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To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR alpha, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation.

We previously reported that mice lacking the RARgamma gene and one or both alleles of the RARbeta gene (i.e., RARbeta+/-/RARgamma-/- and RARbeta-/-/RARgamma-/- mutants) display a severe and fully penetrant interdigital webbing (soft tissue syndactyly), caused by the persistence of the fetal interdigital mesenchyme (Ghyselinck et al., 1997, Int. J. Dev. Biol. 41, 425-447). In the present study, these compound mutants were used to investigate the cellular and molecular mechanisms involved in retinoic acid (RA)-dependent formation of the interdigital necrotic zones (INZs). The mutant INZs show a marked decrease in the number of apoptotic cells accompanied by an increase of cell proliferation. This marked decrease was not paralleled by a reduction of the number of macrophages, indicating that the chemotactic cues which normally attract these cells into the INZs were not affected. The expression of a number of genes known to be involved in the establishment of the INZs, the patterning of the autopod, and/or the initiation of apoptosis was also unaffected. These genes included BMP-2, BMP-4, Msx-1, Msx-2, 5' members of Hox complexes, Bcl2, Bax, and p53. In contrast, the mutant INZs displayed a specific, graded, down-regulation of tissue transglutaminase (tTG) promoter activity and of stromelysin-3 expression upon the removal of one or both alleles of the RARbeta gene from the RARgamma null genetic background. As retinoic acid response elements are present in the promoter regions of both tTG and stromelysin-3 genes, we propose that RA might increase the amount of cell death in the INZs through a direct modulation of tTG expression and that it also contributes to the process of tissue remodeling, which accompanies cell death, through an up-regulation of stromelysin-3 expression in the INZs. Approximately 10% of the RARbeta-/- /RARgamma-/- mutants displayed a supernumerary preaxial digit on hindfeet, which is also a feature of the BMP-7 null phenotype (Dudley et al., 1995, Genes Dev. 9, 2795-2807; Luo et al., 1995, Genes Dev. 9, 2808-2820). BMP-7 was globally down-regulated at an early stage in the autopods of these RAR double null mutants, prior to the appearance of the digital rays. Therefore, RA may exert some of its effects on anteroposterior autopod patterning through controlling BMP-7 expression.

Vitamin A and its derivatives (collectively referred to as retinoids) are required for many fundamental life processes, including vision, reproduction, metabolism, cellular differentiation, hematopoesis, bone development, and pattern formation during embryogenesis. There is also considerable evidence to suggest that natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects in human diseases such as cancer. Therefore it is not surprising that a significant amount of research was dedicated to probe the molecular and cellular mechanisms of retinoid action during the past decade. One of the cellular mechanisms retinoids have been implicated in is the initiation and modulation of apoptosis in normal development and disease. This review provides a brief overview of the molecular basis of retinoid signaling, and focuses on the retinoid-regulation of apoptotic cell death and gene expression during normal development and in pathological conditions in vivo and in various tumor cell lines in vitro.

Tissue transglutaminase is a multifunctional enzyme that accumulates to high levels in cells undergoing apoptosis. Retinoids act as an acute and direct regulator of tissue transglutaminase gene transcription. The studies reported here were carried out to elucidate the molecular mechanisms involved in the regulation of tissue transglutaminase expression. We have isolated and characterized the mouse tissue transglutaminase gene promoter and 3.8 kb of 5'-flanking DNA. A large fragment of the promoter that includes both the core promoter and 3.8 kb of 5'-flanking DNA shows retinoid-dependent transcriptional activity when stably transfected into HeLa cells. In these stably transfected HeLa cells both the endogenous tissue transglutaminase gene and transfected mouse tissue transglutaminase promoter are activated by all-trans retinoic acid and by retinoic acid receptor (RAR)-specific and retinoid X receptor (RXR)-specific retinoids. In embryos made transgenic with a transglutaminase promoter-beta-galactosidase reporter gene, the transgene shows specific patterns of expression during limb development. The transglutaminase transgene is expressed in cartilage, the cells of the apical ectodermal ridge, and in regions of apoptotic cell death of the interdigital mesenchyme. It appears that cis-acting elements responsible for the complex retinoid regulation, tissue- and apoptosis-specific expression are embedded within the proximal 3.8 kb of DNA flanking the 5'-end of the mouse tissue transglutaminase gene.

Stably transfected Balb-C 3T3 fibroblasts (clone 5), overexpressing a catalytically active tissue transglutaminase, showed a basal adenylate cyclase activity lower than control cells (clone 1). Several modulators of the adenylate cyclase activity (forskolin, Mn2+ and pertussis toxin) showed the existence of a marked negative control on the adenylate cyclase activity present in clone 5 cells. Very interestingly, this same marked negative control was also found in a Balb-C 3T3 fibroblast clone stably transfected with a mutagenized human tissue transglutaminase (mut277 cys > ser) virtually devoid of transglutaminase catalytic activity (clone Ser). Conversely, a significant increase of the adenylate cyclase activity was observed in bovine aortic endothelial cells after the lowering of tissue transglutaminase expression levels by the transfection of an eukaryotic expression vector containing the gene for tissue transglutaminase in antisense orientation. All these findings suggest a possible role for type II tissue transglutaminase as a negative modulator of the adenylate cyclase activity in different cell types, beside its transglutaminase enzyme activity.

