Identification of a novel membrane protein, HP59, with therapeutic potential as a target of tumor angiogenesis.

CM101, a polysaccharide isolated from the culture medium of Group B streptococcus, a neonatal pathogen, targets pathological angiogenesis and inhibits tumor growth in mice and humans. CM101 also targets neonatal lung and adult sheep lung endothelial cells. A gene encoding a transmembrane protein that interacts with CM101 was isolated from a sheep lung endothelial cell cDNA library. The gene, termed sp55, encodes a 495-amino acid polypeptide. COS-7 cells transfected with a vector containing sp55 express the SP55 protein-bound CM101 in a concentration-dependent manner. Stably transfected CHO cells also bound CM101. The corresponding human gene, hp59, was isolated from a human fetal lung cDNA library and had a predicted identity to SP55 of 86% over 495 amino acids. HP59 protein was shown by immunohistochemistry to be present in the pathological tumor vasculature of the lung, breast, colon, and ovary, but not in the normal vasculature, suggesting that the protein may be critical to pathological angiogenesis. The hp59 gene and/or the HP59 protein was not expressed in a variety of normal tissues, but was significantly expressed in human fetal lung, consistent with the pathophysiology of Group B streptococcus infections in neonates. Mice immunized with HP59 and SP55 peptides showed significant attenuation of tumor growth. Immunization effectively inhibited both the tumor angiogenesis and vasculogenesis processes, as evidenced by lack of both HP59- and CD34-positive vessels. These results and the immunohistochemistry data suggest a therapeutic potential for the CM101 target protein HP59 both as a drug target and as a vaccine against pathoangiogenesis.

Ethanol increases endothelial nitric oxide production through modulation of nitric oxide synthase expression.

Moderate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO.

Identification of authentic estrogen receptor in cultured endothelial cells. A potential mechanism for steroid hormone regulation of endothelial function.

BACKGROUND: Estrogen plays a major role in the delayed expression of coronary heart disease (CHD) in women, and recent data indicate that postmenopausal estrogen therapy reduces the incidence of CHD by > 40%. The mechanism or mechanisms through which estrogen exerts this benefit are unknown, although effects on blood pressure, carbohydrate and lipid metabolism, and coagulation have been suggested. We hypothesized that at least part of the effect of estrogen in reducing the incidence of CHD is due to an effect on endothelial cell function. METHODS AND RESULTS: In the present study, we examined human aortic and umbilical vein endothelial cells and bovine aortic endothelial cells for the presence of estrogen receptors (ERs) through immunoblot and mRNA analyses. Electrophoretic mobility shift assays were also performed to determine the DNA-binding properties of the putative ERs. Nuclear extracts from all three endothelial cell types were found to contain a 67-kD protein that reacted with anti-ER monoclonal antibodies specific to different domains of the ERs. Each of these types of cells expresses proteins that bind specifically to consensus estrogen-responsive elements. Finally, Northern blots verified that endothelial cells express abundant amount of mRNA for the ER. CONCLUSIONS: These data indicate that endothelial cells constitutively possess the potential for transcriptional regulation of target genes by estrogen. The evolutionary conservation of this receptor in bovine and human endothelial cells suggests a common mechanism for estrogen regulation of endothelial function.

Biosynthesis of extracellular low-molecular-mass papain and trypsin inhibitors by Streptomyces sp. 22: effect of cultural conditions.

The production of extracellular inhibitors of papain and trypsin by Streptomyces sp. 22 was studied under different cultural conditions including complex and defined media, temperatures ranging from 18 degrees C to 37 degrees C and a variety of sole carbon and nitrogen sources. In complex nutritionally rich medium, maximal specific growth rates were obtained at 37 degrees C, whereas the highest specific production rates for both papain and trypsin inhibitors were registered at 18 degrees C. Studies on the effect of different carbon and nitrogen sources in defined media underline the importance of the nitrogen source as a strong regulator of the biosynthesis of both inhibitors. Enhanced formation of the inhibitory compounds occurred in the presence of casein. The dynamics of the formation of both inhibitors in defined media showed close association with growth. However, a partial separation of production phases for papain and trypsin inhibitors was observed in complex medium. The results imply differences in the regulation of biosynthesis of the two inhibitors.

Histone H1 in the centromeric heterochromatin of Glyptotendipes barbipes larval polytene chromosomes.

