Search

Tissue transglutaminase (tTG) is upregulated in various cells undergoing apoptosis. To investigate the transcriptional regulation of tTG a mouse strain carrying a beta-galactosidase reporter gene under the control of a 3.8 kilobase fragment of the tTG promoter was characterised. The transgene construct was shown to be expressed in the apoptotic regions of the mouse embryo. Here we report that the regulation of the transgene is also apoptosis-linked in adult animals. The transgene is induced in endocrine apoptosis involving mammary gland involution and corpus luteum regression. Induction of the reporter gene is detectable during in vivo but not in vitro apoptosis of thymocytes induced by the glucocorticoid receptor, the nur77, p53 and the retinoid receptor gamma mediated pathways. Additionally, the lacZ expression mimics the activation of the endogenous promoter in tissues characterised by high apoptotic turnover. These results suggest that the apoptosis-specific transcriptional regulation of tTG is mediated through elements of a 3.8 kb promoter and may require cosignals available only in tissue environment. Cell Death and Differentiation (2000) 7, 1225 - 1233.

The cross-linking enzyme tissue transglutaminase (tTG) participates in a variety of cellular functions. To assess its contribution to extracellular and intracellular processes during development we cloned the cDNA for chicken heart tissue transglutaminase and localized the sites of transglutaminase expression by in situ hybridization and immunohistochemistry. Compared with the chicken red blood cell transglutaminase cDNA, the heart cDNA encodes a transglutaminase with an amino-terminal truncation. The truncated enzyme retains full catalytic activity and is GTP-inhibitable. Tissue transglutaminase expression was observed in developmentally transient structures in embryonic chicken limb at day 7.5 of incubation suggesting that its expression is dynamically regulated during limb morphogenesis. The major morphogenetic events of the limb associated with transglutaminase expression were cartilage maturation during skeletal development, interdigital apoptosis, and differentiation of skeletal muscle. Maturation of the cartilage during endochondral ossification was characterized by intra- and extracellular transglutaminase accumulation in the zone of hypertrophic chondrocytes. Only intracellular enzyme could be detected in mesenchymal cells of the prospective joints, in apoptotic cells of the interdigital web, and in skeletal muscle myoblasts. An apparently constitutive expression of tissue transglutaminase was found in vascular endothelial cells corresponding to the adult expression pattern. The dynamic pattern of transglutaminase expression during morphogenesis suggests that tissue remodeling is a major trigger for transglutaminase induction.

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.

The cardiac response to increased work includes a reactivation of fetal genes. The response to a decrease in cardiac work is not known. Such information is of clinical interest, because mechanical unloading can improve the functional capacity of the failing heart. We compared here the patterns of gene expression in unloaded rat heart with those in hypertrophied rat heart. Both conditions induced a re-expression of growth factors and proto-oncogenes, and a downregulation of the 'adult' isoforms, but not of the 'fetal' isoforms, of proteins regulating myocardial energetics. Therefore, opposite changes in cardiac workload in vivo induce similar patterns of gene response. Reactivation of fetal genes may underlie the functional improvement of an unloaded failing heart.

Tissue transglutaminase is a calcium-dependent, protein cross-linking enzyme that is highly expressed in cells undergoing apoptosis. The expression of tissue transglutaminase is regulated by a variety of molecules including retinoids, interleukin-6, and transforming growth factor-beta1 (TGF-beta1). Retinoid and interleukin-6 inductions of tissue transglutaminase expression are mediated by specific cis-regulatory elements located within the first 4.0 kilobase pairs of the promoter of the gene. The present studies were designed to identify the molecular mechanisms mediating the regulation of tissue transglutaminase gene expression by TGF-beta family members. Transient transfection of Mv1Lu cells with transglutaminase promoter constructs demonstrated that 0.2 nM TGF-beta1 maximally induced the activation of the promoter through a 10-base pair TGF-beta1 response element (TRE; GAGTTGGTGC) located 868 base pairs upstream of the transcription start site. This same element mediated an inhibitory activity of TGF-beta1 on the transglutaminase promoter in MC3T3 E1 cells. The TRE through which TGF-beta1-regulated the activity of the transglutaminase promoter was necessary and sufficient for bone morphogenetic protein 2- (BMP) and BMP4-dependent inhibition of the tissue transglutaminase promoter. The TGF-beta1, BMP2, and BMP4 regulation of the transglutaminase promoter activity was similar to the responses we observed for the endogenous transglutaminase activity of Mv1Lu and MC3T3 E1 cells. For BMP2 and BMP4, this regulation was paralleled by a decrease in tissue transglutaminase mRNA in MC3T3 E1 cells. The results of these experiments suggest that TGF-beta1, BMP2, and BMP4 regulation of mouse tissue transglutaminase gene expression requires a composite TRE located in the 5'-flanking DNA.