Retinoids induce terminal differentiation and subsequent apoptosis in the human myeloid leukemia (HL-60) cell line. We have previously shown that in HL-60 cells, ligand activation of retinoic acid receptors (RARs) is sufficient to induce differentiation but ligand activation of retinoid X receptors (RXRs) is necessary for the retinoid-induced apoptosis of these cells. In the present studies we have characterized the effect of retinoids on the expression of two apoptosis-linked gene products, BCL-2 and tissue transglutaminase. BCL-2 is a membrane-associated protein whose expression has been linked to the suppression of apoptosis in many cells. Tissue transglutaminase is a protein cross-linking enzyme that accumulates in many cells undergoing apoptotic cell death. Our data suggest that ligand activation of RARs in HL-60 cells results in a global suppression of BCL-2 expression whereas ligand activation of both RARs and RXRs triggers the selective accumulation of tissue transglutaminase in the apoptotic HL-60 cells.

Tissue transglutaminase belongs to a family of calcium-dependent enzymes, the transglutaminases that catalyze the covalent cross-linking of specific proteins by the formation of epsilon (gamma-glutamyl)lysine isopeptide bonds. The goal of this study has been the isolation and characterization of the human tissue transglutaminase gene promoter. Genomic DNA clones, spanning the 5' region of the gene, were isolated and the structure of the 5'-end of the human tissue transglutaminase gene was determined. 1.74 kilobases of flanking DNA were sequenced and were found to contain a TATA box element (TATAA), a CAAT box element (GGACAAT), a series of potential transcription factor-binding sites (AP1, SP1, interleukin-6 response element), and a glucocorticoid response elements. Transient transfection experiments showed that this DNA fragment included a functional promoter, which is constitutively active in multiple cell types.

The Alpha Retinoic Acid Receptor (RARalpha) is a Retinoic acid A receptor subtype expressed ubiquitously in embryonic and post-embryonic stages. RARalpha especially plays an important role in cardiac development due to cardiac malformation and cardiac failure related deaths in RARalpha KO mice.

The Beta Retinoic Acid Receptor (RARbeta) is a Retinoic acid A receptor subtype expressed in embryonic and post-embryonic stages. RARbeta especially plays an important role in development of central nervous system. Additionally, RARbeta expression is down-regulated in several forms of cancer.

Genetic defects of the CD95 (Fas/Apo-1) receptor/ligand system, has recently been involved in the development of human and murine autoimmunity. We investigated whether a deregulation of the ;tissue' transglutaminase (tTG), a multifunctional enzyme which is part of the molecular program of apoptosis, may act as a cofactor in the development of autoimmunity. We found that MRLlpr/lpr, which are characterized by a defect in the CD95 receptor and suffer of a severe systemic lupus erythematosus-like disease, produce large amounts of circulating tTG autoantibodies. This phenomenon is paralleled by an abnormal accumulation of an inactive enzyme protein in the accessory cells of lymphoid organs. To investigate the molecular mechanisms by which tTG inhibition may contribute to the development of autoimmunity we generated a cell culture model system consisting of L929 cells stably transfected with a full length tTG cDNA. When L929 cells were killed by Tumor Necrosis Factor alpha (TNFalpha) a pronounced release of DNA and Lactate Dehydrogenase (LDH) was observed. Overexpression of tTG in these cells largely prevented the leakage of macromolecules determined by TNFalpha treatment, an effect which is abolished by inactivating the enzyme cross-linking activity by a synthetic inhibitor. These in vitro observations provided the basis to explain the increased levels of plasmatic LDH we detected in MRLlpr/lpr mice. These data suggest that lack of an active tTG may represent a cofactor in the development of autoimmunity.

Retinoids exert their biological effects by binding to and activating nuclear receptors that interact with responsive elements on DNA to promote gene transcription. There are two families of retinoid receptors, the retinoic acid receptor (RAR) family and the retinoid X receptor (RXR) family, which are each further divided into three subclasses: RAR alpha, beta, gamma and RXR alpha, beta, gamma. Herein we describe the synthesis and structure-activity relationships of a new series of diaryl sulfide retinoid analogs that specifically bind and transactivate the RXRs. Furthermore, the sulfoxide and sulfone derivatives of these analogs are partial agonists which activate the RXRs only at high concentrations. Thus, these compounds possess a potential site of metabolic deactivation and may have less prolonged systemic effects than other compounds with arotinoid-like structures. We show also that these compounds have activity in nontransfected cells as demonstrated by their ability to induce TGase activity in HL-60 cells. Finally, we corroborate our earlier report that RXR-specific agonists may possess reduced teratogenic toxicity compared to RAR-specific agonists since these compounds are much less potent inhibitors of chondrogenesis than RAR-specific agonists such as TTNPB.

Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines.

