Highly selective hydrosilylation of olefins and acetylenes by platinum(0) complexes bearing bulky N-heterocyclic carbene ligands.

Platinum complexes bearing bulky N-heterocyclic carbene (NHC) ligands, i.e., [Pt(IPr*)(dvtms)] (where, IPr* = 1,3-bis{2,6-bis(diphenylmethyl)-4-methylphenyl}imidazol-2-ylidene) and [Pt(IPr*OMe)(dvtms)] (where, IPr*OMe = 1,3-bis{2,6-bis(diphenylmethyl)-4-methoxyphenyl}imidazol-2-ylidene, dvtms = divinyltetramethyldisiloxane) catalyse nearly quantitatively and highly or completely the selective hydrosilylation of terminal olefins as well as terminal or internal acetylenes.

Selective synthesis of E-vinylsilanes and E,E-divinylsilanes via platinum-catalyzed hydrosilylation of alkynes with secondary silanes.

Platinum-N-heterocyclic carbene complex with the formula [Pt(IPr*OMe)(dvtms)] (where IPr*OMe = 1,3-bis{2,6-bis(diphenylmethyl)-4-methoxyphenyl}imidazol-2-ylidene, dvtms = divinyltetramethyldisiloxane) exhibits a high catalytic activity towards the ß-E-selective hydrosilylation of terminal acetylenes with many secondary silanes (46 examples). Depending on the ratio of the reagent concentrations, the products of mono- or disubstitution are selectively obtained. Moreover, the one-pot sequential hydrosilylation of two different alkynes with secondary silane was also achieved over the same platinum catalyst.

The Influence of Sex and Season on Conspecific Spatial Overlap in a Large, Actively-Foraging Colubrid Snake.

Understanding the factors influencing the degree of spatial overlap among conspecifics is important for understanding multiple ecological processes. Compared to terrestrial carnivores, relatively little is known about the factors influencing conspecific spatial overlap in snakes, although across snake taxa there appears to be substantial variation in conspecific spatial overlap. In this study, we described conspecific spatial overlap of eastern indigo snakes (Drymarchon couperi) in peninsular Florida and examined how conspecific spatial overlap varied by sex and season (breeding season vs. non-breeding season). We calculated multiple indices of spatial overlap using 6- and 3-month utilization distributions (UD) of dyads of simultaneously adjacent telemetered snakes. We also measured conspecific UD density values at each telemetry fix and modeled the distribution of those values as a function of overlap type, sex, and season using generalized Pareto distributions. Home range overlap between males and females was significantly greater than overlap between individuals of the same sex and male home ranges often completely contained female home ranges. Male home ranges overlapped little during both seasons, whereas females had higher levels of overlap during the non-breeding season. The spatial patterns observed in our study are consistent with those seen in many mammalian carnivores, in which low male-male overlap and high inter-sexual overlap provides males with greater access to females. We encourage additional research on the influence of prey availability on conspecific spatial overlap in snakes as well as the behavioral mechanisms responsible for maintaining the low levels of overlap we observed.

Systems level-based RNAi screening by high content analysis identifies UBR5 as a regulator of estrogen receptor-α protein levels and activity.

Estrogen receptor-α (ERα) is a central transcription factor that regulates mammary gland physiology and a key driver in breast cancer. In the present study, we aimed to identify novel modulators of ERα-mediated transcriptional regulation via a custom-built siRNA library screen. This screen was directed against a variety of coregulators, transcription modifiers, signaling molecules and DNA damage response proteins. By utilizing a microscopy-based, multi-end point, estrogen responsive biosensor cell line platform, the primary screen identified a wide range of factors that altered ERα protein levels, chromatin remodeling and mRNA output. We then focused on UBR5, a ubiquitin ligase and known oncogene that modulates ERα protein levels and transcriptional output. Finally, we demonstrated that UBR5 also affects endogenous ERα target genes and E2-mediated cell proliferation in breast cancer cells. In conclusion, our multi-end point RNAi screen identified novel modulators of ERα levels and activity, and provided a robust systems level view of factors involved in mechanisms of nuclear receptor action and pathophysiology. Utilizing a high throughput RNAi screening approach we identified UBR5, a protein commonly amplified in breast cancer, as a novel regulator of ERα protein levels and transcriptional activity.

An extended posterior approach to the hip and pelvis for complex acetabular reconstruction that preserves the gluteal muscles and their neurovascular supply.