One of several postulated roles for tissue transglutaminase (tTG) is the stabilization and assembly of extracellular matrix via peptide cross-linking. We previously determined that tTG activity increased in an animal model of hepatic fibrogenesis and in human liver disease. To further study the role of tTG in liver disease, we initiated investigations into the effect of a proinflammatory mediator, tumor necrosis factor (TNF)-alpha, on tTG activity in cultured liver cells. Treatment of human Hep G2 cells with 1 ng/ml TNF-alpha increased [14C]putrescine cross-linking to cellular proteins. An increase in tTG mRNA content was observed 1 h after addition of TNF-alpha, and levels of tTG mRNA remained elevated after 24 h. Hep G2 cells, transiently transfected with a luciferase reporter containing 1.67 kb of the human tTG promoter, showed an increase in reporter activity after addition of TNF-alpha. Gel shift experiments using nuclear extracts from TNF-alpha-treated cells and oligonucleotides containing the tTG nuclear factor (NF)-kappa B motif revealed increased binding, concordant with mRNA data. Transient transfections with a truncated reporter construct lacking the tTG NF-kappa B sequence showed an attenuated response to TNF-alpha treatment. Similar responses were seen in stably transfected HeLa cells. Primary hepatocytes isolated from a transgenic mouse line containing the mouse tTG promoter driving the beta-galactosidase reporter, show similar time-dependent increases in promoter activity when treated with TNF-alpha. Furthermore, Hep G2 cells are incapable of upmodulating tTG promoter reporter activity in the presence of TNF-alpha when those cells overexpress a transdominant, negative mutant NF-kappa B subunit. Because TNF-alpha expression is upregulated in hepatic inflammation, the data suggest TNF-alpha-mediated increases in tTG expression may play an important role in the process of hepatic fibrogenesis.

Retinoic acid receptors (RAR), thyroid hormone receptors (TR), peroxisome proliferator activated receptors (PPARs) and the orphan receptor, LXR, bind preferentially to DNA as heterodimers with a common partner, retinoid X receptor (RXR), to regulate transcription. We investigated whether RXR-selective agonists replicate the activity of ligands for several of these receptors? We demonstrate here that RXR-selective ligands (referred to as rexinoids) function as RXR heterodimer-selective agonists, activating RXR: PPARgamma and RXR:LXR dimers but not RXR:RAR or RXR:TR heterodimers. Because PPARgamma is a target for antidiabetic agents, we investigated whether RXR ligands could alter insulin and glucose signalling. In mouse models of noninsulin-dependent diabetes mellitus (NIDDM) and obesity, RXR agonists function as insulin sensitizers and can decrease hyperglycaemia, hypertriglyceridaemia and hyperinsulinaemia. This antidiabetic activity can be further enhanced by combination treatment with PPARgamma agonists, such as thiazolidinediones. These data suggest that the RXR:PPARgamma heterodimer is a single-function complex serving as a molecular target for treatment of insulin resistance. Activation of the RXR:PPARgamma dimer with rexinoids may provide a new and effective treatment for NIDDM.

