hybridization experiments were capable of discriminating between the virus types. Sequence analysis of the viral U3 region revealed a unique enhancer organization not found in other immunodeficiency viruses. The data indicated that the new isolate is more closely related to HIV-1 than to HIV-2 but clearly differs in a number of important respects.     

Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells

Human umbilical vein endothelial cells in culture produce two chemotactic polypeptides when stimulated with LPS. The chemotactic factors could be purified to apparent homogeneity by HPLC techniques and were identified as 7.5-kDa and 15-kDa polypeptides by SDS-PAGE under nonreducing conditions. Both factors are potent chemotaxins for human neutrophils demonstrating half-maximal chemotaxis at 2 ng/ml and g ng/ml, respectively. In addition both peptides elicited release of azurophilic granule constituents when neutrophils were pretreated with cytochalasin B. Cross-desensitization experiments by using human neutrophils revealed cross-reactivities between both chemotaxins, not, however, with C5a or FMLP, indicating that both endothelial cell-derived neutrophil activating peptides (ENAP) are homologous. In addition, the 7.5-kDa factor (beta-ENAP) proved to be the quantitatively dominating and more potent chemotaxin as compared to the 15 kDa factor (alpha-ENAP). beta-ENAP shows biochemical and biologic similarities to monocyte- and lymphocyte-derived neutrophil-activating peptides MONAP and LYNAP, which recently were purified and sequenced.     

Nitrosylated high density lipoprotein is recognized by a scavenger receptor in rat liver

In order to assess the presence of specific recognition sites for high density lipoprotein (HDL) in vivo, HDL was nitrosylated with tetranitromethane and the decay and liver uptake were compared with that of native HDL. The association of intravenously injected nitrosylated HDL (TNM-HDL) with liver was greatly increased as compared to native HDL. Using a cold cell isolation method, it became evident that the liver endothelial cells were responsible for the increased uptake of the modified HDL. The involvement of the endothelial cells in the uptake of TNM-HDL from the circulation could also be demonstrated morphologically by using the fluorescent dye dioctadecyl-tetramethyl-indocarbocyanine perchlorate (Dil) to label HDL. In vitro competition studies with isolated liver endothelial cells indicated that unlabeled modified HDL and acetylated LDL displaced iodine-labeled TNM-HDL, while no competition was seen with LDL and a slight displacement was seen with unlabeled native HDL. Nonlipoprotein competitors of the scavenger receptor such as fucoidin and polyinosinic acid blocked the interaction of TNM-HDL with the liver endothelial cells. Also the degradation of TNM-HDL was blocked by low concentrations of chloroquine. It can be concluded that a scavenger receptor on liver endothelial cells is involved in the clearance of tetranitromethane-modified HDL, which excludes the possibility of using TNM-HDL in vivo to assess the non-receptor-dependent uptake of HDL. The use of nitrosylated HDL in vitro as a low affinity control is limited to cell types that do not possess scavenger receptors, because cell types with scavenger receptors will recognize and internalize TNM-HDL by a high affinity scavenger pathway.     

Muscle architecture and fibre characteristics of rat gastrocnemius and semimembranosus muscles during isometric contractions

Rat gastrocnemius medialis (GM) and semimembranosus (SM) muscles have a very different morphology. GM is a very pennate muscle, combining relatively short muscle fibre length with sizable fibre angles and long muscle and aponeurosis lengths. SM is a more parallel-fibred muscle, combining a relatively long fibre length with a small fibre angle and short aponeurosis length. The mechanisms of fibre shortening as well as angle increase are operational in GM as well as SM. However, as a consequence of isometric contraction, changes of fibre length and angle are greater for GM than for SM at any relative muscle length. These differences are particularly notable at short muscle lengths: at 80% of optimum muscle length, fibre length changes of approximately 30% are coupled to fibre angle changes of 15 degrees in GM, while for SM these changes are 4% and 0.6 degrees, respectively. A considerable difference was found for normalized active slack muscle length (GM approximately 80 and SM approximately 45%). This is explained by differences of degree of pennation as well as factors related to differences found for estimated fibre length-force characteristics. Estimated normalized active fibre slack length was considerably smaller for SM than for GM (approximately 40 and 60%, respectively). The most likely explanation of these findings are differences of distribution of optimum fibre lengths, possibly in combination with differences of myofilament lengths and/or fibre length distributions.