We investigated the detailed anatomy of the gluteus maximus, gluteus medius and gluteus minimus and their neurovascular supply in 22 hips in 11 embalmed adult Caucasian human cadavers. This led to the development of a surgical technique for an extended posterior approach to the hip and pelvis that exposes the supra-acetabular ilium and preserves the glutei during revision hip surgery. Proximal to distal mobilisation of the gluteus medius from the posterior gluteal line permits exposure and mobilisation of the superior gluteal neurovascular bundle between the sciatic notch and the entrance to the gluteus medius, enabling a wider exposure of the supra-acetabular ilium. This technique was subsequently used in nine patients undergoing revision total hip replacement involving the reconstruction of nine Paprosky 3B acetabular defects, five of which had pelvic discontinuity. Intra-operative electromyography showed that the innervation of the gluteal muscles was not affected by surgery. Clinical follow-up demonstrated good hip abduction function in all patients. These results were compared with those of a matched cohort treated through a Kocher-Langenbeck approach. Our modified approach maximises the exposure of the ilium above the sciatic notch while protecting the gluteal muscles and their neurovascular bundle.

Effects of vitamin E on cytotoxicity of amiodarone and N-desethylamiodarone in isolated hamster lung cells.

Amiodarone (AM) is a potent and efficacious antidysrhythmic agent that can cause potentially life-threatening pulmonary fibrosis. Vitamin E has been demonstrated to decrease AM-induced pulmonary fibrosis in vivo in hamsters. In the present in vitro study, we investigated the effects of vitamin E on cell death induced by AM and its primary metabolite, N-desethylamiodarone (DEA), in freshly isolated hamster lung cells. Following incubation for 24 or 36 h, 300 microM vitamin E decreased (P<0.05) 100 microM AM-induced cytotoxicity (0.5% trypan blue uptake) in alveolar macrophages by 11.7+/-3% or 21.4+/-12%, respectively, but did not decrease cytotoxicity in fractions enriched with alveolar type II cells or non-ciliated bronchiolar epithelial (Clara cells) or in isolated unseparated cells (cell digest). Vitamin E had no effect on 50 microM DEA-induced cytotoxicity. Vitamin E did not alter cellular levels of AM or DEA in any cell fraction. Lipid peroxidation (assessed by isoprostane formation) was increased (P<0.05) in cell digest, alveolar type II cell and Clara cell enriched fractions incubated with 500 microM carbon tetrachloride (CCl(4)) for 4 h but not in enriched fractions of cells exposed to 100 microM AM or 50 microM DEA. No AM-induced loss of viability was observed at this time point, but DEA decreased (P<0.05) Clara cell viability by approximately 25%. These results demonstrate cell type selective protection against AM-induced cytotoxicity by vitamin E, and suggest that lipid peroxidation does not initiate AM- or DEA-induced cytotoxicity in isolated hamster lung cells.

Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism.

Hypocalcemia, rickets, and osteomalacia are major phenotypic abnormalities in vitamin D receptor (VDR)-null mice. In an attempt to understand the abnormal regulation of calcium metabolism in these animals, we examined the expression of calbindins (CaBP) as well as calcium handling in the intestine and kidney of VDR null mice. In adult VDR-null mice, intestinal and renal CaBP-D9k expression was reduced by 50 and 90%, respectively, at both the mRNA and protein levels compared with wild-type littermates, whereas renal CaBP-D28k expression was not significantly changed. Intestinal calcium absorption was measured by the rate of (45)Ca disappearance from the intestine after an oral dose of the isotope. (45)Ca absorption was similar in VDR-null and wild-type mice, but the amount of (45)Ca accumulated in the serum and bone was 3-4 times higher in wild-type mice than in VDR-null mice. Despite the hypocalcemia, the urinary excretion of calcium in VDR-null mice was not different from that in wild-type mice. Moreover, 1 wk of a high-calcium diet treatment that normalized the serum ionized calcium level of VDR-null mice increased the urinary calcium level of these mutant mice to twofold higher than that of wild-type mice on the same diet, suggesting impaired renal calcium conservation in VDR-null mice. These data demonstrate that renal CaBP-D9k, but not CaBP-D28k, is highly regulated by the VDR-mediated action of 1,25-dihydroxyvitamin D(3). Furthermore, the results also suggest that impaired calcium conservation in the kidney may be the most important factor contributing to the development of hypocalcemia in VDR-null mice, and CaBP-D9k may be an important mediator of calcium reabsorption in the kidney.

