Low incidence of heparin-induced skin lesions in orthopedic surgery patients with low-molecular-weight heparins.

BACKGROUND: Heparins are widely prescribed for prevention and therapy of arterial and venous thromboembolic diseases. Heparin-induced skin lesions are the most frequent adverse effect of subcutaneous heparin treatment in non-surgical patients (7.5%-39.8%); no data exist on surgical patients. Commonly, they are due to a delayed-type hypersensitivity reaction (DTH), but may also be a manifestation of life-threatening heparin-induced thrombocytopenia (HIT). Lesions of both entities resemble initially. The risk of HIT is highest among heparin-anticoagulated orthopedic surgery patients. OBJECTIVE: To determine incidence and causes of heparin-induced skin lesions in major orthopedic surgery patients. METHODS: In a prospective cohort study, consecutive patients with subcutaneous low-molecular-weight heparin (LMWH) treatment were examined for cutaneous adverse effects. Further diagnostics (skin biopsy, clinical/laboratory assessment for thrombosis, bleeding, HIT, cross-allergies) were performed. RESULTS: Six of 316 enrolled patients (1.9%; 95% CI: 0.4%-3.4%) developed heparin-induced skin lesions. All were caused by a DTH reaction, and none was due to HIT or other rare heparin-associated skin diseases. Therapeutic use (dosage) of LMWH was identified as only risk factor (odds ratio: 3.1, 95% CI: 1.4-4.9; P = .00141). In addition to DTH, 5 thromboembolic, 4 major bleeding complications but no cases of HIT or cross-allergies were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Orthopedic surgery patients have-unlike non-surgical patients-a low risk for heparin-induced skin lesions during LMWH treatment; all lesions were due to a DTH reaction. The risk for DTH differs considerably between individual patient cohorts. No association with HIT was observed. These data help to tailor anticoagulatory treatment individually and to increase patient safety.

A 55 kDa protein of transitional endoplasmic reticulum from rat liver binds retinol.

A transitional endoplasmic reticulum fraction from rat liver that responds to retinol by increased formation of 50-70 nm diameter transition vesicles bound retinol. Separation on SDS-PAGE with analysis by fluorography indicated the dominant binding component at 55 kDa. Binding was found with transitional vesicles formed from the transitional endoplasmic reticulum, and the endoplasmic reticulum fractions from which vesicles are derived but not with Golgi apparatus, plasma membranes or cytosol.

Identification of antitumor sulfonylurea binding proteins of HeLa plasma membranes.

Plasma membranes of cultured HeLa S cells bound the tritiated antitumor sulfonylurea [3H]LY181984 with high affinity (Kd of about 25 nM). The number of binding sites, estimated to represent 30 to 35 pmol/mg protein, would represent a low abundance protein of the total plasma membrane proteins. The binding proteins appeared to contain one or more thiols in the binding site as high affinity binding of [3H]LY181984 was reduced by treatment with the covalent thiol blocking reagent, N-ethylmaleimide (NEM), or by oxidation with dilute hydrogen peroxide but was protected by glutathione or dithiothreitol. Elimination of binding of [3H]LY181984 by NEM was prevented by excess unlabeled LY181984 (an active sulfonylurea) but less so by excess LY181985 (an inactive sulfonylurea). The binding proteins were specifically labeled with thiol reagents following reaction of unprotected thiols with unlabeled thiol reagents. Binding proteins at ca. 34 kDa were labeled. Plasma membrane proteins after solubilization with SDS under strongly reducing conditions still bound sulfonylurea. [3H]LY181984 binding to plasma membrane proteins resolved on SDS-PAGE correlated as well with proteins in the 30-40 kDa range.

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus.

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.

GTP hydrolysis by transitional endoplasmic reticulum from rat liver inhibited by all-trans-retinol.

GTP hydrolysis by an endoplasmic reticulum fraction from rat liver enriched in part-rough, part-smooth transition elements was inhibited by all-trans-retinol half maximally at a concentration of about 10 micrograms/ml. Similar results were obtained with GTPase activity partially purified by ion-exchange (DE-52) chromatography. The inhibition was non-competitive and given by both retinol and retinaldehyde but not by retinoic acid or alpha-tocopheryl acetate. The hydrolysis of other nucleoside di- and triphosphates was much less affected by retinol. The activity was inhibited by detergents but at much higher concentrations than by retinol. The results suggest that enhancement of cell-free transfer from endoplasmic reticulum to Golgi apparatus by retinol observed previously at low concentrations of cytosol may be mediated through an interaction with GTP.