Tissue transglutaminase (tTG) expression was found to be induced in rat liver following in vivo retinoic acid (RA) treatment (Piacentini et al. (1988) Biochem. J. 253, 33-38). Here we show that the increased enzyme expression in rat liver is at least partially the result of the action of RA in parenchymal cells. In fact, (a) when hepatocytes are isolated from RA-treated animals their transglutaminase protein content is much higher than in similarly isolated control cells; (b) higher tTG protein level is also found by immunoelectronmicroscopy in the hepatocytes of the RA-treated rats as compared with the very low amount detected in the controls; (c) RA induces tTG in hepatocytes under culture conditions as well. One of the functions of tTG is to form a protein polymer in dying apoptotic cells by epsilon(gamma-glutamyl)lysine and, specifically gamma-glutamylpolyamine cross-links (Fesus et al. (1989) FEBS Lett. 245, 150-154). Noteworthy, after in vivo and in vitro RA-treatment we could not determine any increase (there was even a slight decrease) in the number of the cross-linked apoptotic envelopes. In keeping with this is the significant reduction of protein bound gamma-glutamylpolyamine detected in hepatocytes exposed to RA in culture. These findings suggest that the RA-induced tTG in parenchimal cells is an inactive form.

Human umbilical vein endothelial cells contain high levels of mRNA for the beta-retinoic acid receptor, and very low levels of alpha-retinoic acid receptor transcripts. The cells responded to retinoic acid with a significant induction of tissue transglutaminase expression but no alterations in the expression of beta-retinoic acid receptor transcripts. The physiological implications of the constitutive expression of this receptor in endothelial cells is discussed.

Fresh serum and retinoids induce the expression of tissue transglutaminase in cultured mouse resident peritoneal macrophages. Analogues of cyclic AMP, such as dibutyryl cyclic AMP, and agents that increase intracellular cyclic AMP levels enhance the induction. Dibutyryl cyclic AMP alone has little effect on transglutaminase expression, but it increases the sensitivity of macrophages to low concentrations of either serum or retinoic acid. Dibutyryl cyclic AMP potentiates the transglutaminase-inducing activity of both free retinoic acid and retinoic acid bound to the serum retinol-binding protein. Pretreating macrophages with dibutyryl cyclic AMP or retinoic acid does not prime the cells to respond to the other agent; instead, both agents must be present simultaneously to obtain the synergistic induction of transglutaminase. Our studies suggest that the modulation of intracellular cyclic AMP levels may have pronounced effects on retinoic acid-induced gene expression in myeloid cells.

Treatment of Chinese hamster ovary cells with dansylcadaverine or N-(6-aminohexyl)-5-chloro-1-naphthylenesulfonamide (W7) reduced cell attachment in a reversible, dose-dependent manner. The concentration of dansylcadaverine required to produce 50% inhibition of adhesion was significantly higher than that of W7, 300 μM and 50 μM, respectively. Concentrations of dansylcadaverine and W7 which produced decreased adhesion also antagonized calmodulin-dependent activation of phosphodiesterase. Chlorpromazine, another calmoldulin antagonist also decreased cell attachment. Dansylcadaverine and W7 both interfere with cellular transglutaminase activity, but several other transglutaminase antagonists, such as methylamine, butylamine, putrescine and bacitracin, had no effect on CHO cell attachment. We conclude that naphthylsulfonamides such as dansylcadaverine and W7 may inhibit the attachment of CHO cells by a mechanism which could involve inhibition of calmodulin-dependent processes, although further studies are required to show a direct role of calmodulin in cell adhesion.

Retinoids (structural and functional analogs of vitamin A) are potent antiproliferative agents whose mode of action is poorly understood. It has been suggested that the molecular events that underscore their action involve alterations in gene expression, but no gene has yet been shown to be directly regulated by these molecules. Several years ago, we found that retinoic acid caused an accumulation of the enzyme tissue transglutaminase in murine peritoneal macrophages and in human promyelocytic leukemia (HL-60) cells. We now report that this induction is caused by an increase in the mRNA for this enzyme. Retinoic acid is the only mediator of this induction, since its effects do not depend on the presence of serum proteins. The induction of tissue transglutaminase mRNA is not due to an increase in its stability but to an increase in the relative transcription rate of its gene. We present a model to correlate the retinoid induction of tissue transglutaminase with retinoid effects on cellular growth and differentiation.

Retinoic acid has been shown to induce large accumulations of tissue transglutaminase in cultured myeloid cells. Addition of retinoic acid to mouse resident peritoneal macrophages increased the level of tissue transglutaminase mRNA within 30-60 min. Retinoic acid also increased tissue transglutaminase mRNA levels in human promyelocytic leukemia (HL-60) cells. These studies show that retinoic acid can induce acute alterations in specific gene expression in both normal and leukemic myeloid cells.

A tank system containing sediments and brine chosen to simulate an evaporative shallow-water organic-rich sedimentary basin was allowed to evolve for ten months. Early diagenetic reactions between nesquehonite and brine resulted in a marked increase in alkalinity. The first major change was the precipitation of monohydrocalcite spherules which formed a crust between supernatant liquid and sediment. Cores of the nesquehonite layer showed dissolution of the nesquehonite and the formation of huntite and protohydromagnesite, followed by the formation of Mg-rich dolomite, and then dolomite of stoichiometric proportions. Calcite precipitated with dolomite within the nesquehonite layer. A second crust, precipitated within a layer of decaying filamentous algae, between the nesquehonite and calcite layers was composed of calcite, dolomite and halite. Precipitation within the nesquehonite layer was controlled by reactions between brine and nesquehonite, leading to dissolution of nesquehonite and hydrolysis of carbonate. The formation of huntite and dolomite appears to have been aided by a high CO 2− 3 concentration in solution, which was effected by the high salinity, and maintained by the continued dissociation of nesquehonite.