Disruption of mitochondrial function and cellular ATP levels by amiodarone and N-desethylamiodarone in initiation of amiodarone-induced pulmonary cytotoxicity.

Amiodarone (AM), a potent antidysrhythmic agent, can cause potentially life-threatening pulmonary fibrosis. In the present investigation of mechanisms of initiation of AM lung toxicity, we found that 100 microM AM decreased mitochondrial membrane potential in intact hamster lung alveolar macrophages and preparations enriched in isolated alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells, following 2 h of incubation. This was followed by a drop in cellular ATP content (by 32--77%) at 4 to 6 h, and 30 to 55% loss of viability at 24 h. Supplementation of incubation media with 5.0 mM glucose or 2.0 mM niacin did not reduce AM-induced ATP depletion or cell death in macrophages, and the mitochondrial permeability transition inhibitor cyclosporin A (1.0 microM) did not affect AM cytotoxicity. At 50 microM, the AM metabolite N-desethylamiodarone (DEA) produced effects similar to those of AM, but more rapidly and extensively, with the Clara cell-enriched preparation being particularly susceptible. In isolated whole lung mitochondria, DEA was accumulated to a greater extent than AM. Both AM and DEA inhibited complex I- and complex II-supported respiration, but DEA inhibited complex II to a greater degree than AM. These results demonstrate that AM and DEA disrupt mitochondrial membrane potential prior to ATP depletion and subsequent lung cell death, that DEA is more potent than AM, and that the mitochondrial permeability transition is not involved in mitochondrial perturbation by AM. This suggests that AM- and DEA-induced perturbations of mitochondrial function may initiate AM-induced pulmonary toxicity.

Randomised crossover trial of transdermal fentanyl and sustained release oral morphine for treating chronic non-cancer pain.

OBJECTIVES: To compare patients' preference for transdermal fentanyl or sustained release oral morphine, their level of pain control, and their quality of life after treatment. DESIGN: Randomised, multicentre, international, open label, crossover trial. SETTING: 35 centres in Belgium, Canada, Denmark, Finland, the United Kingdom, the Netherlands, and South Africa. PARTICIPANTS: 256 patients (aged 26-82 years) with chronic non-cancer pain who had been treated with opioids. MAIN OUTCOME MEASURES: Patients' preference for transdermal fentanyl or sustained release oral morphine, pain control, quality of life, and safety assessments. RESULTS: Of 212 patients, 138 (65%) preferred transdermal fentanyl, whereas 59 (28%) preferred sustained release oral morphine and 15 (7%) expressed no preference. Better pain relief was the main reason for preference for fentanyl given by 35% of patients. More patients considered pain control as being "good" or "very good" with fentanyl than with morphine (35% v 23%, P=0.002). These results were reflected in both patients' and investigators' opinions on the global efficacy of transdermal fentanyl. Patients receiving fentanyl had on average higher quality of life scores than those receiving morphine. The incidence of adverse events was similar in both treatment groups; however, more patients experienced constipation with morphine than with fentanyl (48% v 29%, P<0.001). Overall, 41% of patients experienced mild or moderate cutaneous problems associated with wearing the transdermal fentanyl patch, and more patients withdrew because of adverse events during treatment with fentanyl than with morphine (10% v 5%). However, within the subgroup of patients naive to both fentanyl and morphine, similar numbers of patients withdrew owing to adverse effects (11% v 10%, respectively). CONCLUSION: Transdermal fentanyl was preferred to sustained release oral morphine by patients with chronic non-cancer pain previously treated with opioids. The main reason for preference was better pain relief, achieved with less constipation and an enhanced quality of life.

Single- and multifrequency models for bioelectrical impedance analysis of body water compartments.

The 1994 National Institutes of Health Technology Conference on bioelectrical impedance analysis (BIA) did not support the use of BIA under conditions that alter the normal relationship between the extracellular (ECW) and intracellular water (ICW) compartments. To extend applications of BIA to these populations, we investigated the accuracy and precision of seven previously published BIA models for the measurement of change in body water compartmentalization among individuals infused with lactated Ringer solution or administered a diuretic agent. Results were compared with dilution by using deuterium oxide and bromide combined with short-term changes of body weight. BIA, with use of proximal, tetrapolar electrodes, was measured from 5 to 500 kHz, including 50 kHz. Single-frequency, 50-kHz models did not accurately predict change in total body water, but the 50-kHz parallel model did accurately measure changes in ICW. The only model that accurately predicted change in ECW, ICW, and total body water was the 0/infinity-kHz parallel (Cole-Cole) multifrequency model. Use of the Hanai correction for mixing was less accurate. We conclude that the multifrequency Cole-Cole model is superior under conditions in which body water compartmentalization is altered from the normal state.

