Forces generated during stretch in the heart of the lobster Homarus americanus are anisotropic and are altered by neuromodulators.

Mechanical and neurophysiological anisotropies mediate three-dimensional responses of the heart of ITALIC! Homarus americanus Although hearts ITALIC! in vivoare loaded multi-axially by pressure, studies of invertebrate cardiac function typically use uniaxial tests. To generate whole-heart length-tension curves, stretch pyramids at constant lengthening and shortening rates were imposed uniaxially and biaxially along longitudinal and transverse axes of the beating whole heart. To determine whether neuropeptides that are known to modulate cardiac activity in ITALIC! H. americanusaffect the active or passive components of these length-tension curves, we also performed these tests in the presence of SGRNFLRFamide (SGRN) and GYSNRNYLRFamide (GYS). In uniaxial and biaxial tests, both passive and active forces increased with stretch along both measurement axes. The increase in passive forces was anisotropic, with greater increases along the longitudinal axis. Passive forces showed hysteresis and active forces were higher during lengthening than shortening phases of the stretch pyramid. Active forces at a given length were increased by both neuropeptides. To exert these effects, neuropeptides might have acted indirectly on the muscle via their effects on the cardiac ganglion, directly on the neuromuscular junction, or directly on the muscles. Because increases in response to stretch were also seen in stimulated motor nerve-muscle preparations, at least some of the effects of the peptides are likely peripheral. Taken together, these findings suggest that flexibility in rhythmic cardiac contractions results from the amplified effects of neuropeptides interacting with the length-tension characteristics of the heart.

Regulation of apoptosis during mammary involution by the p53 tumor suppressor gene.

Regulation and functions of the p53 tumor suppressor gene have been studied extensively with respect to its critical role in maintaining the stability of genomic DNA following genotoxic insults. However, p53 is also induced by physiologic stimuli resulting in cell cycle arrest and apoptosis. In other situations, the activity of p53 must be repressed to prevent inappropriate removal of cells. The mammary gland provides a valuable system in which to study the mechanisms by which the expression and biological responses to p53 can be regulated under a variety of physiological circumstances. The pro-apoptotic role of p53 in the secretory mammary epithelium may be especially relevant to lactation in livestock. We have utilized p53-deficient mice to establish the molecular targets of p53 in the mammary gland and biological consequences when it is absent. The p21/WAF1 gene (Cdkn1a) is a transcriptional target gene of the p53 protein that responds to elevated levels of p53 during milk stasis providing an endogenous reporter of p53 activity. Abrogation of p53 resulted in delayed involution of the mammary epithelium, demonstrating the physiological role of p53 in regulating involution. Though delayed, stromal proteases were induced in the mammary gland by 5 d postweaning, providing a p53-independent mechanism that resulted in removal of the residual secretory epithelium. These processes can be interrupted by treatment with hydrocortisone. These data establish p53 as a physiological regulator of involution that acts to rapidly initiate apoptosis in the secretory epithelium in response to stress signals, but also indicate the presence of compensatory pathways to effect involution. Additional mechanisms involving intracellular stress signaling pathways (e.g., Stat3) and stromal-mediated pathways have been identified and, together with p53 pathways, may be used to identify animals with greater persistency of lactation.

Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome.

Breast cancer is the most frequent tumor type among women in the United States and in individuals with Li-Fraumeni syndrome. The p53 tumor suppressor gene is altered in a large proportion of both spontaneous breast malignancies and Li-Fraumeni breast cancers. This suggests that loss of p53 can accelerate breast tumorigenesis, yet p53-deficient mice rarely develop mammary tumors. To evaluate the effect of p53 loss on mammary tumor formation, the p53(null) allele was back-crossed onto the BALB/c genetic background. Median survival was 15.4 weeks for BALB/c-p53(-/-) mice compared to 54 weeks for BALB/c-p53(+/-) mice. Sarcomas and lymphomas were the most frequent tumor types in BALB/c-p53(-/-) mice, whereas 55% of the female BALB/c-p53(+/-) mice developed mammary carcinomas. The mammary tumors were highly aneuploid, frequently lost the remaining wild-type p53 allele, but rarely lost BRCA1. Although mammary tumors were rarely detected in BALB/c-p53(-/-) female mice, when glands from BALB/c-p53(-/-) mice were transplanted into wild-type BALB/c hosts, 75% developed mammary tumors. The high rate of mammary tumor development in the BALB/c background, but not C57Bl/6 or 129/Sv, suggests a genetic predisposition toward mammary tumorigenesis. Therefore, the BALB/c-p53(+/-) mice provide a unique model for the study of breast cancer in Li-Fraumeni syndrome. These results demonstrate the critical role that the p53 tumor suppressor gene plays in preventing tumorigenesis in the mammary gland.

