Osmosis: a macroscopic phenomenon, a microscopic view.

At present, physical chemistry employs the tools of thermodynamics to treat osmosis across a semipermeable membrane. We propose a model in terms of momentum transfer, the inherent asymmetry of which leads quantitatively to the van't Hoff relationship; qualitatively, the solute molecules can be looked upon as micropumps that suck solvent through the pores in the membrane.

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation.

A synthetic heparin-mimicking polyaromatic anionic compound RG-13577 (polymer of 4-hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr approximately 5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of (14)C-RG-13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor-related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG-13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild-type and heparan sulfate-deficient Chinese hamster ovary (CHO) cells, as well as normal- and LDL receptor negative- human skin fibroblasts bind RG-13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor-related protein (LRP) expressed a markedly reduced binding of RG-13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG-13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG-13577, suggesting that this inhibition is mediated by RG-13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and thereby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis.

Endothelial cell proliferation activity in benign prostatic hyperplasia and prostate cancer: an in vitro model for assessment.

PURPOSE: Urinary excretion of several pro-angiogenic and antiangiogenic substances has been correlated with malignant tumor growth. The aim of this study was to assay angiogenic activity in urine from patients with cancer of the prostate and benign prostatic hyperplasia (BPH). MATERIALS AND METHODS: Urine specimens from 22 healthy male volunteers (control), 33 patients with BPH and 29 with organ confined prostate cancer were analyzed for angiogenic activity in a bovine capillary endothelial cell proliferation assay. In parallel the concentration of basic fibroblast growth factor and vascular endothelial growth factor was determined by enzyme immunoassay in the corresponding urine specimens. RESULTS: Urine samples from patients with BPH and prostate cancer increased bovine capillary endothelial cell proliferation by 13.1% and 15.1%, respectively, whereas urine from the control group showed a significantly lower angiogenic activity, increasing endothelial cell proliferation by only 0.7% (p = 0.001). Urinary basic fibroblast growth factor and vascular endothelial growth factor were highest in patients with BPH and lowest in the group with prostate cancer (p = 0.0001). CONCLUSIONS: Urine from patients with BPH and prostate cancer stimulates endothelial cell proliferative activity. The degree of endothelial cell stimulation does not correlate with the concentration of basic fibroblast growth factor or vascular endothelial growth factor. Whether the observed pro-angiogenic activity is due to an increased production or release of (an) other angiogenic factor(s) and/or loss of (an) angiogenesis inhibitor(s), deserves further investigation.

Antiproliferative activity to glomerular mesangial cells and receptor binding of a heparin-mimicking polyaromatic anionic compound.

Proliferation of mesangial cells (MC) is a key feature in the pathogenesis of numerous renal diseases involving the glomerulus. Heparin, one of several compounds capable of suppressing MC proliferation, did not prove beneficial in the treatment of human glomerular diseases. In a search for a superior antiproliferative agent, a synthetic polyaromatic "heparin mimicking" compound (RG-13577, polymer of 4-hydroxyphenoxy acetic acid, M(r) approximately 5800), previously reported to inhibit the proliferation of vascular smooth muscle cells, was applied. RG-13577 exhibits approximately 1% of the anticoagulant activity of heparin and is nontoxic in animal experiments. Proliferation of primary rat MC was almost completely inhibited in the presence of 10 to 25 micrograms/ml RG-13577, and 50% inhibition was obtained at 1 to 5 micrograms/ml RG-13577. The cells resumed their normal growth rate after removal of RG-13577 from the culture medium. Under the same conditions, heparin exerted only a small inhibitors effect. RG-13577 inhibited signaling (i.e., tyrosine phosphorylation) and MC proliferation induced by both basic fibroblast growth factor and platelet-derived growth factor. RG-13577 binds to a naturally produced extracellular matrix, and the bound molecule retained its antiproliferative effect toward MC. 14C-Labeled RG-13577 also binds to cultured MC in a specific and saturable manner. Binding of 14C-RG-13577 was reduced by 80 to 90% in the presence of excess unlabeled RG-13577, apolipoprotein E, or lactoferrin, but there was no effect with heparin. Furthermore, the antiproliferative effect of RG-13577 was abolished in the presence of lactoferrin. It is proposed that compound RG-13577 inhibits MC proliferation through neutralization of growth-promoting factors, primarily heparin-binding growth factors, and possibly through binding to specific cell surface receptors, most likely the LDL receptor-related protein. RG-13577 and related polyanionic compounds may be applied to inhibit MC proliferation in glomerular diseases.

