Polychlorinated biphenyls (PCBs) decrease the placental syncytiotrophoblast volume and increase Placental Growth Factor (PlGF) in the placenta of normal pregnancy.

INTRODUCTION: Polychlorinated biphenyls (PCBs) are a class of biologically active, highly stable compounds. Exposure risks include consumption of fatty fish, meat, dairy products and human breast milk, as well as environmental and occupational settings. Numerous reports have described PCB-dependent adverse effects on human fetal growth, including increased risk for IUGR, changes in endocrine function and hormone metabolism, and immunosuppressive and neurological deficits. Here we test the prediction that in utero PCB exposure adversely effects placental morphology, potentially leading to placental insufficiency en route to fetal growth restriction. METHODS: PCB homologs (10) were measured in the maternal and fetal blood of a small cohort of normotensive pregnancies (22) by gas chromatography-mass spectrometry. PCB levels were compared with angiogenesis associated proteins Placental Growth Factor (PlGF) and sFlt-1, determined by ELISA, and the total estimated syncytiotrophoblast (ST) volume. RESULTS: Significant associations between PCB exposure and both PlGF and ST volume were identified. DISCUSSION: PCB effects on placenta morphology and predicted function are discussed. CONCLUSION: These results demonstrate that the human placenta, including ST, is a target of PCB toxicity, and that current environmental PCB exposure levels are a risk to reproductive health.

Synergistic actions of formamidine insecticides on the activity of pyrethroids and neonicotinoids against Aedes aegypti (Diptera: Culicidae).

Formamidines are unique insecticides and acaricides that elicit multiple effects in controlling insects. Here, we tested two formamidines, amitraz, and chlordimeform, for their synergistic actions on type II pyrethroids and neonicotinoids to increase their larvicidal actions on the fourth instars of Aedes aegypti L. An organophosphate insecticide was used as a negative control. After 24 h, the synergism of formamidines was highest on imidacloprid, followed by two type II pyrethroids, deltamethrin and fenvalerate. After 48 h, the synergism of formamidines on imidacloprid decreased, remained unchanged on type II pyrethroids, and increased noticeably on two of the newer type neonicotinoids, dinotefuran and thiamethoxam. By 72 h, synergism of formamidines on dinotefuran reached the maximum, while that on imidacloprid was at a minimum. Both formamidines did not show synergistic effects on permethrin or fenitrothion. In all cases, the synergistic effects of amitraz on the two major classes of larvicides were greater than for chlordimeform. These results indicate that amitraz is a promising synergist that shows the potential to increase the efficacy of certain members of type II pyrethroids as well as neonicotinoids to control Ae. aegypti larvae.

Spontaneous arterial baroreflex control of the heart rate during head-down tilt in heat-stressed humans.

The purpose of this study was to elucidate the effect of raised body temperature per se during acute heat stress on the spontaneous arterial baroreflex control of heart rate (fc) in humans. To investigate whether unloading of cardiopulmonary baroreceptors during whole-body heating would alter the arterial baroreflex control of fc, we controlled loading of the cardiopulmonary baroreceptors by head-down tilt (HDT) at angles of 5 degrees, 10 degrees, 15 degrees, and 30 degrees during heat stress produced by hot-water-perfused suits. The sensitivity of the arterial baroreceptor-cardiac reflex was calculated from the spontaneous changes in beat-to-beat arterial pressure and fc. As an index of cardiopulmonary baroreceptor loading, the left atrial diameter (LAD) was measured by echocardiography. During whole-body heating, the LAD decreased with the rising body core temperature and increased with the HDT. The decreased LAD during heating almost recovered to the normothermic control level by 10 degrees HDT. In the supine position, cardiac baroreflex sensitivity remained unchanged during heating. Arterial pressure, fc and cardiac baroreflex sensitivity were not changed by HDT ranging from 5 degrees to 30 degrees during heating. These results suggest that cardiac baroreflex sensitivity remain unchanged during graded loading of the cardiopulmonary baroreceptors in heat-stressed humans. Also, we conclude that the sensitivity of the spontaneous arterial baroreflex controlling the fc is not influenced by raised body temperature per se during acute heat stress.