A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development.

Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53(null) mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53(null) mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53(null) mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53(null) and p53wt mammary epithelium. The treatment groups were: (1) untreated; (2) continuous hormone stimulation with pituitary isografts; (3) multiple pregnancies; (4) DMBA alone; and (5) DMBA+pituitary isografts. The tumor incidences in p53(null) vs p53wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53(null) mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53(null) mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.

Regulation of p53 and its targets during involution of the mammary gland.

Post-lactational involution of the mammary gland provides a system in which to study the expression and function of genes that regulate apoptosis in the context of a normal tissue. The functions of the p53 tumor suppressor gene have been extensively studied as a mediator of apoptosis in response to DNA damage, but its regulation in normal physiologic processes has been poorly characterized. Expression of p53 mRNA was shown to be among the first genes to be induced in mammary tissue following weaning of neonates. Although involution proceeds in the absence of a functional p53 gene, it is delayed compared to normal individuals. Therefore, involution can be viewed as biphasic with initial responses being sensitive to p53, whereas secondary responses being p53-independent. These observations can be exploited to determine the subset of genes that are p53-responsive and that mediate the effects of p53 in normal mammary tissue.

Cloning and characterization of a new intercellular adhesion molecule ICAM-R.

The human intercellular adhesion molecules ICAM-1, ICAM-2 and their counter-receptors, the beta 2 or leukointegrins, mediate a variety of homotypic and heterotypic leukocyte and endothelial cell-cell adhesions central to immunocompetence. It has been found that cell-cell adhesion which is dependent on expression of the leukocyte function-associated antigen LFA-1 is not always blocked completely by antibodies raised against ICAM-1 and ICAM-2. Other leukointegrin ligands therefore probably exist, such as a glycoprotein of M(r) 124K that binds LFA-1 and has been designated ICAM-3 on the basis of this function. We have molecularly cloned a new member of the ICAM family, ICAM-R, which is related to ICAM-1 and ICAM-2. The complementary DNA encoding ICAM-R is 1,781 base pairs long and the protein has five extracellular immunoglobulin-family type domains. The mature cell-surface form of the ICAM-R protein has an M(r) which varies from 116 to 140K in a cell type-specific fashion. Overall identities in protein sequence with ICAM-1 and ICAM-2 are 48% and 31% respectively, with the degree of similarity varying between individual domains. The high level of expression of ICAM-R on resting leukocytes of all lineages and its lack of expression on either resting or cytokine-activated endothelial cells indicates a pattern of expression distinct from ICAM-1 and ICAM-2. In common with ICAM-1 and ICAM-2, ICAM-R is a ligand for the beta 2-integrin CD11a/LFA-1 (CD18).

Cyclic AMP efflux is regulated by occupancy of the adenosine receptor in pig aortic smooth muscle cells.

Cultured pig aortic smooth muscle cells respond to extracellular adenosine by activating adenylate cyclase and by initiating the efflux of cAMP. In the presence of extracellular adenosine, efflux is first order with respect to intracellular cAMP concentration up to at least 125 pmol/10(6) cells. The apparent first-order rate constant for the efflux of cAMP increases in a dose-dependent manner in response to extracellular adenosine or 5-N-ethylcarboxamide adenosine. The EC50 for adenosine for promoting cAMP efflux is 12 microM. For cells stimulated with 5-N-ethylcarboxamide adenosine, the EC50 is 5 microM. When extracellular adenosine is removed, efflux stops abruptly. Cellular cAMP content falls but is still in a range that supports cAMP efflux when agonist is present. Efflux is not affected by H8 (N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride), an inhibitor of cAMP-dependent protein kinase. These data suggest that in pig aortic smooth muscle cells, the efficiency of cAMP efflux is regulated by A2 receptor occupancy.