Anionic- and lipophilic-mediated surface binding inhibitors of human leukocyte elastase.

We report the synthesis of a series of diphenylmethane-based oligomers containing anionic and lipophilic functionalities that are potent inhibitors of human leukocyte elastase (HLE). The enzyme inhibition is regulated by the size of the oligomer, as well as, the number of charges. Lipophilicity is an important element in determining potency and specificity against other basic enzymes. Compounds whose scaffolds contain three phenoxyacetic acid groups and three alkyl ethers are competitive and specific inhibitors of HLE with Ki = 20 nM. The mechanism of action of this class of compounds is believed to involve multidendate interactions with the surface of HLE near the active site which prevents substrate access to the catalytic site.

Modulation of fibroblast growth factor-2 receptor binding, dimerization, signaling, and angiogenic activity by a synthetic heparin-mimicking polyanionic compound.

Heparan sulfate (HS) proteoglycans play a key role in cell proliferation induced by basic fibroblast growth factor (FGF-2) and other heparin-binding growth factors. To modulate the involvement of HS, we have used a synthetic, nonsulfated polyanionic aromatic compound (RG-13577) that mimics functional features of heparin/HS. FGF-2-stimulated proliferation of vascular endothelial cells was markedly inhibited in the presence of 5-10 microg/ml compound RG-13577 (poly-4-hydroxyphenoxy acetic acid; Mr approximately 5 kD). Direct interaction between RG-13577 and FGF-2 was demonstrated by the ability of the former to compete with heparin on binding to FGF-2. RG-13577 inhibited FGF-2 binding to soluble- and cell surface-FGF receptor 1 (FGFR1). Unlike heparin, RG-13577 alone failed to mediate dimerization of FGF-2. Moreover, it abrogated heparin-mediated dimerization of FGF-2 and FGFR1, as well as FGF-2 mitogenic activity in HS-deficient F32 lymphoid cells. The antiproliferative effect of compound RG-13577 was associated with abrogation of FGF-2-induced tyrosine phosphorylation of FGFR1 and of cytoplasmic proteins involved in FGF-2 signal transduction, such as p90 and mitogen-activated protein kinase. A more effective inhibition of tyrosine phosphorylation was obtained after removal of the cell surface HS by heparinase. In contrast, tyrosine phosphorylation of an approximately 200-kD protein was stimulated by RG-13577, but not by heparin or FGF-2. RG-13577 prevented microvessel outgrowth from rat aortic rings embedded in a collagen gel. Development of nontoxic polyanionic compounds may provide an effective strategy to inhibit FGF-2-induced cell proliferation associated with angiogenesis, arteriosclerosis, and restenosis.

Microsatellite spreading in the human genome: evolutionary mechanisms and structural implications.

Microsatellites are tandem repeat sequences abundant in the genomes of higher eukaryotes and hitherto considered as "junk DNA." Analysis of a human genome representative data base (2.84 Mb) reveals a distinct juxtaposition of A-rich microsatellites and retroposons and suggests their coevolution. The analysis implies that most microsatellites were generated by a 3'-extension of retrotranscripts, similar to mRNA polyadenylylation, and that they serve in turn as "retroposition navigators," directing the retroposons via homology-driven integration into defined sites. Thus, they became instrumental in the preservation and extension of primordial genomic patterns. A role is assigned to these reiterating A-rich loci in the higher-order organization of the chromatin. The disease-associated triplet repeats are mostly found in coding regions and do not show an association with retroposons, constituting a unique set within the family of microsatellite sequences.

Rapid DNA fragmentation from hypoxia along the thick ascending limb of rat kidneys.

Extensive DNA fragmentation, a marker for programmed cell death, was selectively and rapidly induced by hypoxia in the thick ascending limbs of rat kidneys. In isolated perfused kidneys, DNA breaks were present in medullary tubules as early as after 10 minutes of local hypoxia and were prevented by reduction of metabolic work. In a model of radiocontrast-induced acute renal failure, DNA breaks were detected selectively along thick ascending limbs as early as 15 minutes following insult, preceding overt morphological damage. Hypoxia induces rapid DNA fragmentation along thick ascending limbs, where programmed cell death could play an important role in nephron injury and kidney failure.