Purpose/Objective(s): Traditional clonogenic survival and high throughput colorimetric assays are inadequate for drug screens to identify novel radiation sensitizers. To bridge this gap in knowledge, we have developed a method which we coin the High Throughput Clonogenic Survival Screen (HCS) that will allow high volume screening of drug libraries to identify potent radiation sensitizers, particularly for K-ras mutant lung cancer, a current unmet need. Materials/Methods: Drugs at various concentrations or vehicle were added to cells seeded overnight in 96 well plates at low densities, and after 6 hours, plates were irradiated at various doses. Plates were kept in culture 4-6 days depending on the cell line. After incubation, colonies were stained with cresyl-violet, imaged on a high content screening system, and colony count determined using a proprietary image analysis software. Colonies achieving 50 or more cells were tallied. Traditional clonogenic survival assays (tCSA) were performed by seeding cells into 6-well plates overnight in triplicate and adding drugs or vehicle for 6 hours prior to irradiation. Media was changed after 3 days, and plates were kept in culture for 10-14 days. Drug screens were done using the K-ras mutant lung cancer cell line H460 and the Custom Clinical Collection (145 compounds). Results: We compared the HCS method to tCSA and found that HCS recapitulates a similar cell survival curve and survival fraction at 2 Gy (SF2) values for several cancer cell types. As a proof-of-principle, we tested a known sensitizer vorinostat (SAHA) in both the tCSA and HCS assays. As expected, 1 mM vorinostat caused a significant radiation sensitizing effect using tCSA, and was recapitulated, albeit at a lower concentration (100 nM), using the HCS assay. Drug screens (repeated 3 times) identified several compounds that were cytotoxic to the K-ras mutant H460 cells alone at 1 mM (inhibitors to Src/Abl, IGF1R, ROCK1, AKT, PI3K, c-MET) and at low nM concentrations (HSP90 and HDAC inhibitors). The entire class of HDAC inhibitors (5 drugs) had some sensitizing effect to radiation (a leftward shift in the IC50 curve by w10 fold). However, many drugs such as inhibitors to PARP, SRC, Chk1/2, DNA-PK, JNK, and MEK1/2, exhibited significant radiation sensitizing effect with minimal to no activity by themselves. We validated the sensitizing effect of the potent MEK1/2 inhibitor trametinib using tCSA, and found the effect was specific for K-ras mutant and not K-ras wild type cells. Conclusions: High throughput drug screening for novel radiation sensitizers for K-ras mutant lung cancer is feasible using the HCS approach. This is an enabling technology that will allow synthetic lethal drug screens with radiation and accelerate the translation of drugs into clinical testing. Author Disclosure: S.H. Lin: None. J. Zhang: None. U. Giri: None. C. Stephan: None. M.M. Sobieski: None. N. Nguyen: None. P. Davies: None. R. Komaki: None. S.S. Yoo: None. J.V. Heymach: None.

Vitamin A and its derivatives (retinoids) have been known to cause premature epiphyseal closure in humans as an unwanted side effect of chronic treatment. The purpose of the present study was to determine if guinea pigs could serve as an animal model of retinoid-induced epiphyseal plate closure, and to utilize this model to study the mechanism. Weanling male Hartley guinea pigs were treated ip via osmotic pump for up to 14 days with vehicle or 0.50 to 5.5 mg/kg/day of the retinoic acid receptor (RAR)-selective agonist AGN 190121. Histopathological examination of the proximal tibia of AGN 190121-treated guinea pigs revealed a dose-dependent disruption of the epiphyseal plate. The natural retinoids all-trans-retinoic acid and 13-cis-retinoic acid also induced epiphyseal plate closure in guinea pigs when administered by ip injection for 10 days. Prominent histological features of retinoid-induced epiphyseal closure included the loss of basophilic staining in the extracellular matrix of epiphyseal plate chondrocytes and the invasion of the epiphyseal plate by osteoclasts. To determine if the epiphyseal closure detected histologically was reversible, guinea pigs were treated for 6 days with the RAR-selective agonist (E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propen- 1-y1]benzoic acid (TTNPB) or vehicle, and groups of guinea pigs were euthanized on Day 7 or 57. TTNPB but not vehicle treatment caused histological evidence of epiphyseal closure at both time points, and significant bone elongation between Day 7 and Day 57 was detected only in vehicle-treated animals. Epiphyseal closure and other toxic effects of TTNPB were blocked by cotreatment of guinea pigs with a fivefold molar excess of AGN 193109, an RAR antagonist. Taken together, these data demonstrate the utility of the guinea pig as an animal model of retinoid-induced epiphyseal closure and suggest that RAR activation is necessary and sufficient for this activity.