Effects of heptachlor epoxide on components of various signal transduction pathways important in tumor promotion in mouse hepatoma cells. Determination of the most sensitive tumor promoter related effect induced by heptachlor epoxide.

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE; 0, 0.1, 1, 10, and 50 microM) on several cellular tumor promoter-sensitive parameters were studied in mouse 1c1c7 hepatoma cells in an effort to identify the most sensitive biomarker for OC promoter exposure and the critical pathway and target of OC promoters. The levels of Ca2+ in the endoplasmic reticulum (ER) store, connexin43 (Cx43), PLCgamma(1), nPKCvarepsilon, and AP-1 DNA binding in nucleus were studied to screen for effects induced by submicromolar HE levels. While all the parameters tested elicited effects, particulate PLCgamma(1) and AP-1 DNA binding were found to be the most sensitive parameters affected by HE on both dose and temporal bases. Their levels were increased with 10- to 100-fold lower HE concentrations than were required to affect nPKCvarepsilon or Cx43. Further, with the lower HE dosages, particulate PLCgamma(1) and nuclear AP-1 were positively modulated by HE after 1 h versus 3 or 72 h for nPKCvarepsilon and Cx43. Ca2+ store depletion was probably the third most sensitive parameter, after AP-1 and PLCgamma(1). These results suggest the tyrosine kinase growth factor receptor pathway is the probable critical pathway for HE-induce tumor promotion with the critical target most likely being upstream of PLCgamma(1) and AP-1. This work also demonstates that upon exposure to a tumor promoter such as HE, many hepatocellular effects or changes result, suggesting that a cellular-program shift occurs similar to that described by the resistant hepatocyte model after exposure to a carcinogen or enzyme inducer.

Mutant Caldesmon lacking cdc2 phosphorylation sites delays M-phase entry and inhibits cytokinesis.

Caldesmon is phosphorylated by cdc2 kinase during mitosis, resulting in the dissociation of caldesmon from microfilaments. To understand the physiological significance of phosphorylation, we generated a caldesmon mutant replacing all seven cdc2 phosphorylation sites with Ala, and examined effects of expression of the caldesmon mutant on M-phase progression. We found that microinjection of mutant caldesmon effectively blocked early cell division of Xenopus embryos. Similar, though less effective, inhibition of cytokinesis was observed with Chinese hamster ovary (CHO) cells microinjected with 7th mutant. When mutant caldesmon was introduced into CHO cells either by protein microinjection or by inducible expression, delay of M-phase entry was observed. Finally, we found that 7th mutant inhibited the disassembly of microfilaments during mitosis. Wild-type caldesmon, on the other hand, was much less potent in producing these three effects. Because mutant caldesmon did not inhibit cyclin B/cdc2 kinase activity, our results suggest that alterations in microfilament assembly caused by caldesmon phosphorylation are important for M-phase progression.

Increased ErbB-2 tyrosine kinase activity, MAPK phosphorylation, and cell proliferation in the prostate cancer cell line LNCaP following treatment by select pesticides.

The oncogene erbB-2 codes for a receptor tyrosine kinase that functions as a key mitotic signal in a variety of cell types. Amplification or overexpression of erbB-2 occurs in many forms of cancer, such as of the breast, colon, and prostate, and is an indicator of poor prognosis in those diseases. In the human prostate cancer cell lines LNCaP and PC-3, erbB-2 kinase was activated by pesticides of different chemical classes: (1) the organochlorine insecticides beta-hexa-chlorocyclohexane (beta-HCH), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), and heptachlor epoxide; (2) the pyrethroid insecticide trans-permethrin, and (3) the fungicide chlorothalonil. o,p'-DDT also causes phosphorylation of mitogen-activated protein kinase (MAPK) and cellular proliferation of the androgen-dependent LNCaP line. However, no proliferative effect was observed in the androgen-independent PC-3 line. The proliferative effect of o,p'-DDT in LNCaP could not be blocked by the androgen receptor antagonist p,p'-dichlorodiphenyldichloroethene (p,p'-DDE), indicating that this effect of o,p'-DDT does not occur through direct interaction with the androgen receptor. Together these data demonstrate a putative mechanism for the action of certain pesticides in hormonal carcinogenesis.