Rapid effects of 1,25(OH)2 vitamin D3 on signal transduction systems in colonic cells.

Previous work from our laboratory demonstrated that 1,25(OH)2D3 rapidly stimulated hydrolysis of membrane polyphosphoinositides (PI) in rat colonocytes and in Caco-2 cells, generating the second messengers DAG and IP3. [Ca2+]i subsequently increased due to IP3-mediated release of intracellular Ca2+ stores, and to Ca2+ influx through a receptor-mediated Ca channel. Studies examining purified antipodal plasma membranes and experiments using Caco-2 cell monolayers found that 1,25(OH)2D3 influenced PI turnover only in the basolateral (BLM) and not brush border (BBM) membranes. Vitamin D analogues with poor affinity for the vitamin D receptor were found to effectively stimulate PI turnover, suggesting the presence of a unique vitamin D receptor in the BLM. Studies from our laboratory have demonstrated saturable, reversible binding of 1,25(OH)2 D3 to colonocyte BLM. Recently, we found that 1,25(OH)2D3 activated the tyrosine kinase c-src in colonocyte BLM by a heterotrimeric guanine nucleotide binding protein (G-protein)-dependent mechanism, with subsequent phosphorylation, translocation to the BLM, and activation of PI-specific phospholipase C gamma. Due to the rise in [Ca2+]i and DAG, two isoforms of protein kinase C (PKCalpha and PKCbeta2), but not other isoforms were activated by 1,25(OH)2D3 in rat colonocytes. Recent studies demonstrated that the seco-steroid translocated the beta2 isoform to the BLM, but not the BBM. In contrast, the alpha isoform did not translocate to either antipodal plasma membrane, but modulated IP3-mediated Ca2+ release from the endoplasmic reticulum. Preliminary studies have shown that 1,25(OH)2D3 also activated phosphatidylcholine phospholipase D (PLD) in Caco-2 cells, generating phosphatidic acid and contributing to the sustained rise in DAG. PLD stimulation occurred by both PKC-dependent and -independent mechanisms. Inhibitors of G-proteins, c-src, and PKC blunted the seco-steroid-mediated activation of PLD. Cells stably transfected with sense PKCalpha showed increased 1,25(OH)2D3-stimulated PLD activation, whereas transfectants with antisense PKCalpha had an attenuated response. In addition, 1,25(OH)2D3 also regulated PLD by activating the monomeric G-protein rho A by a mechanism independent of the G-protein/ c-src/PKC pathway.

Expression of G protein alpha subunits in normal rat colon and in azoxymethane-induced colonic neoplasms.

BACKGROUND & AIMS: Heterotrimeric G proteins are important in growth-regulating signal transduction. The aim of this study was to characterize the relative expression of G protein alpha subunits in rat colonocytes, colonocyte antipodal plasma membranes, and colonic neoplasms. METHODS: Antipodal plasma membranes were prepared from isolated colonocytes. Azoxymethane was administered to rats to induce colonic neoplasms. K-ras mutations in the neoplasms were determined by oligonucleotide hybridization and confirmed by primer mediated-restriction fragment length polymorphism. Colonocyte and tumor homogenates or membranes were probed for Galpha subunits by Western blotting with isoform-specific antibodies. RESULTS: The expressions of Galphai2, alphai3, and alphaq/11 were significantly enriched in the basolateral compared with brush border fraction of colonic antipodal plasma membranes. In neoplasms without K-ras mutations, the expression of Galphai2 increased 4-fold, Galphas(long) increased 2.5-fold, and Galphai3 increased 1.5-2-fold. Expression did not differ among tumor grades. K-ras mutations were associated with lowered expression of G proteins, especially Galphao. CONCLUSIONS: In colonocytes, Galpha subunits are localized primarily in basolateral plasma membranes. The increased expressions of Galphai2 and, to a lesser degree, Galphai3 and Galphas(long) in tumors was independent of tumor grade but was modulated by the presence of K-ras mutations.

Differential susceptibilities of isolated hamster lung cell types to amiodarone toxicity.

