The response of pulmonary vascular endothelial cells to monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro and in vivo.

Monocrotaline pyrrole (MCTP) causes pulmonary vascular endothelial cell (EC) injury followed by progressive pulmonary vascular leak in vivo and the inhibition of EC proliferation in vitro. It was hypothesized that MCTP inhibits cell proliferation in vitro by interfering with cell cycle progression in a cycle phase-specific manner. Furthermore, it was proposed that early alterations in MCTP-induced lung injury leading to hypertension were associated with a similar inhibition of EC proliferation. Subconfluent cultures of bovine pulmonary artery endothelial cells (BECs) were synchronized with aphidicolin (APH), a reversible G1-S phase inhibitor. Upon removal of APH, BECs were exposed to MCTP (5 micrograms/ml) or its vehicle for a 4-h interval corresponding to either the G1-S, S-G2, or G2 through mitosis (M) phases of the cell cycle. Fluorescence-activated cell sorting (FACS) was used to identify MCTP-induced changes in cell cycle progression in BECs, and the transit of S phase cells through the cycle was characterized through the incorporation of bromodeoxyuridine (BrdU). Synchronized BECs exposed to MCTP between mid-S-G2 or G2 through M were briefly delayed in G2-M at 12 h but underwent cell division by 24 h. By contrast, BECs treated with MCTP immediately after release from APH block became arrested in G2-M at 24 h and showed evidence of continued DNA synthesis and hypertetraploidy, but they did not divide. In vivo, MCTP (3.5 mg/kg i.v.) administration caused an increase in arterial EC BrdU incorporation between Days 3 and 7, but no increase in EC density. During this same interval, pulmonary vascular permeability increased and persisted. In summary, MCTP inhibits cell proliferation in a cell cycle phase-dependent manner in vitro. The results suggest that a similar mechanism could occur in vivo and may be associated with delayed EC repair, a process that could contribute to persistent pulmonary vascular leak.

Common links in the structure and cellular localization of Rhizobium chitolipooligosaccharides and general Rhizobium membrane phospholipid and glycolipid components.

Several common links between the structural chemistry of the chitolipooligosaccharides of Rhizobium and the general rhizobial membrane lipid and lipopolysaccharide chemistry of these bacteria have been uncovered. Aspects of common chemistry include sulfation, methylation, and the position and extent of fatty acyl chain unsaturation. We find that bacteria which are known to synthesize sulfated chitolipooligosaccharides (such as Rhizobium meliloti strains and the broad-host-range Rhizobium species strain NGR234) also have sulfated lipopolysaccharides. Their common origins of sulfation have been demonstrated by using mutants which are known to be impaired in sulfating their chitolipooligosaccharides. In such cases, there is a corresponding diminution or complete lack of sulfation of the lipopolysaccharides. The structural diversity of the fatty acids observed in the chitolipooligosaccharides is also observed in the other membrane lipids. For instance, the doubly unsaturated fatty acids which are known to be predominant components of R. meliloti chitolipooligosaccharides were also found in the usual phospholipids and glycolipids. Also, the known functionalization of the chitolipooligosaccharides of R. sp. NGR234 by O- and N-methylation was also reflected in the lipopolysaccharide of this organism. The common structural features of chitolipooligosaccharides and membrane components are consistent with a substantial degree of biosynthetic overlap and a large degree of cellular, spatial overlap between these molecules. The latter aspect is clearly demonstrated here since we show that the chitolipooligosaccharides are, in fact, normal membrane components of Rhizobium. This increases the importance of understanding the role of the bacterial cell surface chemistry in the Rhizobium/legume symbiosis and developing a comprehensive understanding of the highly integrated membrane lipid and glycolipid chemistry of Rhizobium.

Paradoxical increase in DNA cross-linking in a human ovarian carcinoma cell line resistant to cyanomorpholino doxorubicin.