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.

A 92-kD transglutaminase (TGase K), expressed in human cultured keratinocytes and stratum corneum, catalyzes a critical step in the formation of the cornified envelope of terminal differentiation. A rabbit polyclonal antibody to TGase K was used to isolate overlapping cDNA clones from a human keratinocyte cDNA expression library. The cDNA clones were sequenced and unequivocally identified as TGase K by comparison to the N-terminal amino acid sequences of two cyanogen bromide fragments from the purified enzyme. The mRNA for Tgase K is expressed in cultured keratinocytes but not in A431 squamous carcinoma cells, in fibroblasts, or in other non-epithelial tissues and cells. Although TGase K protein expression is limited to the upper layers of normal epidermis, the mRNA is generally present throughout the epidermis, suggesting the possibility of post-transcriptional regulation. Precocious expression of TGase K protein occurs in psoriasis, and quantitative Northern blot analysis of TGase K mRNA from normal and involved epidermal biopsies from psoriasis patients suggests that TGase K mRNA levels are increased in psoriatic lesions. By using quantitative laser scanning confocal microscopy (LSCM) and in situ hybridization, the increase of the TGase K mRNA was in the range of 3-7 times in the psoriatic epidermis and was significantly higher compared with normal skin and with paired adjacent skin. Quantitative LSCM provides a powerful and direct method for analysis of gene expression in skin.

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.

Objective We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. Study Design Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. Results No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. Conclusion Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

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.

Monitoring minimal residual disease (MRD) in chronic myeloid leukemia after imatinib therapy can help in determining therapeutic strategy. Sensitive methods based on the detection of the BCR/ABL fusion gene or its fusion transcript by FISH or RT-PCR, respectively, are used to monitor residual leukemia in peripheral blood or bone marrow cells. While sensitive, these methods require an invasive procedure to obtain bone marrow. The patchy nature of the residual disease may cause some of the reported variation in detecting MRD. We hypothesized that plasma RNA level reflects total body disease and is unaffected by the tendency of some myeloid cells not to circulate. We measured the BCR/ABL fusion transcript by real-time quantitative RT-PCR using RNA isolated from blood plasma. We used a quantitative RT-PCR assay capable of measuring both the b2a2 and b3a2 BCR/ABL fusion transcripts by targeting exon 13 of BCR with the 5′ and exon 2 of ABL by the 3′ primer. RNA isolated from 10–30 μl of frozen plasma was subjected to real-time quantitative RT-PCR using the Taqman technology. Quantification was achieved by extrapolation from a standard curve generated from a ten-fold dilution series of a synthetic oligonucleotide corresponding to the (+) strand of the target sequence. To normalize the fusion mRNA levels we also measured β-actin mRNA from the same samples. Assays were run in duplicate. The results of plasma RT-PCR were correlated with results of conventional cytogenetic analysis, FISH, and quantitative RT-PCR of bone marrow suspension (“bone marrow PCR”). Plasma and bone marrow samples were obtained at 3, 6, 9, 12 and 15 months after imatinib therapy. Ninety-five plasma samples were analyzed. There was a strong positive correlation between the results of plasma RT-PCR vs. positive cytogenetics (n=95, p

ALTERED EXPRESSION OF TYROSINE KINASE RECEPTORS HER2/NEU AND GP130 FOLLOWING LEFT VENTRICULAR ASSIST DEVICE (LVAD) PLACEMENT IN PATIENTS WITH HEART FAILURE I.P. Uray, J.H. Connelly, O. Frazier, H. Taegtmeyer, P.J. Davies; University of Texas at Houston Medical School, Houston, TX; St. Luke’s Hospital, Houston, TX; Texas Heart Institute, Houston, TX; University of Texas Health Science Center, Houston, TX , USA

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.