Treatment of cardiac dysrhythmias with the iodinated benzofuran derivative amiodarone (AM) is limited by pulmonary toxicity. The susceptibilities of different lung cell types of male Golden Syrian hamsters to AM-induced cytotoxicity were investigated in vitro. Bronchoalveolar lavage and protease digestion to release cells, followed by centrifugal elutriation and density gradient centrifugation, resulted in preparations enriched with alveolar macrophages (98%), alveolar type II cells (75-85%), and nonciliated bronchiolar epithelial (Clara) cells (35-50%). Alveolar type II cell and Clara cell preparations demonstrated decreased viability (by 0.5% trypan blue dye exclusion) when incubated with 50 microM AM for 36 h, and all AM-treated cell preparations demonstrated decreased viability when incubated with 100 or 200 microM AM. Based on a viability index ((viability of AM-treated cells/viability of controls) x 100%), the Clara cell fraction was significantly (p<0.05) more susceptible than all of the other cell types to 50 microM AM. However, AM cytotoxicity was greatest (p<0.05) in alveolar macrophages following incubation with 100 or 200 microM AM. There was no difference between any of the enriched cell preparations in the amount of drug accumulated following 24 h of incubation with 50 microM AM, whereas alveolar macrophages accumulated the most drug during incubation with 100 microM AM. Thus, the most susceptible cell type was dependent on AM concentration. AM-induced cytotoxicity in specific cell types may initiate processes leading to inflammation and pulmonary fibrosis.

High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

High-molecular-weight proteins often blot onto nitrocellulose membranes poorly following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, resulting in low levels of detection on immunoblots, and hence difficulty in analyzing rare proteins. Moreover, optimizing conditions for the transfer of high-molecular-weight proteins to nitrocellulose frequently results in the inefficient transfer or loss of lower molecular weight proteins. This problem is particularly vexing during the analysis of large proteins which may be processed to one or more smaller biologically active forms. Using radiolabeled protein standards and phosphorimaging technology, we have quantitated the efficacy of many different protein gel electrophoresis and blotting protocols. Here we report novel gel and blotting conditions which significantly improve the transfer and retention of high-molecular-weight proteins, without sacrificing the efficient transfer of lower molecular weight proteins. Using this newly described procedure, we have detected a rare 500-kDa protein in immunoblots which was previously not detectable with any of the commonly used blotting procedures. Since the improved conditions offer increased sensitivity across a spectrum of protein sizes, they should be widely applicable.

1,25 dihydroxyvitamin D3 stimulates phospholipase C-gamma in rat colonocytes: role of c-Src in PLC-gamma activation.

Our laboratory has previously demonstrated that 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) rapidly stimulated polyphosphoinositide (PI) hydrolysis, raised intracellular Ca2+, and activated two Ca2+-dependent protein kinase C (PKC) isoforms, PKC-alpha and -betaII in the rat large intestine. We also showed that the direct addition of 1,25(OH)2D3 to isolated colonic membranes failed to stimulate PI hydrolysis, but required secosteroid treatment of intact colonocytes, suggesting the involvement of a soluble factor. Furthermore, this PI hydrolysis was restricted to the basal lateral plasma membrane of these cells. In the present studies, therefore, we examined whether polyphosphoinositide-phospholipase C-gamma (PI-PLC-gamma), a predominantly cytosolic isoform of PI-PLC, was involved in the hydrolysis of colonic membrane PI by 1,25(OH)2D3. This isoform has been shown to be activated and membrane-associated by tyrosine phosphorylation. We found that 1,25(OH)2D3 caused a significant increase in the biochemical activity, particulate association, and the tyrosine phosphorylation of PLC-gamma, specifically in the basal lateral membranes. This secosteroid also induced a twofold increase in the activity of Src, a proximate activator of PLC-gamma in other cells, with peaks at 1 and 9 min in association with Src tyrosine dephosphorylation. 1,25(OH)2D3 also increased the physical association of activated c-Src with PLC-gamma. In addition, Src isolated from colonocytes treated with 1,25(OH)2D3, demonstrated an increased ability to phosphorylate exogenous PLC-gamma in vitro. Inhibition of 1,25(OH)2D3-induced Src activation by PP1, a specific Src family protein tyrosine kinase inhibitor, blocked the ability of this secosteroid to stimulate the translocation and tyrosine phosphorylation of PLC-gamma in the basolateral membrane (BLM). Src activation was lost in D deficiency, and was reversibly restored with the in vivo repletion of 1,25(OH)2D3. These studies demonstrate for the first time that 1,25(OH)2D3 stimulates PLC-gamma as well as c-Src in rat colonocytes, and indicate that PLC-gamma is a direct substrate of secosteroid-activated c-Src in these cells.