Retinoic acid rapidly induces the accumulation of a specific enzyme, tissue transglutaminase (EC 2.3.2.13), in mouse macrophages. We have used the induction of tissue transglutaminase to study the regulation of gene expression by retinoic acid. In this study we report that pertussis toxin can inhibit retinoic acid-induced expression of tissue transglutaminase in mouse resident peritoneal macrophages. This inhibition is paralleled by the ADP-ribosylation of 41,000-dalton macrophage membrane protein.

Tissue transglutaminase accumulates rapidly and to very high levels (1-2% of cellular protein) in mouse peritoneal macrophages cultured in mouse serum. The induction is due to accelerated synthesis of the enzyme (150-fold increase) that occurs within 90 min of exposure of the cells to a heat-labile constituent of serum or plasma. The induction is reversible and is not reproduced by known activators of macrophage function such as lipopolysaccharide, muramyl dipeptide, and tuftsin. In animals, elevated levels of tissue transglutaminase are also found in inflammatory macrophages elicited by thioglycolate broth.

The levels and activity of tissue transglutaminase were studied in human peripheral blood monocytes during differentiation into macrophages in vitro. The enzyme was present at low levels in freshly isolated monocytes (less than 20 ng/mg cell protein) but increased 50-fold during 10 d of adherent culture in autologous serum, reaching levels of 0.1% of total cellular protein. The rate of appearance of tissue transglutaminase in monocytes was accelerated by low levels of lipopolysaccharide. The half-life of disappearance of transglutaminase from human monocytes was 11 and 7 h in 2-d-old and 10-d-old cells, respectively. Treatment of 1-day-old monocytes with actinomycin D for 24 h blocked the increase in transglutaminase levels. These results indicated that the induction of gene transcription and protein synthesis was responsible for the increased transglutaminase levels and activity observed with cultured human monocytes. The induction of tissue transglutaminase may be a component in the in vivo differentiation of human monocytes into macrophages.

Aragonite, calcite, and monohydrocalcite synthetic ooids similar in morphology and internal structure to natural radial (quiet-water) ooids, have been prepared from seawater containing organic matter. Products showing a limited degree of concentric banding surrounding a relatively large spherulitic nucleus were also obtained and are termed synthetic “proto-ooids”. The ability of various types of organic matter to promote the formation of synthetic ooids decreases in the order: humic acids > proteinaceous matter > fulvic acids > amino acids, amino sugars, algal mucilage and amino-acid—sugar condensates. A detailed investigation of synthetic ooid formation in solutions containing humic and fulvic acids shows that this ability does not parallel the extent to which the organic matter is sorbed by carbonate surfaces, but is related to a specific type of organo—carbonate interaction involving the formation of organic membranes. The membranes themselves are considered to form by association of individual organic molecules through metal “bridging” and hydrophobic/hydrophilic interactions — processes which are known to contribute to the stability of biological membranes. Major features of a proposed mechanism of formation for the synthetic ooids are: (1) formation and precipitation of Ca and Mg humates and fulvates from the corresponding Na salts added to the solution; (2) aggregation of the higher-molecular-weight components into membranes; (3) inhibition of carbonate precipitation by the interaction of lower-molecular-weight components of the organic matter with potential nuclei; (4) activation of the nuclei, by deposition of an organic membrane having more hydrophilic character than that presented to the solution by the low-molecular-weight organic matter; (5) dentritic (spherulitic) crystallization of carbonate needles induced by a high degree of supersaturation of the solution and strong sorption of organic matter on the carbonate surfaces; (6) strong inhibition of carbonate precipitation by the combined effects of sorption of Mg(II) ions and low-molecular-weight organic matter on the growing carbonate surfaces, (7) regeneration of the inactive carbonate surface by the deposition of another organic membrane. The experimental data provide strong evidence that periodic fluctuations in the physical and/or chemical composition of the parent solution are not necessary for the formation of radial carbonate ooids. Accordingly, the possibility that natural quiet-water ooids result from the influence of specific organo—carbonate interactions on carbonate precipitation under almost constant physical and chemical conditions deserved serious consideration.

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.

The culture of peritoneal macrophages in serum-containing media induces a dramatic increase in the expression of the enzyme tissue transglutaminase. The transglutaminase-inducing activity of serum is abolished by extraction of lipids and fully restored by re-addition of physiological concentrations (1-100 nM) of trans-retinoic acid. Induction of the enzyme is detectable within a 90-min exposure of macrophages to retinoic acid and is completely blocked by actinomycin D, suggesting that the retinoid rapidly increases the rate of transglutaminase gene expression. Delipidized serum is required to elicit the transglutaminase-inducing activity of retinoic acid and this effect is decreased if the serum is depleted of the serum retinol-binding protein. Our studies suggest that retinoic acid and serum retinol-binding protein can directly regulate macrophage gene expression and specifically induce the synthesis of tissue transglutaminase.