Antiproliferative activity to vascular smooth muscle cells and receptor binding of heparin-mimicking polyaromatic anionic compounds.

Proliferation of bovine aortic smooth muscle cells (SMCs) induced by thrombin, basic fibroblast growth factor, or serum is inhibited by anionic, nonsulfated aromatic compounds that mimic many of the effects of heparin. Among these compounds are aurintricarboxylic acid (ATA) and a newly synthesized polymer of 4-hydroxyphenoxy acetic acid (compound RG-13577). Iodinated- or 14C-labeled compound RG-13577 binds to cultured SMCs in a highly specific and saturable manner. Scatchard analysis of the binding data revealed the presence of an estimated 1 x 10(7) binding sites per cell with an apparent dissociation constant of 3 x 10(-6) mol/L. Binding of radiolabeled RG-13577 was efficiently competed for by related aromatic anionic compounds and by apolipoprotein E, but not by heparin, heparan sulfate, suramin, or various purified growth factors and extracellular matrix proteins. Receptor cross-linking of SMC-bound 125I-RG-13577 revealed a single species of high M(r) (approximately 280 kD) cell surface receptors detected in the absence but not the presence of excess unlabeled compound RG-13577. Binding was susceptible to downregulation and restoration of receptor levels in a manner similar to that of hormone and growth factor receptors. We suggest that the antiproliferative activity of compound RG-13577 and related compounds is initiated by binding to specific growth-inhibiting cell surface receptors. Heparin-mimicking compounds may be applied to inhibit SMC proliferation associated with atherosclerosis and restenosis.

Extended duration of vertical position might impair bone metabolism.

Bone-remodelling is markedly influenced by vectors of gravitational forces. Sleep-deprivation, common during military training, involves a change in the normal balance between horizontal and vertical forces enacting on the skeleton. Stress fractures are likewise prevalent among army recruits. In order to investigate the impact of sleep-deprivation on bone-metabolism, three groups of young, healthy volunteers were selected to exercise the following: 63 h of sleeplessness (17 participants, group A); vertical sleep in a seated position for three consecutive nights (9 participants, group B); controls who slept 6 h a night horizontally (14 participants, group C). During periods of wakefulness, all participants were kept in an upright position. Twenty-four hours' urine collection was strictly observed from two days prior to the experiment until two days after it (1 week). Changes in levels of the most characteristic bone-metabolites, calcium and hydroxyproline indicate an increased bone-resorption in the two experimental groups, but not in controls. The calcium excreted in the fasting urine peaked significantly at 72 h after the beginning of the experiment (+ 170% in group A; + 68% in group B, relative to the basal level). Qualitatively, similar results were obtained with hydroxyproline. On an individual basis, approximately 40% of the participants in either group responded by exceeding urinary-calcium elevation. A comparison of pre-test bone-density between responders and non-responders, reveals a significantly lower bone-density (-5%) in calcium and hydroxyproline excretors. These results suggest a pre-disposition to bone-resorption associated with responsiveness to changes in the balance between gravitational forces.

Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate.

PrPSc is the only known component of the scrapie prion. The difference between PrPSc and its normal isoform PrPc is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin-like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin-like molecules, excluding HS, can inhibit the synthesis of PrPSc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrPSc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrPc into PrPSc remains to be established.

Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues.

Heparin, among the best studied glycosaminoglycans, is well known for its involvement in a variety of physiological processes. Many proteins, whose activities are modulated via heparin binding, were identified, and the consequences of their interaction with heparin were characterized. However, in the absence of solid structural information regarding heparin-protein complexes, the mechanism by which heparin operates at the molecular level is still obscure. The structure of such a complex is hereby explored via the identification of a common motif in heparin binding sequences. To avoid ambiguity we included in our data base only sequences that have been shown experimentally to be directly involved in heparin binding. Then, a comparison of the spatial distribution of basic residues was conducted among those peptides for which three-dimensional structures were defined. Using computer graphics techniques we were able to identify a unique distribution shared by all of these segments. Two basic amino acids (most frequently arginine) are located at about 20 A apart, facing opposite directions of an alpha-helix. Other basic amino acids are dispersed between these two residues, facing one side, while nonpolar residues face the opposite side, forming an amphipathic structure. The distribution of basic amino acids in other heparin binding sequences that preserves the same spatial arrangement seems to be compatible with a beta-strand structure. The 20-A interval accommodates a glycosaminoglycan pentasaccharide, and the spatial distribution of the basic residues suggests an intertwinement of the heparin-protein complex. The dynamics of such an interaction may provide a clue regarding the ensuing change in protein activity.