Down-regulation of particulate protein kinase Cepsilon and up-regulation of nuclear activator protein-1 DNA binding in liver following in vivo exposure of B6C3F1 male mice to heptachlor epoxide.

The effects of in vivo administration of the cyclodiene tumor promoter heptachlor epoxide on mouse liver protein kinase C were studied in male B6C3F1 mice by protein kinase C activity assays and Western blotting under conditions known to increase the incidence of hepatocellular carcinoma because protein kinase C is thought to be critical in phorbol ester-induced tumor promotion. Under these test conditions, 20 ppm dietary heptachlor epoxide for 1-20 days increased cytosolic and decreased particulate total protein kinase C activities, while 10 ppm had no effect. Further, total cytosolic and particulate protein kinase C activities were decreased within 1 hour by 10 mg/kg intraperitoneal (i.p.) heptachlor epoxide. Western blotting showed that conventional protein kinase Calpha and beta isoforms were unaffected by heptachlor epoxide. Particulate novel protein kinase Cepsilon, however, was selectively down-regulated by 1, 10, and 20 ppm dietary heptachlor epoxide, whereas the cytosolic isoform was decreased by 1 and 10 ppm heptachlor epoxide for 10 days. The high-dose treatment for 24 hours also decreased particulate novel protein kinase Cepsilon but increased the cytosolic titer. These results demonstrate that this isoform is unique in its sensitivity to heptachlor epoxide. Activator protein-1 DNA binding, a critical factor in tumor promotion, was substantially increased at 3 and 6 hours with 3.7 mg/kg (i.p.) heptachlor epoxide and at 3 and 10 days with 20 ppm dietary heptachlor epoxide. The effects of heptachlor epoxide on protein kinase C and activator protein-1 are similar to those caused by phorbol ester treatments and correlate well to heptachlor levels found to induce tumors in mice. However, heptachlor epoxide did not initially activate protein kinase C with in vivo treatments or with in vitro treatments of a plasma membrane fraction aimed at demonstrating direct activation, as has been shown for phorbol esters. The ability of heptachlor epoxide to down-regulate particulate novel protein kinase Cepsilon correlates to dosages used in in vivo tumor promotion studies. However, this may represent a negative feedback response rather than a causative effect.

Role of the actin bundling protein fascin in growth cone morphogenesis: localization in filopodia and lamellipodia.

Growth cones at the distal tips of growing nerve axons contain bundles of actin filaments distributed throughout the lamellipodium and that project into filopodia. The regulation of actin bundling by specific actin binding proteins is likely to play an important role in many growth cone behaviors. Although the actin binding protein, fascin, has been localized in growth cones, little information is available on its functional significance. We used the large growth cones of the snail Helisoma to determine whether fascin was involved in temporal changes in actin filaments during growth cone morphogenesis. Fascin localized to radially oriented actin bundles in lamellipodia (ribs) and filopodia. Using a fascin antibody and a GFP fascin construct, we found that fascin incorporated into actin bundles from the beginning of growth cone formation at the cut end of axons. Fascin associated with most of the actin bundle except the proximal 6--12% adjacent to the central domain, which is the region associated with actin disassembly. Later, during growth cone morphogenesis when actin ribs shortened, the proximal fascin-free zone of bundles increased, but fascin was retained in the distal, filopodial portion of bundles. Treatment with tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which phosphorylates fascin and decreases its affinity for actin, resulted in loss of all actin bundles from growth cones. Our findings suggest that fascin may be particularly important for the linear structure and dynamics of filopodia and for lamellipodial rib dynamics by regulating filament organization in bundles.