Physiological concentrations of retinoic acid can induce acute alterations in the expression of the enzyme tissue transglutaminase in cultured macrophages. The induction of this enzyme offers a probe to study the mechanism of retinoid action in both normal and leukemic cells.

Field and laboratory studies suggest that different types of ooids form during quiet and agitated water conditions. Both types have been synthesized in the laboratory. Quiet water types exhibit a radial orientation of carbonate crystals, whereas in those formed in agitated conditions, a tangential orientation is prevalent. Successful laboratory formation of quiet water ooids was accomplished in supersaturated seawater solutions containing humic acids. Negative results were obtained from strictly inorganic solutions, and from those containing simple amino acids, single proteins, mixtures of proteins or mucopolysaccharides, soil and sediment extracts. Partly successful results were obtained using an organic extract from Bahamian ooids. The organic parameters most important in quiet water ooid formation are molecular weight, the presence of carboxyl groups and an ability to participate in hydrophobic/hydrophilic interactions, all of which are critical to membrane formation. Membranes form concentric shells which act as growth surfaces for carbonate and also induce the periodicity in carbonate precipitation. Ooids exhibiting a tangential orientation of batten-like crystals have been synthesized under conditions of agitation, supersaturation and without the intervention of organic processes during the precipitation. Complete growth may be divided into agitation, resting and sleeping stages In the agitation stage, quartz nuclei induce an inorganic, heterogeneous nucleation from a supersaturated solution, which finally ceases as a result of Mg2+ and possibly H+ poisoning of the carbonate surfaces. No further precipitation occurs until the crystal surfaces are reactivated by removal of Mg2+ and H+ during the resting stage. Following a series of agitation and resting stages, precipitation is inhibited by a degree of poisoning which is not totally removed during the resting stage. For further growth, a new substrate is required and is provided by the development of organic membranes around the grains. This occurs when the grains are buried in the subsurface, the period of organic growth constituting the sleeping stage. Only 2% of an ooid's life is spent growing in the agitated environment, while 95% of its life is spent accreting organic membranes in the subsurface. Our experimental work indicates that ooids of Bahamian type are inorganic precipitates. The tangential arrangement of battens is the result of suspension in an environment where the degree of turbulence is sufficient to induce grain to grain contact of sufficient strength and frequency to inhibit any crystal growth other than tangential. The role of organics is to provide a substrate for further growth after precipitation has slowed to a point when no further accretion is occurring.

Filamin, a protein recently identified in chicken gizzard (Wang, K., Ash, F., and Singer, S. J. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 4483-4486), has been purified free of other components and its molecular properties have been examined. Filamin has a sedimentation constant (S020,w) of 8.86 S and a partial specific volume of 0.734 ml/g. Sedimentation equilibrium experiments give a value of 498,000 for the molecular weight of native filamin. From these data a frictional ratio of 2.32 has been calculated. On sodium dodecyl sulfate gel electrophoresis, filamin migrates as a single protein band with an estimated molecular weight of 240,000. Filamin is a soluble protein and under a variety of conditions tested does not by itself form filaments or precipitate from solution. However, filamin binds to rabbit skeletal muscle F-actin, and the complex is readily sedimented by centrifugation to yield a gelatinous pellet containing actin and filamin. These results indicate that filamin is a dimeric protein with a moderate degree of asymmetry that binds to actin. The results also suggest that the distribution of filamin in cells is derived from its interaction with polymerized actin.

GEBELEIN and Hoffman1 drew attention to the association of dolomite with stromatolitic sequences, and suggested that the precipitation of dolomite is related to the presence of organic material. Their experiments confirmed previous results2,3 that large quantities of magnesium may be absorbed from seawater by organic material. Magnesium-rich algal filaments may form sites for the precipitation of high magnesium calcite with 17–20 mol % MgCO3 and although dolomite has not been precipitated in such conditions, it is possible that high-Mg calcite could be diagenetically altered to dolomite because of the high Mg–Ca ratio associated with the organic material1.

Publisher Summary This chapter presents properties and procedure for purification of Avian and mammalian filamins. Filamin can be purified from both smooth muscle and nonmuscle cells. However, chicken gizzard is a particularly convenient source because large quantities of fresh tissue are readily obtainable and inexpensive. Two general problems are encountered during filamin purification: proteolytic degradation, and aggregation of the protein. To reduce proteolysis, we routinely work with fresh tissues, carry out all steps at 0–4 °, move as expeditiously as possible through the early steps of purification, and include EDTA in all buffers. Aggregation can be minimized also by working with fresh tissues in the cold, by including reducing agents in the buffers, and by keeping the ionic strength of buffers above 100 mM KCI until all the actin is removed from the preparation. Purification of filamin is monitored by SDS-polyacrylamide gel electrophoresis on 5% linear polyacrylamide slab gels.