Immunofluorescent analysis with antibodies against histone H1 failed to detect H1 in the centromeric heterochromatin blocks of the polytene chromosomes of Glyptotendipes barbipes larvae. Centromeric regions were dissected microsurgically and acid-extracted. Electrophoresis in SDS and acid-urea gels revealed a band comigrating with H1 of calf thymus and of Gl. barbipes salivary gland nuclei. ELISA dot assay of the extracted material gave a positive reaction with anti-H1 monoclonal antibodies and with anti-H1 affinity-purified polyclonal antibodies. This shows that the centromeric heterochromatin contains histone H1 but packed in a way which prevents the H1 antigenic determinants from reacting in situ with the specific antibodies.

Accessibility of an epitope common to all histone H3 variants in folded and unfolded chromatin as studied by a monoclonal antibody.

In a recent publication the isolation and some characteristics of an anti-histone 3 monoclonal antibody, 1GB3 were described (Muller et al. FEBS Lett. 182: 459-464, 1985). We now report that the epitope recognized is phylogenetically conserved and located in the N-terminal part of H3, most likely between residues 40 and 50. Using the ELISA technique we found this region to be accessible in chromatin to the monoclonal antibody. The effect of non-ionic detergents on the adsorbtion of chromatin on microtiter plates was studied in this context. Immunological analysis of the reaction of the monoclonal antibody with chromatin by immunoinhibition and immunosedimentation shows that the H3 epitope is accessible in both folded and unfolded chromatin fibre as well as in high- and low-molecular weight oligonucleosomes.

Differences in the mode of iodination of H2a variants in chromatin.

Modification of H2a variants with radioactive iodine was used to study under different ionic conditions the accessibility of their tyrosine residues in chromatin, in monosomes and when free in solution. The modification of tyrosine 57 in the hydrophobic part of H2a was found responsible for the appearance of new fractions with a reduced electrophoretic mobility in the presence of Trition X 100, detected only by autoradiography (radioactive "ghosts"). At low ionic strength a very small number of molecules were iodinated in chromatin, the modification affecting only their hydrophobic region. At moderate ionic strength the tyrosine residues near the N-terminal region of the molecule were predominantly modified. In chromatin the accessibility of the tyrosine residues of H2a1 was much greater than that of H2a2, a difference not observed with free histones.

A comparison of histone variants in different rat tissues.

The histone patterns of rat liver, spleen, kidney and brain were compared by means of a two-dimensional electrophoresis in the presence of Triton X-100. In addition, a two-step procedure was introduced for quantitative estimation of different histone variants. Tissue differences wee found in the relative content of the different variants of H2a, H3 and the subfraction H1O. It was also found that the high salt induced H2a specific proteolytic activity was very high in spleen, very low in liver and kidney and absent in brain.

Analysis of the high mobility group proteins associated with salt-soluble nucleosomes.

Two methods have recently been described for the isolation of monomer nucleosomes enriched in transcribed sequences which depend on their solubility in 0.1 M NaCl (Levy, W.B. and Dixon (1978), Nucleic Acid Res., 5, 4155-4163) or solutions containing divalent metal ions (Bloom, K.S. and Anderson, J.N. (1978), Cell, 15, 141-150). Using these procedures the proteins associated with such nucleosomes from rabbit thymus, calf liver and hen oviduct nuclei were isolated and analysed. Increased amounts of proteins HMG14 AND HMG17 and small amounts of HMG1 and HMG2 were found associated with the four core histones H2A, H2B, H3 and H4 in these nucleosomes. HMG14 and HMG17 were found to be enriched 2 - 7 fold, suggesting an involvement of these two proteins with transcribed sequences. 0.1 M NaCl-soluble monomer nucleosomes prepared by the method of Levy and Dixon were analysed by polyacrylamide gel electrophoresis and found to be composed of principally two types of particle: 1. Core particles of 145 base pairs of DNA associated with the four core histones only. 2. Nucleosomes with 160 base pairs of DNA associated with the four core histones, increased amounts of HMG14 and 17, and no H1. Small amounts of HMG1 and HMG2 are also detected. These results suggest that HMG14 and HMG17 might be interacting with the 15 base pair linker DNA. A model is presented for the structure of transcriptionally active chromatin.