Role of myosin light chain phosphorylation in the regulation of cytokinesis.

Phosphorylation of regulatory light chain (RMLC) of myosin II at Ser19/Thr18 is likely to play important roles in controlling the morphological changes seen during cell division of cultured mammalian cells. Phosphorylation of RMLC regulates the activity of myosin II, an essntial motor for cytokinesis, and phosphorylation of RMLC shows dramatic changes during mitosis. Two exzymes, myosin phosphatase and kinase, control phosphorvlation of RMLC. Myosin phosphatase is activated during mitosis, apparently as a result of mitosis-specific phosphorylation of the myosin phosphatase targeting subunit (MYPT). This activation of myosin phosphatase is likely to result in RMLC dephosphorylation, causing the disassemly of stress fibers and focal adhesions during prophase. The phosphorylation of MYPT is lost in cyotokinesis, which would decrease myosin phosphatase activity. At the same time, ROCK (Rho-kinase) probably phosphorylates MYPT at its inhibitory sites, further decreasing the activity of myosin phosphatase. These changes in MYPT phosphorylation would raise RMLC phosphorylation, leading to the activation of myosin II for cyotokinesis. RMLC phosphorylation is also regulated by several RMLC kinases including ROCK (Rho-kinase), MLCK and citron kinase, all of which are localized at cleavage furrows. Future studies should examine whether these multiple kinases are redundant or whether they control distinct aspects of cell division.

Posttransplant infusion of donor-specific blood induces immunological unresponsiveness in rat hepatic allografts.

BACKGROUND: We previously reported that pretransplant donor-specific blood transfusion (DST) induces CD45RC-CD4+ T cells, Th2-like effector cells, and prolongs rat hepatic allograft survival. Our study investigated the effects of posttransplant DST on rat hepatic allograft survival. METHODS: Three days after transplantation, LEW (RT1(1)) recipient rats with ACI (RT1a) livers were injected i.v. with freshly heparinized donor-specific blood. The time kinetics of CD45RC-CD4+ and CD45RC+CD4+ T cell subsets in hepatic infiltrates were examined. RESULTS: Posttransplant DST significantly prolonged rat hepatic allograft survival. Interferon (IFN)-gamma, interleukin (IL)-12, and IL-18 mRNA levels in hepatic allografts of untreated recipients were significantly greater than in recipients treated with posttransplant DST. However, hepatic allografts of recipients treated with posttransplant DST showed significantly higher IL-4, IL-10, and transforming growth factor (TGF)-beta mRNA levels than untreated recipients. The ratio of CD45RC-CD4+ T cells to CD45RC+CD4+ T cells was significantly higher in hepatic allografts treated with posttransplant DST than in untreated animals. Immunostaining with anti-rat dendritic cell (OX-62) monoclonal antibody revealed that OX-62+ cells were distributed to the splenic red pulp of animals treated with posttransplant DST and to the splenic white pulp in untreated animals. Most OX62+ cells isolated from the spleen of recipients treated with posttransplant DST expressed donor RT1Ba class II major histocompatibility complex antigens, suggesting that OX-62+ cells were of donor origin. CONCLUSION: Posttransplant DST was associated with persistent infiltration of CD45RC-CD4+ T cells, Th2-like effector cells, in rat hepatic allografts, causing immunologic unresponsiveness and establishment of microchimerism in the spleen.

Expression of the actin-bundling protein fascin in cultured human dendritic cells correlates with dendritic morphology and cell differentiation.