NADH-activated cell-free transfer between Golgi apparatus and plasma membranes of rat liver.

This report concerns development of a cell-free system from rat liver to study transport of membrane constituents from the Golgi apparatus to the plasma membrane. Highly purified Golgi apparatus as donor and a mixture of sheets and vesicles as plasma membrane acceptor fractions were combined to analyze requirements for lipid and protein transport. In the reconstituted system, the Golgi apparatus donor was in suspension. To measure transfer, membrane constituents of the donor membranes were radiolabeled with [3H]acetate (lipids) or [3H]leucine (proteins). The plasma membrane vesicles were used as the acceptor and were unlabeled and immobilized on nitrocellulose for ease of recovery and analysis. The reconstituted cell-free transfer was dependent on temperature, but even at 37 degrees C, the amount of transfer did not increase with added ATP, was not specific for any particular membrane fraction or subfraction nor was it facilitated by cytosol. ATP was without effect both in the presence or absence of a cytosolic fraction capable of the support of cell-free transfer in other systems. In contrast to results with ATP, NADH added to the reconstituted system resulted in an increased amount of transfer. A further increase in transfer was obtained with NADH plus a mixture of ascorbate and dehydroascorbate to generate ascorbate free radical. The transfer of labeled membrane constituents from the Golgi apparatus to the plasma membrane supported by NADH plus ascorbate radical was stimulated by a cytosol fraction enriched in less than 10 kDa components. This was without effect in the absence of NADH/ascorbate radical or with ATP as the energy source. Specific transfer was inhibited by both N-ethylmaleimide and GTP gamma S. The findings point to the possibility of redox activities associated with the trans region of the Golgi apparatus as potentially involved in the transport of membrane vesicles from the Golgi apparatus to the cytoplasmic surface of the plasma membrane.

Retinoic acid and calcitriol inhibition of growth and NADH oxidase of normal and immortalized human keratinocytes.

Plasma membranes were isolated by aqueous two-phase partition from normal human keratinocytes (HKc) and from human keratinocytes immortalized with human papillomavirus type 16 DNA (HKc/HPV16). The NADH oxidase of plasma membrane vesicles of normal HKc was stimulated by epidermal growth factor whereas that of HKc/HPV16 was not. The NADH oxidase of the plasma membranes from both normal HKc and HKc/HPV16 was inhibited by calcitriol (1 alpha-1,25-dihydroxy vitamin D-3) and retinoic acid. However, with plasma membranes from HKc/HPV16 the NADH oxidase was more susceptible to inhibition by retinoic acid than were membranes from normal HKc. Similarly, clonal growth of HKc/HPV16 was inhibited by retinoic acid at lower concentrations than normal HKc whereas inhibition of clonal growth of normal HKc and HKc/HPV16 by calcitriol showed similar dose-dependencies. Comparing normal HKc and HKc/HPV16, the results demonstrate parallel inhibition of clonal growth and NADH oxidase by both retinoic acid and calcitriol of HKc/HPV16 but not of normal HKc. These results suggest that an increased sensitivity of the plasma membrane NADH oxidase of HKc/HPV16 to retinoic acid may be related to the increased sensitivity of these cells to growth control by retinoic acid. In addition, since plasma membrane NADH oxidase of HKc/HPV16 shows altered responsiveness to growth modulators such as EGF, retinoic acid and calcitriol, it appears that HKc/HPV16 express an NADH oxidase with different characteristics than those of normal HKc.

Inhibition by brefeldin A of NADH oxidation activity of rat liver Golgi apparatus accelerated by GDP.

Reduced pyridine nucleotide has been reported to enhance cell-free transfer of membrane material from a radiolabeled Golgi apparatus donor fraction from rat liver to an acceptor fraction consisting of inside-out plasma vesicles immobilized on nitrocellulose [(1992) Biochim. Biophys. Acta 1107, 131]. As part of a continuing effort to identify NADH-requiring enzymes in the Golgi apparatus which may be important to membrane trafficking, highly purified fractions of Golgi apparatus from rat liver were tested for their ability to oxidize NADH and the inhibition of the oxidation of NADH by brefeldin A. The isolated Golgi apparatus fractions were found to oxidize NADH with a specific activity comparable to that of the plasma membrane of rat liver. The activity was inhibited by brefeldin A and this inhibition was augmented by GDP. At near optimal concentrations of 7 microM brefeldin A and 1 microM GDP, the activity was > 90% inhibited. Brefeldin A inhibition of NADH oxidation by the Golgi apparatus was time-dependent and GDP appeared to accelerate the inhibition by brefeldin A.