Filamin is a high molecular weight (240 000) actin-binding protein. It has been isolated from alveolar macrophages [l] , leukemic leukocytes [2] and smooth muscle [3,4] and identified immunochemically in rat and mouse fibroblasts [3,5]. Although the protein is present in substantial amounts in some tissues (l-2% of the protein of macrophage and smooth muscle cells), little is known of its function in the cell. Stossel and Hartwig [ 1,6,7] have shown that when macrophage filamin and actin are mixed together at 25°C they form a gel. Shizuta et al. [4] also observed gel formation when chicken-gizzard filarnin and actin were combined. Although little information is available on what effect filamin might have on actin function and what factors may regulate the interaction of filamin and actin, a known function of actin is activation of the Mg-ATPase activity of myosin or heavy meromyosin (HMM) (for review see ref. [S] ), We have studied the effects of filamin on F-actin activation of HMM ATPase and myosin ATPase and have found that filamin markedly reduced actin activation of these activities.

The possibility that prostaglandins might be responsible for the development of the pre-ovulatory refractoriness to the stimulation by LH of cyclic AMP accumulation in vitro in the Graafian follicle was examined. Isolated rabbit Graafian follicles were obtained at estrus and at 0.5, 5 and 9 hours after an ovulatory dose of LH. The follicles were incubated in vitro in the presence of [8-3H]adenine and the accumulation of [8-3H]cyclic AMP measured. Follicles from estrous animals responded to the in vitro addition of LH with a marked increase of cyclic AMP accumulation and lost this response as the time of ovulation approached. Animals pretreated with indomethacin, which inhibits the usual pre-ovulatory rise of follicular prostaglandin levels, showed essentially the same loss of responsiveness. Indomethacin alone was without effect. It is concluded that prostaglandins are not the major factor in the development of refractoriness to LH stimulation in vitro which has been observed in pre-ovulatory follicles.

We have used inhibitors of receptor-mediated endocytosis to investigate the mechanism and function of epidermal growth factor uptake by cultured cells. When rhodamine-labeled epidermal growth factor is bound to cell surface receptors on confluent monolayers of BALB/c 3T3 cells, it rapidly collects in cell surface clusters and is internalized. The clustering of occupied receptors requires Ca 2+ and is inhibited by primary alkylamines; both of these properties are shared by the enzyme transglutaminase (R-glutaminyl-peptide:amine γ-glutamyl-yltransferase, EC 2.3.2.13). In Chinese hamster ovary cell extracts, methylamine inhibits 25-50% of the transglutaminase activity with a K i of 0.2 mM, and it inhibits the remaining transglutaminase activity with a K i of 20 mM. Clustering is almost completely inhibited by 10 mM methylamine. The polypeptide antibiotic bacitracin inhibits clustering of rhodamine-labeled epidermal growth factor or α 2 -macroglobulin at 0.7 mM, and it inhibits approximately 40% of the transglutaminase activity in Chinese hamster ovary cells with a K i of 0.03 mM. Fluorescent ligands bound to cell surface receptors in the presence of bacitracin form clusters within 30 min after bacitracin is removed from the culture medium. These results indicate that a transglutaminase-like enzyme may be required for the clustering and subsequent internalization of occupied receptors. The effects of 10 mM methylamine and 0.7 mM bacitracin on epidermal growth factor stimulation of DNA synthesis were examined. The stimulation of DNA synthesis by epidermal growth factor was increased 2- to 7-fold in the presence of methylamine or bacitracin. Alone, methylamine or bacitracin increased DNA synthesis 1.1- to 3-fold. The stimulation of DNA synthesis resulting from the simultaneous presence of the hormone and the clustering inhibitor was always greater than the sum of the stimulations produced by the hormone and the clustering inhibitors alone. The potentiation of epidermal growth factor activity by clustering inhibitors suggests that the hormone acts at the cell surface. We propose that rapid internalization of occupied receptors via coated pits may be a mechanism to limit the response to hormones.

Filamin is a high molecular weight (subunit Mr 250 000) actin-binding protein isolated from smooth muscle. The protein forms a gel when mixed with solutions of F-actin. A proteolytic fragment of filamin, heavy merofilamin (subunit Mr 240 000), generated by the action of Ca2+-activated protease binds to actin but does not produce gelation. We have studied the self-association properties of filamin and heavy merofilamin by direct examination in the electron microscope and by equilibrium sedimentation distribution studies in the ultracentrifuge. Filamin self-associates reversibly to form dimers; the free energy of dimerization is approximately 7 kcal/mol. Further association to form tetramer and multimer appears to be irreversible. Warming of filamin solutions accelerates aggregation. Heavy merofilamin does not appear to self-associate but is entirely monomeric. These studies suggest that filamin produces gelation of F-actin by binding to actin and then self-associating to cross-link actin filaments into a gel.