The hydrolysis of extracellular adenine nucleotides by arterial smooth muscle cells. Regulation of adenosine production at the cell surface.

The extracellular reaction sequence ATP----ADP----AMP----adenosine participates in regulating the time course of cellular response during crisis or signaling events, such as thrombus formation or neurotransmission. We have investigated the whole time course of hydrolysis of ATP to adenosine by recirculating adenine nucleotide substrates over smooth muscle cells attached to polystyrene beads. Kinetic parameters were estimated for each reaction by fitting observed time courses to models of the pathway. In spite of the inhibition of 5'-nucleotidase by ADP, adenosine was produced very rapidly by smooth muscle cells. Comparisons of the apparent Km values of ADPase and 5'-nucleotidase (determined from experiments in which each substrate was used as the initial substrate with Km values observed when each substrate was supplied from the upstream reaction) suggest that the local concentrations of substrate supplied from the preceding reactions are very much higher than those in the bulk phase. This enhancement of efficiency overcomes the effect of the feed-forward inhibition to give rise to very rapid adenosine production from ADP or ATP. These observations are in marked contrast to our previous findings with endothelial cells (Gordon, E. L., Pearson, J. D., and Slakey, L. L. (1986) J. Biol. Chem. 261, 15496-15504), on which feed-forward inhibition causes a profound lag in adenosine production from adenine nucleotides and on which there are no apparent surface effects on substrate delivery.

Monoclonal antibodies to gp110 and gp41 of human immunodeficiency virus.

Six mouse hybridomas secreting monoclonal antibodies specific for the glycoproteins of human immunodeficiency virus were developed. All six antibodies reacted by radioimmunoprecipitation with the glycoprotein precursor of 150,000 daltons as well as one of the proteolytic processing products of 110,000 daltons (gp110) or 41,000 daltons (gp41). Recombinant polypeptides spanning the env coding region were used to locate epitopes on the glycoprotein molecule. The panel of antibodies detected two distinct epitopes of gp41 and one epitope of gp110. We used the antibodies in indirect immunofluorescence assays to evaluate 13 clinical isolates of human immunodeficiency virus from diverse geographic regions, and we found that the gp110 epitope was recognized on all tested isolates, whereas the two gp41 epitopes were detected on 10 of 13 and 4 of 13 isolates.

Time course of release into the medium of newly synthesized proteins by cultured aortic endothelial cells. Role of serum in preventing proteolytic degradation.

Endothelial cells from pig aortas were labeled with 35S-methionine, and the soluble proteins that were released into the culture medium were examined by SDS-PAGE. Proteins were collected during labeling, and during the intervals 0 to 3, 3 to 6, 6 to 12, and 12 to 24 hours of chase after a 1-hour labeling period, and during the intervals 0 to 12, 12 to 24, and 24 to 48 hours of chase after a 24-hour labeling period. Release of radiolabeled soluble protein from cells into the medium continued over the longest time periods examined. If the cells were labeled for 1 hour, characteristic patterns of proteins appeared in the medium during the labeling period, early (0- to 3-hour postlabeling) and late (6- to 24-hour postlabeling). The time course of release of fibronectin (Fn) and of angiotensin-converting enzyme (ACE) was determined by using immunoprecipitation. ACE appeared only after 6 hours of chase. Fn appeared throughout the chase period. The effects of serum and of the protease inhibitors alpha-2 macroglobulin, pepstatin, and leupeptin on protein release were examined. In the absence of serum, endothelial cell culture medium contained substantial protease activity capable of completely degrading most of the released proteins; a major effect of serum was to protect newly released protein from degradation.

A micromethod for the quantitation by radioimmunoassay of cyclic AMP in samples containing immuno-cross reactive compounds and other interfering substances.