Stimulation of vitamin A(1) acid signaling by the HIV protease inhibitor indinavir.

HIV protease inhibitors (PIs) are effective drugs for the treatment of AIDS. However, PI therapy is sometimes associated with side-effects including increased plasma lipids and altered body fat distribution, although fat redistribution may occur in some patients not treated with PIs. Overdosage with vitamin A(1) acid (all-trans-retinoic acid, ATRA) or its metabolites may cause similar changes in lipid metabolism. Moreover, the PI indinavir and retinoids have been associated with nail, skin, and hair defects, suggesting that indinavir and retinoids may exert their effects through similar molecular mechanisms. This hypothesis was tested by examining the effects of PIs on retinoid signaling in vitro. Mesenchymal stem cells (C3H10T1/2) were cultured in the presence of various PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) and synthetic retinoids, and the metabolic response was assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP). Of the PIs tested, only indinavir stimulated ATRA-dependent ALP activity and altered stem cell morphology; the effects of indinavir occurred in the presence of ATRA, but not in its absence. Moreover, indinavir increased the effects of ATRA on lipid accumulation during fat cell differentiation. AGN 193109 (4-[[5,6-dihydro-5, 5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), a retinoic acid receptor (RAR) antagonist, inhibited the synergistic effects of indinavir and ATRA, indicating that indinavir increases RAR signaling. However, indinavir did not potentiate ALP activity in the presence of the RAR agonist CH55 (3,5-di-tert-butylchalcone 4'-carboxylic acid). Unlike ATRA, CH55 does not bind to cytosolic retinoic acid binding protein (CRABP), suggesting that CRABP may regulate the effects of indinavir on RAR signaling. These observations support the proposal that altered retinoid signaling promotes some of the adverse reactions associated with indinavir therapy, such as altered lipid metabolism.

Effects of troglitazone and metformin on glucose and lipid metabolism: alterations of two distinct molecular pathways.

Troglitazone and metformin are antidiabetic agents that belong to the thiazolidinedione and biguanide classes of drugs, respectively. To evaluate how these drugs influence fuel utilization, we compared their effects on several pathways regulating carbohydrate and lipid metabolism in vitro. Both drugs stimulated glucose transport and utilization in C3H10T1/2 cells, a cell line capable of differentiating into adipocytes when treated with thiazolidinediones. However, we observed that these drugs had a number of different in vitro effects. Unlike metformin, troglitazone stimulated beta3-adrenergic receptor-mediated lipolysis, lipogenesis, and transcriptional activity of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Further, by using a mitochondrial-specific fluorescent dye, we found troglitazone to be more effective than metformin at increasing mitochondrial mass. In contrast to troglitazone, metformin was more effective at increasing mitochondrial fatty acid beta-oxidation, peroxisomal fatty acid beta-oxidation, and anaerobic respiration (i.e. lactate production). Additionally, metformin stimulated and troglitazone inhibited both aerobic respiration and basal lipolysis. Insulin enhanced the effects of troglitazone, but not those of metformin, on these cells. Taken together, the data show that troglitazone and metformin affect two distinct metabolic pathways: one that is anabolic (i.e. troglitazone) and the other that is catabolic (i.e. metformin). Further, these observations suggest that the metabolic activity of mitochondria may be lower in cells treated with troglitazone than with metformin.

Drug-antibody conjugates with anti-HIV activity.

Human immunodeficiency virus (HIV)-specific peptide antibody-brefeldin A conjugates and antibody-glaucarubolone conjugates directed to cell surface viral glycoprotein epitopes were prepared and tested for antiviral activity. A selective response was observed both on survival of cell lines permanently infected with lentiviruses and on HIV infectivity. With human peripheral blood mononuclear cells (PBMCs), the conjugate also was effective in reducing virus titers. The effectiveness of an HIV-specific peptide antibody-brefeldin A conjugate was enhanced by combination with 3'-azido-3'-deoxythymidine (AZT) and was effective against AZT-resistant isolates in combination with AZT. The conjugates reduced virus production in MOLT-4 cells and in HIV-1-infected PBMCs without affecting the viability of uninfected cells.

HIV protease inhibitors block adipogenesis and increase lipolysis in vitro.