The cyanomorpholino analog of doxorubicin (MRA-CN) is a potent cytotoxic agent which is known to cross-link DNA. A human ovarian carcinoma cell line, ES-2, was grown in increasing concentrations of MRA-CN from 0.1 to 0.5 nM. The resultant resistant subline, ES-2R, was 4-fold resistant to MRA-CN. DNA damage and repair in response to MRA-CN were compared in the parental and resistant cell lines using alkaline elution. DNA cross-links were detectable after 3-h incubation of the cells at 37 degrees C in MRA-CN at concentrations greater than or equal to 1.0 nM. Paradoxically, 2-fold more cross-links were detected in the ES-2R cells as compared with the ES-2 cells. This paradoxical difference in cross-links between the 2 cell lines was observed to increase with time of exposure to 2.5 nM of MRA-CN. Non-protein-associated DNA strand breaks were also detected in the 2 cell lines after exposure to 2.5 nM of the drug. The ES-2 cells consistently showed twice as many breaks as the ES-2R cells, which could explain the paradoxical higher apparent DNA cross-linking observed with the ES-2R cells after exposure to MRA-CN. Studies of the time course of cross-link repair after exposure to MRA-CN revealed that 75% of the DNA cross-links disappeared in the ES-2R cells by the end of 8 h in drug-free medium. In contrast, cross-links in the ES-2 cells were undetectable after 4 h, which coincided with a progressive increase in DNA strand breaks. The topoisomerase II level in the ES-2 cells was 2- to 4-fold higher than that in the ES-2R cells. However, proteinase K treatment of the lysed cells did not increase the number of apparent strand breaks produced by MRA-CN, suggesting that topoisomerase II may not be involved. These findings indicate that, in addition to DNA cross-linking, MRA-CN causes DNA strand breakage. Resistance to MRA-CN in the ES-2R cells is associated with more apparent DNA cross-linking and less DNA strand breakage, which may be a consequence of differences in DNA repair and/or nonspecific DNA degradation between the resistant and the sensitive cell lines.

Induction of erythroid differentiation in human leukemic K-562 cells by membrane-directed action of adriamycin covalently bound to microspheres.

Covalent coupling of Adriamycin (ADR) to polyglutaraldehyde microspheres focuses the drug action to the plasma membrane which leads to growth inhibition and also to induction of erythroid differentiation in human leukemic K-562 cells without any evidence of cellular internalization of the drug-microsphere complexes. As observed with the free drug, a reduction in cell growth by the coupled drug correlated with a recruitment of differentiating cells. Treatment of sensitive cells with ADR-microspheres results in 40% of cells containing hemoglobins as determined by benzidine staining at 87% growth inhibition. Similar treatment of ADR-resistant cells produces 24% of benzidine-positive cells at 72% growth inhibition. Furthermore, free and coupled forms of ADR stimulate heme synthesis 12- and 20-fold. Hemoglobin analysis of ADR-polymer induced cells demonstrates additional embryonic (Gower-2, X, Portland) and fetal (F) types of hemoglobin in comparison to uninduced cells which synthesize only small amounts of Gower-1 in sensitive cells and Gower-1 plus hemoglobin X in resistant cells. In addition, free and bound forms of Adriamycin differ markedly in the relative proportion of hemoglobin types that they induce. Free and coupled forms of ADR produce an increase in the gamma-globin mRNA synthesis in sensitive K-562 cells. These results demonstrate that both ADR-sensitive and -resistant K-562 cells can be induced to differentiate at the cell surface by ADR-microspheres and that this induction differs qualitatively from that of free ADR.

Semantic priming in the lexical decision task: roles of prospective prime-generated expectancies and retrospective semantic matching.

In semantic priming paradigms for lexical decisions, the probability that a word target is semantically related to its prime (the relatedness proportion) has been confounded with the probability that a target is a nonword, given that it is unrelated to its prime (the nonword ratio). This study unconfounded these two probabilities in a lexical decision task with category names as primes and with high- and low-dominance exemplars as targets. Semantic priming for high-dominance exemplars was modulated by the relatedness proportion and, to a lesser degree, by the nonword ratio. However, the nonword ratio exerted a stronger influence than did the relatedness proportion on semantic priming for low-dominance exemplars and on the nonword facilitation effect (i.e., the superiority in performance for nonword targets that follow a category name rather than a neutral XXX prime). These results suggest that semantic priming for lexical decisions is affected by both a prospective prime-generated expectancy, modulated by the relatedness proportion, and a retrospective target/prime semantic matching process, modulated by the nonword ratio.

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.

Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein.

The principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) is located in the external envelope protein, gp120, and has previously been mapped to a 24-amino acid-long sequence (denoted RP135). We show here that deletion of this sequence renders the envelope unable to elicit neutralizing antibodies. In addition, using synthetic peptide fragments of RP135, we have mapped the neutralizing determinant to 8 amino acids and found that a peptide of this size elicits neutralizing antibodies. This sequence contains a central Gly-Pro-Gly that is generally conserved between different HIV-1 isolates and is flanked by amino acids that differ from isolate to isolate. Antibodies elicited by peptides from one isolate do not neutralize two different isolates, and a hybrid peptide, consisting of amino acid sequences from two isolates, elicits neutralizing antibodies to both isolates. By using a mixture of peptides of this domain or a mixture of such hybrid peptides the type-specificity of the neutralizing antibody response to this determinant can perhaps be overcome.