A procedure for measuring cyclic AMP in samples containing interfering substances, especially high concentrations of other adenine nucleotides, is described. Samples are purified by sequential chromatography on phenyl boronate followed by Dowex - 1. The final elution is with 0.1 normal hydrochloric acid; the samples are immediately ready for acetylation and radioimmunoassay. Cyclic AMP can be detected at 0.5 nanomolar without need to concentrate the sample at any step. The method has been successfully applied to samples containing 100 micromolar ATP, ADP, or AMP in tissue culture medium.

Effect of adenosine on synthesis and release of cyclic AMP by cultured vascular cells from swine.

Both endothelial and smooth muscle cells cultured from swine aorta responded to adenosine by elevating cAMP levels. cAMP was found both intracellularly and released into the incubation medium. Total cAMP in endothelial cells in buffer alone was 2 pmol cAMP/10(6) cells. Total cAMP reached a maximum stimulated level of 2 times control at 640 microM adenosine. The level of cAMP in unstimulated smooth muscle cells was 7 pmol/10(6) cells. Total cAMP was increased 2-fold at 10 microM adenosine and reached a maximum stimulated level of as much as 20 times control at 640 microM adenosine. Kinetics of release of cAMP from smooth muscle cells show that fractional release rates were increased 2 to 7 fold by 10 microM adenosine and 7 to 29 fold by 160 microM adenosine. These experiments indicate that adenosine specifically stimulates release of cAMP as well as synthesis. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) had complex effects on the distribution of cAMP between the cells and medium. The data are consistent with the model that IBMX is a low affinity competitive inhibitor of adenosine, and is itself slightly effective as an agonist for release.

Plasma-derived serum as a selective agent to obtain endothelial cultures from swine aorta.

Endothelial cell and smooth muscle cell cultures from artery wall provide a potential model system for studying cellular processes involved in atherogenesis. To prepare serial subcultures of swine arterial endothelial cells that are free of smooth muscle cells without either selecting a small population or subjecting the cells to cytotoxic conditions, we used swine plasma-derived serum (SPDS) to establish conditions in which endothelial cells have a growth advantage. Endothelial cells were collected by collagenase digestion and smooth muscle cell cultures were prepared by outgrowth from explants of arterial medial segments. Growth rates were compared when each cell type was maintained on SPDS, or fetal bovine serum (FBS), or swine whole serum (SWS). When 20% FBS or SWS were used the doubling times were less than 30 h for both endothelial cells and smooth muscle cells. On 20% SPDS the doubling time for endothelial cells was 32 h, but for smooth muscle cells it was at least 168 h. Using SPDS, we prepare endothelial subcultures from swine aorta that express principally polygonal morphology at confluence. Endothelial cell cultures grown on SPDS have higher angiotensin-converting enzyme than those grown on FBS.

Distribution of membrane marker enzymes in cultured arterial endothelial and smooth muscle cells. The subcellular location of oleoyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase.

The subcellular distribution of oleoyl-CoA:1-acyl-sn-glycero-3-phosphocholine acyltransferase (E.C.2.3.1.23) in cultured swine aorta endothelial cells and smooth muscle cells was investigated. Isolated membrane pellets were centrifuged through linear sucrose gradients, and the distributions of the activities of seven membrane-bound enzymes were measured. The distribution of acyltrasferase activity was similar to that of the endoplasmic reticulum enzymes. Gradient fractions which contained intact mitochondria had very low activities of acyltransferase. Experiments using mixed fractions and measurements made under conditions which inhibit phospholipase A2 showed that no acyltransferase activity from this location was masked by competing activities. When membranes were treated with digitonin, plasma membranes specifically increased in density, facilitating their separation from endoplasmic reticulum membranes. The plasma membranes were free of acyltransferase activity. We conclude that in cultured swine arterial smooth muscle and endothelial cells, acyltransferase is located primarily in the endoplasmic reticulum.

Chromosomal location of a gene defining nicotinamide deamidase in Escherichia coli.

A gene specifying the enzyme nicotinamide deamidase has been mapped, by the interrupted mating technique, at about 33 min on the chromosome of Escherichia coli K-12.