Dendritic cells are key players of the immune system as they efficiently induce primary immune responses by activating naive T cells. We generated human dendritic cells from CD14+ blood precursors and investigated expression of the actin-bundling protein fascin during maturation by western blotting, immunofluorescence, and cytofluorometry. Cells obtained by culture of CD14+ blood precursors in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, which were only weakly positive for the maturation marker CD83, expressed low amounts of fascin. Addition of a cytokine cocktail including tumor necrosis factor alpha, interleukin-1beta, interleukin-6, and prostaglandin E2 induced maturation of the cells and enhanced fascin expression in parallel with CD83 expression. Isolated mature CD83+ cells displayed especially high fascin levels on western blots, as did gated CD83+ dendritic cells in cytofluorometry. Dendritic cells generated from CD34+ blood precursors expressed high levels of fascin as well. Confocal microscopy revealed that location of fascin within the cell was restricted to the area of the submembranous actin cytoskeleton and to the dendritic processes. Suppression experiments using antisense constructs of fascin hint at a retarded morphologic maturation of dendritic cells, supporting the view that fascin expression is pivotal for dendrite formation. Our data suggest that fascin could serve as a marker molecule to monitor the maturation state of in vitro generated dendritic cells for use in clinical trials.

An oncogenic epidermal growth factor receptor signals via a p21-activated kinase-caldesmon-myosin phosphotyrosine complex.

Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformation-specific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosin-associated signaling module may mediate some of the biochemical changes of myosin found in v-ErbB- transformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorage-independent cell growth.

Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts.

ROCK (Rho-kinase), an effector molecule of RhoA, phosphorylates the myosin binding subunit (MBS) of myosin phosphatase and inhibits the phosphatase activity. This inhibition increases phosphorylation of myosin light chain (MLC) of myosin II, which is suggested to induce RhoA-mediated assembly of stress fibers and focal adhesions. ROCK is also known to directly phosphorylate MLC in vitro; however, the physiological significance of this MLC kinase activity is unknown. It is also not clear whether MLC phosphorylation alone is sufficient for the assembly of stress fibers and focal adhesions. We have developed two reagents with opposing effects on myosin phosphatase. One is an antibody against MBS that is able to inhibit myosin phosphatase activity. The other is a truncation mutant of MBS that constitutively activates myosin phosphatase. Through microinjection of these two reagents followed by immunofluorescence with a specific antibody against phosphorylated MLC, we have found that MLC phosphorylation is both necessary and sufficient for the assembly of stress fibers and focal adhesions in 3T3 fibroblasts. The assembly of stress fibers in the center of cells requires ROCK activity in addition to the inhibition of myosin phosphatase, suggesting that ROCK not only inhibits myosin phosphatase but also phosphorylates MLC directly in the center of cells. At the cell periphery, on the other hand, MLCK but not ROCK appears to be the kinase responsible for phosphorylating MLC. These results suggest that ROCK and MLCK play distinct roles in spatial regulation of MLC phosphorylation.

Artificial phosphorylation removes Gelsolin's dependence on calcium.

Gelsolin is one of the best known actin-binding proteins with several distinct activities regulated by calcium. Using a kinase fraction isolated from mitotic HeLa cells, we found that the plasma form of gelsolin can be phosphorylated at a site located within the NH2-terminus region which does not exist in the cytoplasmic form. After this phosphorylation, gelsolin no longer requires Ca2+ for activity; it severs and subsequently caps actin filaments, and nucleates filament formation in Ca2+-free solution. These findings may clarify the mechanism of gelsolin regulation by Ca2+, and indicate that changes in electrical interactions between the NH2- and COOH-terminal ends are important for this regulation. Moreover, since only a single site is phosphorylated, and since the phosphorylated region does not contribute to this protein's own activity, the results suggest that a single chemical charge modification at a site away from the protein's core structure, such as this phosphorylation site, is sufficient to alter the protein's function.

Calcium-channel blocker attenuates Kupffer cell production of cytokine-induced neutrophil chemoattractant following ischemia-reperfusion in rat liver.