AIDS therapies employing HIV protease inhibitors (PIs) are associated with changes in fat metabolism. However, the cellular mechanisms affected by PIs are not clear. Thus, the affects of PIs on adipocyte differentiation were examined in vitro using C3H10T1/2 stem cells. In these cells the PIs, nelfinavir, saquinavir, and ritonavir, reduced triglyceride accumulation, lipogenesis, and expression of the adipose markers, aP2 and LPL. Histological analysis revealed nelfinavir, saquinavir and ritonavir treatment decreased oil red O-staining of cytoplasmic fat droplets. Inhibition occurred in the presence of the RXR agonist LGD1069, indicating the inhibitory effects were not due to an absence of RXR ligand. Moreover, these three PIs increased acute lipolysis in adipocytes. In contrast, two HIV PIs, amprenavir and indinavir, had little effect on lipolysis, lipogenesis, or expression of aP2 and LPL. Although, saquinavir, inhibited ligand-binding to PPARgamma with an IC(50) of 12.7+/-3.2 microM, none of the other PIs bound to the nuclear receptors RXRalpha or PPARgamma, (IC(50)s>20 microM), suggesting that inhibition of adipogenesis is not due to antagonism of ligand binding to RXRalpha or PPARgamma. Taken together, the results suggest that some, but not all, PIs block adipogenesis and stimulate fat catabolism in vitro and this may contribute to the effects of PIs on metabolism in the clinic.

Comparison of the acute pharmacodynamic responses after single doses of ephedrine or sibutramine in healthy, overweight volunteers.

OBJECTIVE: With an increase in the incidence of obesity, tremendous effort has been devoted to the development of weight loss agents and the prospective surrogate markers of both a product's efficacy and safety. The objective of the present study was to compare the pharmacodynamic responses of ephedrine and sibutramine using surrogate markers of weight loss potential and potential adverse events. DESIGN AND SUBJECTS: The study was designed as a 5-way, randomized, double-blinded, placebo-controlled trial with 3 single doses of ephedrine sulfate (0.25, 0.5 and 1 mg x kg(-1)) followed by an open-labeled sibutramine (10 mg) treatment. Healthy, mildly overweight (BMI = 25) subjects were administered the respective treatment and pharmacokinetic and pharmacodynamic measurements (body surface temperature, resting metabolic rate, blood pressure, heart rate, glucose, glycerol, nonesterified fatty acids, triglycerides) were obtained for 8 hours post dose and for an additional 4 measurements during the sibutramine treatment period. RESULTS: Sibutramine treatment significantly increased resting metabolic rate compared to the placebo condition. Ephedrine significantly increased heart rate, systolic blood pressure and glucose but did not significantly affect other measurements. CONCLUSION: Both sibutramine and ephedrine have been shown to have weight loss potential, however, they elicit different metabolic and biochemical responses after a single dose. The nontherapeutic responses from these types of compounds may serve as a screening tool for the development of agents in the treatment of obesity.

Impact of baseline atrial fibrillation cycle length on acute and long-term outcome of persistent atrial fibrillation ablation.

BACKGROUND: A short baseline atrial fibrillation (AF) cycle length (CL) has been associated with a worse outcome after catheter ablation for AF, whereas the impact of a long baseline AFCL is unknown. We investigated the influence of AFCL on acute and long-term success in a large series of patients undergoing catheter ablation for persistent AF. METHODS: Overall, 177 consecutive patients undergoing catheter ablation of persistent AF using a sequential ablation approach were included in the analysis. AFCL was measured in the left atrial appendage (LAA) at baseline and following each ablation step. The primary endpoint was freedom from any atrial arrhythmia off antiarrhythmic drugs (AAD) with a single ablation procedure after 12 months. RESULTS: Mean AFCL was 164 ± 24 ms. A shorter AFCL was associated with longer AF duration, larger LA diameter, and longer procedure duration. Termination to sinus rhythm (SR) was achieved in 57 (32 %) patients. Baseline AFCL was shorter (161 ± 24 ms) in patients without AF termination compared to patients with AF termination (169 ± 23 m, p = 0.03). The primary endpoint was reached less frequently in patients with a short (<155 ms) AFCL (18 vs. 38.5 %, p = 0.006). Patients with an AFCL between 155 and 200 ms had the best outcome compared to patients with AFCL <155 or ≥200 ms (40 vs. 18 %, p = 0.003). CONCLUSIONS: Patients with a baseline AFCL between 155 and 200 ms have the best outcome after a single ablation procedure for persistent AF compared to patients with an AFCL of <155 or ≥200 ms.