Depending on the time of addition, prostaglandin I2 (PGI2; greater than or equal to 10(-9) M) either inhibits or reverses platelet agglutination mediated by human factor VIII-related von Willebrand factor activity (FVIIIvWF) and ristocetin, or bovine FVIIIvWF alone. 6-Keto-PGF1 alpha, the inactive metabolite of PGI2, is without effect, PGI2 inhibition is potentiated by the phosphodiesterase inhibitor, theophylline, and is not the result of PGI2 suppression of ADP release. PGI2 (+/- theophylline) does not inhibit ristocetin-induced binding of purified human 125I-FVIIIvWF multimers to washed platelets or to platelets treated with PGI2 and then formalin fixed (although subsequent agglutination of these platelets is impaired). Washed platelets treated previously with 2-aminoethylisothiouronium bromide (AET), an agent that reduces disulfide bonds and alters platelet membranes, also bind human 125I-FVIIIvWF multimers without agglutinating. We conclude that FVIIIvWF-mediated agglutination requires both functional platelet FVIIIvWF binding sites and platelet-platelet cohesion sites, and that platelet surface cohesion sites are altered by AET and PGI2. PGI2 from adjacent intact endothelial cells may prevent excessive platelet accumulation on exposed subendothelium without suppressing an essential hemostatic process--the binding of platelets to subendothelial FVIIIvWF.

Transglutaminases are a group of enzymes that catalyze the posttranslational modification of protein-bound glutamine residues. It appears that the major physiological role of these enzymes is to crosslink proteins by promoting the formation of isopeptide bonds between protein-bound glutamine and lysine residues. These enzymes also catalyze the covalent conjugation of polyamines to proteins and this latter activity has led to speculation about their involvement in the biology of polyamine action. In this report we would like to present a brief review of the theoretical and experimental evidence for considering transglutaminases as components in polyamine metabolism.

We find that the protease inhibitor N-α-tosyl-L-lysyl chloromethyl ketone (TLCK) inhibits the transformation-specific kinase activity (Collett and Erikson, 1978) associated with p60 src , the avian sarcoma virus (ASV) gene product responsible for the transformation of fibroblasts. TLCK has been shown to induce the phenotypic reversion of ASV-transformed cells to normal (Weber, 1975). Kinase activity was measured in extracts of chick embryo fibroblasts (CEF) transformed by the Schmidt-Ruppin strain of ASV (SR-ASV) with antiserum from rabbits bearing ASV-induced tumors. The immunoprecipitates were incubated with γ- 32 P-ATP under conditions in which the phosphorylation of the IgG heavy chain in the immunoprecipitate was directly proportional to the concentration of cell extract. When ASV-transformed CEF were treated with 0.1 mM TLCK, the kinase activity was reduced by 60% after 2 hr and by 80% after 6 hr, and continued to remain low for up to 40 hr when TLCK was present. When TLCK was removed, the kinase activity rose slowly over a period of many hours, suggesting that the enzyme is irreversibly inactivated by TLCK and new enzyme must be synthesized. The effect of TLCK in vivo is concentration-dependent and specific. Other serine protease inhibitors had no effect on kinase activity. At low concentrations (0.03 mM), TPCK produced partial inhibition (≤20%), but at higher concentrations TPCK was extremely toxic to the cells and therefore could not be tested. The inhibition by TLCK was not due to its ability to inhibit protein synthesis since cycloheximide treatment (1 μg/ml) did not significantly reduce kinase activity. TLCK also inhibited kinase activity when added directly to cell extracts, but about 5 times higher concentrations of TLCK were required to produce 50% inhibition. Under these conditions both TLCK and TPCK were comparable inhibitors, whereas PMSF had no effect. Our finding that the inhibition of the kinase by TLCK in vivo parallels the reversion of cell morphology to normal suggests that the kinase has an important role in transformation and offers a biochemical rationale for treatment of tumors with this agent.

The binding of [3H]prostaglandin E1 to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E1, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E1 binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E1 and have reduced prostaglandin E1 binding Exposure of cells to prostaglandin E1 results both in decreased prostaglandin E1 responsiveness and reduced prostaglandin E1 binding. Activation of adenylate cyclase is correlated to binding of prostaglandin E1 to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.

We have studied the role of ADP-ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL-60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP-ribose)n synthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP-ribose)n synthetase was decreased in HL-60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP-ribose)n synthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N'-methylnicotinamide (N'-Met-NAm), agents which decrease ADP-ribosylation. Treatment of HL-60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N'-Met-NAm was more potent than NAm in inducing maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N'-Met-NAm also potentiated the maturation of HL-60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose-response curve for RA; maturation was observed at 10(-11) M RA in the presence of either 2 mM NAm or 0.2 mM N'-Met-NAm while 10(-9) M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N'-Met-NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N'-Met-NAm decreased by about 75% the amount of endogenous (ADP-ribose)n in a selected fraction of chromosomal proteins which included histone H1 and the nonhistone high mobility group proteins. The results of this study support the concept that ADP-ribosylation of chromosomal proteins influences the regulation of human myeloid cell maturation.

Incubation of subcellular fractions of fibroblasts with [ 32 P]ATP demonstrated 10 phosphoproteins whose phosphorylation can be increased by cyclic AMP or cyclic AMP-dependent protein kinase. One of these phosphoproteins, MW 240,000, resembles the actin binding protein, filamin, and can be selectively precipitated by antibodies to chicken gizzard filamin. Furthermore chicken gizzard filamin can be phosphorylated by skeletal muscle protein kinase and cyclic AMP stimulates this reaction.