Modulation of aflatoxin B1 biotransformation by beta-naphthoflavone in isolated rabbit lung cells.

The cytotoxic and carcinogenic mycotoxin aflatoxin (AF) B1 (AFB1) is biotransformed by the cytochrome P450 monooxygenases (CYP) to a number of relatively nontoxic metabolites, as well as to the ultimate toxic metabolite, AFB1-8,9-epoxide. In a number of tissues and species, AFB1 hydroxylation to the relatively nontoxic metabolite, AFM1, is induced by beta-naphthoflavone (BNF) treatment. Although the liver is the principle target organ for AFB1 toxicity, the mycotoxin is also toxic and carcinogenic to respiratory tissues. To determine if BNF treatment alters the extent of pulmonary AFB1 bioactivation by enhancing detoxification and thereby decreasing epoxidation, the effects of BNF on pulmonary AFB1 metabolism were examined. Rabbit lung cells, isolated by protease digestion and centrifugal elutriation, were incubated with [3H]AFB1. In nonciliated bronchiolar epithelial (Clara) cell-enriched (45-50%) fractions, [3H]AFM1 production (pmol/mg DNA per 2 h) was increased by prior treatment of rabbits with BNF (80 mg/kg per day, 3 and 2 days before cell isolation) as follows: with 1.0 microM [3H]AFB1; control, 10.6 +/- 2.3; BNF, 30.0 +/- 6.4; with 0.10 microM [3H]AFB1; control, 9.4 +/- 4.7; BNF, 20.6 +/- 5.9. With 1.0 microM [3H]AFB1, prior treatment of animals with BNF abolished formation of [3H]aflatoxicol (AFL) but not [3H]AFQ1. The activation (epoxidation) of [3H]AFB1 was measured indirectly as covalent binding to endogenous DNA. With 1.0 microM [3H]AFB1, treatment of rabbits with BNF did not alter DNA binding (pmol/mg DNA per 2 h) in the Clara cell-enriched fraction: control, 103 +/- 41; BNF, 114 +/- 49. However, with 0.10 microM [3H]AFB1, DNA binding in the same fraction was 47% lower in cells from BNF treated animals: control, 17.4 +/- 4.2; BNF, 9.3 +/- 3.9. Formation of 8,9-dihydro-8,9-dihydroxy-AFB1, and the glutathione conjugate of the aflatoxin epoxide (AFB1-GSH) were not detectable at the AFB1 concentration and time point studied, in cells from either BNF-treated or control rabbits. Incubation of isolated, unseparated lung cells from untreated rabbits with 5.0 to 50 microM BNF decreased [3H]AFB1-DNA binding in the presence of 0.1 microM [3H]AFB1 by 35 to 77%, while lower BNF concentrations did not alter DNA binding. In lung cells isolated from BNF treated rabbits, BNF was not detectable (i.e. < 0.5 microM detection limit). Therefore, the amount of BNF present in isolated rabbit lung cells following in vivo treatment with BNF was below that required to directly inhibit AFB1-DNA adduct formation. The decrease in AFB1-DNA binding from rabbits treated with BNF is apparently due to the selective induction of CYP isozymes and related increases in AFM1 formation, and not to direct inhibition of epoxidation or enhanced conjugation of AFB1-8,9-epoxide with glutathione.

Guanylin activates rat colonic particulate guanylate cyclase.

The polypeptide guanylin is an endogenous activator of small intestinal guanylate cyclase. In rat, guanylin mRNA is found predominantly in intestinal tissues, with its highest abundance in the colon. To date, the effect of guanylin on rat colonic particulate guanylate cyclase, however, has not been examined. It was, therefore, of interest to determine whether the addition of guanylin to intact rat colonocytes, or directly to isolated crude colonic membranes, stimulated guanylate cyclase activity. These studies demonstrated that: 1) rat guanylin, in a concentration-dependent manner, rapidly (within min), but transiently, stimulated particulate guanylate cyclase activity when added to intact colonocytes; 2) guanylin also stimulated guanylate cyclase activity when added directly to isolated colonic membranes; and 3) this latter effect of guanylin on guanylate cyclase activity was increased by ATP or ADP and markedly accentuated by ATP gamma S. Taken together, these results demonstrate that guanylin rapidly stimulates rat colonic particulate guanylate cyclase activity and, moreover, that this effect can be modulated by adenine nucleotides.