We investigated the effects of the calcium-channel blocker verapamil hydrochloride on the production of cytokine-induced neutrophil chemoattractant (CINC) following reperfusion injury in rat liver. Ischemia was induced for 30 min by portal vein occlusion. Animals were pretreated with intravenous injection of verapamil hydrochloride (2.5 mg/kg) 5 min before vascular clamp. Verapamil hydrochloride limited increases in the chemoattractant compared with nonpretreated rats. Most cells immunostained for chemoattractant were ED2-positive macrophages in sinusoids. In vitro chemoattractant production by Kupffer cells isolated from animals pretreated with verapamil hydrochloride was significantly lower than by Kupffer cells from nonpretreated animals. Expression of transcripts in liver for chemoattractant peaked 3 hr after reperfusion in nonpretreated animals, while pretreatment with verapamil hydrochloride significantly decreased chemoattractant mRNA levels. In vitro chemoattractant production could be induced in naive Kupffer cells after stimulation with oxygen radicals generated by hypoxanthine and xanthine oxidase, but verapamil hydrochloride prevented these increases. We concluded that the calcium-channel blocker verapamil hydrochloride significantly attenuates chemoattractant release by Kupffer cells after ischemia-reperfusion in the rat liver.

Analysis of difference in vivo effects of TCDD between c-src +/+ mice, c-src deficient, -/+ and -/- B6, 129-Src(tm 1 sor) mice and their wild-type littermates.

Toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were tested on two strains of c-src deficient B6, 129-Src(tm 1 sor) mice, c-src -/+ and c-src -/- and their matched wild-type littermates c-src +/+ mice along with another c-src +/+ mice, from the same genetic lineage, B6, 129-Fos(tm 1 Pa) mice. The most conspicuous effect of c-src deficiency on the toxicity of TCDD appears to be the reduced hepatotoxicity. TCDD-treated c-src deficient mice show only modest degrees of hepatomegaly and triglycerides accumulation as compared to treated wild-type mice.

Ca2+-dependent myosin II activation is required for uropod retraction during neutrophil migration.

Buffering of intracellular Ca2+ transients in human neutrophils leads to reduced motility due to defective uropod detachment on fibronectin and vitronectin-coated surfaces. Since one potential target of a rise in [Ca2+]i is the activation of myosin II, we characterized the role of myosin II during motility. Treatment of neutrophils with a myosin inhibitor (2,3-butanedione monoxime), or myosin light chain kinase inhibitors (ML-7, ML-9, or KT5926) resulted in impaired uropod retraction and a dose-dependent decrease in chemokinesis following stimulation with N-formyl-Met-Leu-Phe (fMLP). Treatment with ML-9 resulted in a redistribution of F-actin and talin to the non-retracted uropods, mimicking the redistribution observed during [Ca2+]i buffering. Impairment of uropod retraction and redistribution of F-actin and talin by myosin II inhibition was only observed on adhesive substrates such as fibronectin and not on poorly adhesive substrates such as human serum-coated glass. At higher concentrations of ML-9, cell polarization was inhibited and pseudopod extension occurred radially. Using an antibody specific for serine 19-phosphorylated regulatory light chain of myosin II, regions of activated myosin II were found at the leading edge as well as the uropod in motile fMLP-stimulated cells. [Ca2+]i depletion caused a 50% decrease in the level of serine 19-phosphorylated myosin II suggesting that activation of myosin II by intracellular Ca2+ transients may be an essential step in establishing a polarized pseudopod and providing the force required for uropod retraction during PMN motility on adhesive surfaces.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced changes in activities of nuclear protein kinases and phosphatases affecting DNA binding activity of c-Myc and AP-1 in the livers of guinea pigs.