We have investigated the regulation of transglutamine activity (ϵ-(γ-glutamyl)lysine crosslinking enzme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of the soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20–70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.

The response of isolated ovarian follicles to gonadotropic stimulation in vitro has been investigated. Large follicles (1–2 mm diameter) dissected from estrous rabbit ovaries were cleared of interstitium and then incubated in Krebs-Ringer bicarbonate buffer containing 100 μCi sodium acetate-l-14C plus gonadotropin or an equivalent concentration of bovine serum albumin as control. Incorporation of radioactivity into estrone and 17β-estradiol was assessed by reverse isotope dilution analysis with recrystallization of radioactive products to constant specific activity. Luteinizing hormone (LH) at concentrations of 2.5, 0.25, 0.025 or 0.0025 μg/ml caused 5-to 13-fold increases in incorporation of radioactivity into estrogens; LH at 0.001 μg/ml caused a doubling of incorporation. Prolactin (100 μg/ml) was without stimulatory activity in the follicular system. The following FSH preparations caused increases in incorporation which could be related to the LH content: i) follicle stimulating hormone (FSH) at conce...

The deduced amino acid sequences for tissue transglutaminases from human endothelial cells and mouse macrophages have been derived from cloned cDNAs. Northern blot analysis of both tissue transglutaminases shows a message size of approximately 3.6-3.7 kilobases. The molecular weights calculated from the deduced amino acid sequences were 77,253 for human endothelial tissue transglutaminase and 76,699 for mouse macrophage tissue transglutaminase. The deduced amino acid sequence for the human endothelial transglutaminase was confirmed by comparison with the amino acid sequence obtained by cyanogen bromide digestion of the human erythrocyte transglutaminase. The amino acid sequences of both human endothelial and mouse macrophage tissue transglutaminases were compared to other transglutaminases. A very high degree of homology was found between human endothelial, mouse macrophage, and guinea pig liver tissue transglutaminase (greater than 80%). Moreover, human endothelial tissue transglutaminase was compared with human Factor XIIIa and a very high degree of homology (75% identity) was found in the active site region.

Hypertriglyceridemia is a frequent complication accompanying the treatment of patients with either retinoids or rexinoids, [retinoid X receptor (RXR)-selective retinoids]. To investigate the cellular and molecular basis for this observation, we have studied the effects of rexinoids on triglyceride metabolism in both normal and diabetic rodents. Administration of a rexinoid such as LG100268 (LG268) to normal or diabetic rats results in a rapid increase in serum triglyceride levels. LG268 has no effect on hepatic triglyceride production but suppresses post-heparin plasma lipoprotein lipase (LPL) activity suggesting that the hypertriglyceridemia results from diminished peripheral processing of plasma very low density lipoproteins particles. Treatment of diabetic rats with rexinoids suppresses skeletal and cardiac muscle but not adipose tissue LPL activity. This effect is independent of changes in LPL mRNA. In C2C12 myocytes, LG268 suppresses the level of cell surface (i.e., heparin-releasable) LPL activity without altering LPL mRNA. This effect is very rapid (t(1/2) = 2 h) and is blocked by the transcriptional inhibitor actinomycin D. These studies demonstrate that RXR ligands can have dramatic effects on the post-translational processing of LPL and suggest that skeletal muscle may be an important target of rexinoid action. In addition, these data underscore that the metabolic consequences of RXR activation are distinct from either retinoic acid receptor or peroxisome proliferator-activated receptor activation.

Background Serotonin (5HT), released from aggregating platelets at sites of vascular injury following coronary angioplasty, is a known mitogen for vascular smooth muscle cells (SMC). Retinoids have been shown to inhibit cellular proliferation possibly due to effects on cytokines, cytokine receptors and proto-oncogene expression. Objective In this study we examined whether all trans retinoic acid (RA) or its derivatives 13-Cis RA and 9-Cis RA would prevent the 5HT induced SMC proliferation as determined by 3 [H]-thymidine incorporation into DNA. Methods Canine aortic primary SMC (in 2nd or 3rd passage) were incubated with different concentration of RA 13-Cis RA or 9-Cis RA and 200 μM of serotonin for 20 hours in serum free medium. Then 3 [H]-thymidine (1 μCi/plate) was added and 3 [H]-thymidine incorporation into SMC was measured 4 hours later. Results The 200μM of serotonin induced approximately a 3–4 fold increase in 3 [H]-thymidine incorporation (5570 ± 242 cpm/10 6 cells to 18202 ± 881 cpm/l0 6 cells) in SMC (n = 5). All three retinoids inhibited serotonin induced SMC proliferation. The dose of retinoids that produced a 50% reduction in DNA (p l 0.0001) synthesis in SMC were 47nM for RA 410nM for 9-Cis RA and 5μM for 13-Cis RA. There was no evidence of cytotoxicity with any of the retinoids to SMC up to a concentration of 1–5 mM. Conclusion Retinoids prevent 5HT induced SMC proliferation at low concentrations. Since 5HT released from platelets at sites of vascular injury may playa role in the development of neointimal proliferation following coronary angioplasty, retinoids may potentially be useful for prevention of restenosis following angioplasty.