We developed a specific antibody to the catalytic subunit (C-subunit) of cyclic AMP-dependent protein kinase and used it to localize C-subunit in cultured cells. C-subunit antigen was purified from bovine cardiac muscle and cross-linked to hemocyanin with glutaraldehyde. Immunized goat serum showed a low titer of antibody after boosting; it was enriched 100-fold by affinity chromatography on catalytic subunit-Sepharose. The antibody immunoprecipitated C-subunit from type I and type II holoenzyme and depleted enzymatic activity from solution. At 12.5 nM antigen, 1 microgram antibody immunoprecipitated 10 ng of C-subunit. Immunoprecipitation of 35S-labeled cell extracts and 125I-antibody detection on nitrocellulose paper revealed that the antibody specifically reacts with C-subunit in Chinese hamster ovary (CHO) whole cell extracts. Using indirect immunofluorescence to localize C-subunit, we found a pattern of diffuse staining in the cytoplasm of CHO cells with little or no nuclear staining. A similar distribution of the enzyme was observed in Swiss 3T3 cells, bovine endothelial tracheal cells, human lung fibroblasts and NRK cells. Treatment of CHO cells with 8-bromo-cyclic AMP produced no change in the pattern or intensity of immunofluorescence. We conclude that the majority of C-subunit is localized in cytoplasm and that in cultured fibroblasts exposure to cyclic AMP analogues causes no apparent redistribution of catalytic subunit.

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.

The receptor-mediated endocytosis of alpha 2-macroglobulin can be inhibited by a diverse group of chemical compounds all of which share the property of being inhibitors of one form of cellular transglutaminase. The present results strongly suggest that protein cross-linking may be essential for receptor-mediated endocytosis of some protein and polypeptide hormones.

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.

The function of intracellular transglutaminases remains to be clarified. In fibroblasts the links between the activity of this enzyme and receptor-mediated endocytosis are complex and open to interpretation. However, the issue cannot be firmly laid to rest until the structural specificity of the alkylamine inhibitors of endocytosis is explained. In macrophages, there is substantial evidence that the enzyme plays some role in receptor-mediated phagocytosis, but what this role is and how it might relate to endocytosis in other types of cells is at present an unresolved issue.

The reefless tract directly behind the ribbon reefs on the outer shelf off Cooktown supports a luxuriant growth of Halimeda that, during the Holocene, has developed into bioherms. These mounded biodies of unconsolidated sediment have formed banks that vary in height between 2 and 20 m. Combined shallow, high-resolution seismic reflection profiles and side-scan sonar have diferentiated three areas of biohermal complexes behind the ribbon reefs of Cooktown. Observations by SCUBA and submersible plus the sedimentology of the bioherms indicate that they are in situ accumulations. Evidence from dating of cores suggests that the Halimeda bioherms began to grow about 10 000 years B.P. and their growth has continued to the present time, even though their tops are presently restricted to a depth of -20 m. It is suggested that the origin and morphology of the bioherms are related to a specific hydrodynamic phenomenon, involving jets of nutrient-rich, upwelled oceanic water intruding onto the outer shelf via the narrow passes between the ribbon reefs, and forming eddies behind the ribbons.

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.

We have purified the high molecular weight actin-binding protein, filamin from guinea pig vas deferens. We find this mammalian filamin is very similar to chicken gizzard filamin in subunit molecular weight, amnio acid composition, actin-binding properties, immunological cross-reactivity, and the ability to be phosphorylated by cyclic AMP-dependent protein kinase. Anti-filamin antibodies cross-react with a high molecular weight macrophage actin-binding protein, and with a high molecular weight protein in platelets and fibroblasts. Furthermore like filamin, these proteins are also phosphorylated and cyclic AMP stimulates their phosphorylation. Anti-filamin antibodies do not cross-react with the erythrocyte membrane protein spectrin or with high molecular weight proteins in brain extracts. We conclude that filamin from avian and mammalian smooth muscle are very similar proteins and furthermore that many, but not all, non-muscle cells contain a protein closely related to filamin.