To study the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on nuclear protein phosphorylation activities, male guinea pigs were treated in vivo with a single 1 microg/kg i.p. injection of TCDD, and the state of protein kinases and phosphatases in the nuclei of the hepatocytes was examined after 1, 10, and 40 days. TCDD was found to cause a rise in nuclear protein tyrosine kinase on day 1, and to a lesser extent on day 10, but this effect diminished almost completely on day 40. TCDD also caused a reduction in nuclear casein kinase II (CKII) activity at all time points. To study the biochemical events taking place at the early stage of the action of TCDD, a short-term in vitro model system was established using explant liver tissues maintained in tissue culture medium. It was found that TCDD caused a rapid reduction of the activity of nuclear CKII with an accompanying increase in the cytosol. Such changes in protein phosphorylation activities were also accompanied by an increase in the DNA binding activity of activator protein 1 (AP-1). The effect of TCDD on nuclear proteins binding to the c-Myc response element DNA was, on the other hand, biphasic: an initial increase of protein binding to the c-Myc response element was followed by suppression. To test the hypothesis that some of the above changes were caused by TCDD-induced changes in protein kinase activity, nuclear proteins isolated from hepatocytes of in vivo treated guinea pigs were incubated with exogenously added Mg2+ and ATP under cell-free conditions. The results showed that this in vitro phosphorylation treatment exacerbated this tendency of increased AP-1 and decreased c-Myc binding to their respective response element DNAs, indicating that kinases and phosphatases present in the isolated nuclear protein preparation were active and capable of modifying protein binding to DNA. Such effects of Mg2+ and ATP on AP-1 were blocked by heparin, indicating that CKII plays an important role in transducing the signal of TCDD into the nucleus.

Rho-kinase/ROCK is involved in cytokinesis through the phosphorylation of myosin light chain and not ezrin/radixin/moesin proteins at the cleavage furrow.

The small GTPase Rho and one of its targets, Rho-kinase (also termed ROK or ROCK), are implicated in various cellular functions including stress fiber formation, smooth muscle contraction, tumor cell invasion and cell motility. We have previously reported that Rho-kinase accumulates at the cleavage furrow during cytokinesis in several cultured cells. Here, using Rho-kinase inhibitors, Y-27632 and HA1077, we found that Rho-kinase is responsible for the phosphorylation of myosin regulatory light chain at Ser19 in the cleavage furrow during cytokinesis. On the other hand, phosphorylation of ezrin/radixin/moesin (ERM) proteins at the cleavage furrow was enhanced by the addition of the above Rho-kinase inhibitors. Treatment with Y-27632 strongly enhanced the accumulation of Rho-kinase but not RhoA and citron kinase at the cleavage furrow. Furthermore, the furrow ingression in cytokinesis was significantly prolonged in the presence of Y-27632. These results suggest that Rho-kinase is involved in the progression of cytokinesis through the phosphorylation of several proteins including myosin light chain at the cleavage furrow.

Reduced interleukin-12, interleukin-18, and interferon-gamma production with prolonged rat hepatic allograft survival after donor-specific blood transfusion.

Interferon-gamma is a key immunoregulatory cytokine involved in acute graft rejection. Immunologic unresponsiveness to organ allografts has been induced by pretransplantation donor-specific blood transfusion, both experimentally and clinically. We investigated interferon-gamma production and intragraft gene expression of type-1 T-helper cytokines such as interleukin-12 and -18 and type-2 T-helper cytokines such as interleukin-10 and transforming growth factor-beta in rats receiving hepatic allografts after such transfusions. The animals were divided into four groups: group I received isografts; group II received allografts; group III received allografts after donor-specific transfusion; and group IV received allografts and was treated with FK 506. Donor blood given seven days prior to transplantation significantly prolonged allograft survival. The serum interferon-gamma concentrations in group II increased, peaking on day 5 and then decreasing. Serum interferon-gamma concentrations in groups I, III, and IV were significantly lower than those observed in group II, as were levels of interleukin-12 and interleukin-18 mRNA in the graft. Transforming growth factor-beta and interleukin-10 mRNA levels in grafts in transfused animals were significantly greater than those in the untreated allograft group. Interleukin-12 and -18 mRNA transcripts in an allogeneic mixed lymphocyte reaction were inhibited by interleukin-10 and transforming growth factor-beta. These results suggest that interleukin-12 and -18 expression in hepatic allografts is inhibited in the immunologically unresponsive state induced by donor-specific transfusion.