Immunohistochemistry of phosphotyrosine residues: identification of distinct intracellular patterns in epithelial and steroidogenic tissues.

Tyrosine kinases are thought to play a major role in the control of cell growth and differentiation. Most of the work on the phosphorylated product was performed, however, on isolated proteins or cultured cell lines. To assess the overall involvement of tyrosine phosphorylation in vivo, a monoclonal antibody (MAb) against phosphotyrosine was applied to conventional histological sections of various tissues. With the immunoperoxidase staining method, two unique patterns of intracellular distribution of phosphotyrosine were identified among a large variety of normal tissues. (a) In many of the epithelia examined, a peripheral staining was observed, either at the apical aspect alone or at the entire contact region between neighboring cells. This pattern of staining seems to coincide with the distribution of a subset of cytoskeletal elements and requires pre-treatment with proteinase K. (b) In most steroidogenic tissues examined, vesicular cytoplasmic staining was evident, which seems to represent steroid-containing granules. In this case, proteolytic pretreatment is not essential and can be harmful. An extensive survey of human ovarian carcinoma biopsies failed to reveal any consistent staining pattern. These findings might indicate the involvement of tyrosine phosphorylation in basic cellular activities such as the assembly of the specialized cytoskeletal components.

Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.

Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.

Neonatal exposure to protoporphyrin-activating lighting as a contributing cause of childhood acute lymphocytic leukemia.

While being a relatively rare disease, acute lymphocytic leukemia (ALL) is the leading form of cancer in children in the developed world today. ALL sharply peaks in incidence at ages three to four years. In the United States there have been persistent, unexplained increases in incidence of ALL in the past two decades. We hypothesize that exposure to photosensitizing lighting immediately after birth may be a contributing cause of ALL. Fluorescent lamps and other light sources with strong illumination, around 400 nanometers, are protoporphyrin-activating. Activation of protoporphyrin produces superoxides and free radicals that can induce breaks in DNA. In newborn nurseries in the US, the intensity of lighting has increased five- to 10-fold over the past two decades. Thus, protoporphyrin-activating light may be a contributing cause of childhood ALL. Additional retrospective and prospective studies should be undertaken of the relationship between exposure of newborns to protoporphyrin-activating illumination and the development of childhood ALL, along with in vitro studies of the hematologic effects of fluorescent lighting. Protoporphyrin-activating lighting is clearly not the sole determinant of ALL, but it could be a completely preventable cause. Inexpensive plastic filters could reduce these exposures substantially.

Selective abrogation of alloreactivity via priming in the presence of aphidicolin, a specific inhibitor of DNA polymerase.

Aphidicolin, a specific and direct inhibitor of eukaryotic DNA polymerase alpha, was used to investigate its impact on immunologic reactions in vitro. Dose response curve of the inhibitory effect was studied in murine and human primary allogeneic responses, as well as the proliferative responses to both PHA and Con A mitogens. The presence of aphidicolin during the allosensitization phase in secondary MLR of mice splenocytes resulted in complete abolishment of the subsequent response directed against the priming alloantigens, whereas alloreactivity to unrelated alloantigen-bearing cells was inhibited to a much lesser degree. The allosensitized aphidicolin-treated cells lost the ability to respond to subsequent PHA stimulation, but were capable of exerting a high responsiveness to Con A. The presence of aphidicolin during the allosensitization phase in secondary MLR of human mononuclear cells resulted in markedly decreased alloreactivity directed against the priming cells, but spared the subsequent response to unrelated alloantigens and to both PHA and Con A mitogenic stimuli. It is suggested that aphidicolin may be used for selective inactivation of proliferating cells without interfering with immunologic functions of other quiescent subsets. Aphidicolin may thus be a useful agent for induction of specific unresponsiveness in experimental models of allogeneic transplantation.