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.

Chicken gizzard filamin has been digested with purified Ca2+-activated protease. The subunits of (Mr = 250,000) of the protein are cleaved asymmetrically into two fragments, heavy merofilamin, Mr = 240,000, and light merofilamin, Mr = 9,500. Digestion is complete at substrate to enzyme ratios of 100:1 and requires Ca2+ concentrations in excess of 0.3 mM. Heavy merofilamin binds to F-actin as evidenced by cosedimentation with F-actin, by direct observation under the electron microscope, and by its ability to inhibit actin activation of heavy meromyosin ATPase. Heavy merofilamin does not form a gel when mixed with actin, except at very low concentrations of KCl. Thus, actin binding and gelation are separable activities of filamin. We speculate that Ca2+-stimulated proteolysis may play a role in the regulation of actin-filamin interactions.

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.

An operational procedure is developed for the optical rectification and gridding of satellite photographs. The procedure is described and examples are shown to illustrate both the advantages and disadvantages of the optical rectification system over earlier methods which rectified the geographical grid rather than the Tiros photograph. The problems involved in this rectification system are largely those of achieving dynamic similarity between the film projection system with its spherical section, and the Tiros system which was actually used in obtaining the film (the vidicon camera which photographed the true earth and its cloud cover at an angle). Finally, a relatively simple computer program is devised for the determination of the satellite tilt angle and the latitude and longitude of the TPP when only the TSP co-ordinates, and the satellite altitude are known. (Author)

Tissue transglutaminase (tTgase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification of proteins via epsilon(gamma-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signaling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

α2-MACROGLOBULIN (α2M), insulin and epidermal growth factor (EGF) have a common mechanism for internalisation1. We have proposed a model for this mechanism1–3 in which the ligands bind to diffusely distributed receptors followed by the collection of the mobile ligand–receptor complexes over coated pits. These coated pits pinch off from the plasma membrane to form endocytic vesicles. The diffuse initial distribution of the α2M receptors has been shown by binding α2M to fixed cells and observing the location of α2M by an electron microscopic immunocytochemical method2. The mobility of the ligand–receptor complexes has been demonstrated using fluorescence photobleaching recovery4, and the clustering of such complexes has been demonstrated by fluorescence microscopy1,3,5. Electron microscopic immunocytochemical localisation has been used to show that the clustering occurs over coated pits which pinch off to form endocytic vesicles2. The mechanism proposed for the endocytosis of α2M, insulin and EGF is similar to that described for the internalisation of low density lipoprotein (LDL), except that unoccupied LDL receptors may cluster in coated pits before binding LDL6. It seems likely that many molecules, including other polypeptide hormones and the lysosomal hydrolases7 which bind to specific receptors on the cell surface, are internalised by a similar mechanism. To determine the role of clustering and internalisation in hormone action it would be useful to have an agent that inhibits these processes. It has been found that the internalisation of lysosomal enzymes is noncompetitively inhibited by some amines and ammonia (E. Neufeld, personal communication). It has also been reported that ammonia prevents the internalisation of diptheria toxin by HeLa cells8. In view of these effects, we examined the possibility that amines and ammonia would block the clustering of α2M and EGF, and we report here that these agents are effective in blocking clustering and internalisation.

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.

Studies of modem coral reefs have shown that Holocene reef growth is relatively thin, and that in many instances it has developed on an older limestone surface1–4. This surface is commonly associated with leaching and pedogenic processes, indicative of subaerial exposure, and has been termed the solution unconformity5. Investigations into reef growth history have interpreted the pre-Holocene substrate as an older reef surface2–4,6–8 that may or may hot have undergone karst erosion during periods of lower sea level6,9. Uranium-series dating of corals directly below the uppermost solution unconformity at Eniwetok10 and Mururoa11 has shown that previous reef growth, before the Holocene, occurred ∼120 kyr ago. We present here 230Th/234U dates that confirm a last interglacial age for reefal limestones directly beneath the uppermost solution unconformity in the southern Great Barrier Reef.