Thiazolidinediones inhibit alkaline phosphatase activity while increasing expression of uncoupling protein, deiodinase, and increasing mitochondrial mass in C3H10T1/2 cells.

Although there are a number of cell lines committed to differentiate into brown adipocytes, the stem-cell origin of brown fat remains unclear. To address this problem, we explored the effects of various pharmacological agents on differentiation of C3H10T1/2 cells, a pluripotent stem-cell line of mesodermal origin. Histochemical and biochemical analysis revealed that, when these cells were treated with retinoic acid, they expressed the osteoblastic marker alkaline phosphatase. Upon addition of thiazolidinediones and insulin, these cells accumulated lipid and expressed the adipocyte marker aP2, indicating differentiation into adipocytes. Treatment during the growth phase with thiazolidinediones resulted in maximal lipogenesis indicating a need for clonal expansion for efficient adipogenic differentiation. Further analysis revealed that addition of thiazolidinediones to the cells increased (1) the lipolytic response of the cells to beta3-agonists, (2) the expression of uncoupling protein (UCP), (3) the expression of mRNA for type II iodothyronine 5'-deiodinase (5'D-II), and (4) mitochondrial staining. These results suggest the anti-diabetic effects of thiazolidinediones may, in part, involve increased brown adipocyte differentiation. Moreover, this is the first direct evidence indicating that brown adipocytes and osteoblasts may arise from the same stem cell.

Modulation of guanine triphosphate nucleotide binding to p21ras immunoprecipitates of rat liver plasma membranes by agents affecting redox state.

GTP-gamma-[35S] and GTP-gamma-[32P] or GTP-alpha-[32P] bound to plasma membranes of rat liver was immunoprecipitated using anti p21V-H-ras. Binding was enhanced approximately 2-fold by incubation with an exogenous electron acceptor, potassium ferricyanide (but not with potassium ferrocyanide), or oxidized ubiquinone10 and was inhibited or unaffected by incubation with reduced pyridine nucleotides (NADH or NADPH) or reduced ubiquinone10. The results suggest a mechanism of guanine nucleotide exchange that is responsive to oxidation-reduction.

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver.

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the 16 degrees C compartment of the endoplasmic reticulum in rat liver and cultured hamster kidney cells.

In many systems transfer between the endoplasmic reticulum and the Golgi apparatus is blocked at temperatures below 16 degrees C. In virus-infected cells in culture, a special membrane compartment is seen to accumulate. Our studies with rat liver show a similar response to temperature both in situ with slices and in vitro with isolated transitional endoplasmic reticulum fractions. With isolated transitional endoplasmic reticulum fractions, when incubated in the presence of nucleoside triphosphate and a cytosol fraction, temperature dependent formation of vesicles occurred with a Q10 of approximately 2 but was apparent only at temperatures greater than 12 degrees C. A similar response was seen in situ at 12 degrees C and 16 degrees C where fusion of transition vesicles with cis Golgi apparatus, but not their formation, was blocked and transition vesicles accumulated in large numbers. At 18 degrees C and below and especially at 8 degrees C and 12 degrees C, the cells responded by accumulating smooth tubular transitional membranes near the cis Golgi apparatus face. With cells and tissue slices at 20 degrees C neither transition vesicles nor the smooth tubular elements accumulated. Those transition vesicles which formed at 37 degrees C were of a greater diameter than those formed at 4 degrees C both in situ and in vitro. The findings show parallel responses between the temperature dependency of transition vesicle formation in vitro and in situ and suggest that a subpopulation of the transitional endoplasmic reticulum may be morphologically and functionally homologous to the 16 degrees C compartment observed in virally-infected cell lines grown at low temperatures.

Intracellular membrane flow: reconstitution of transition vesicle formation and function in a cell-free system.

Transfer of membrane between endoplasmic reticulum and Golgi apparatus in situ is considered to occur via 60-nm transition vesicles derived from part-rough, part-smooth transition elements of the endoplasmic reticulum. A procedure is described for the isolation of a fraction enriched in these transition elements from rat liver. The isolated fraction generates small vesicles morphologically resembling transition vesicles when incubated with nucleoside triphosphate at 37 degrees C. In the cell-free system consisting of a donor fraction enriched in transition elements and an acceptor fraction consisting of intact Golgi apparatus immobilized on nitrocellulose strips, transfer in vitro of radiolabeled membranes was demonstrated. Nucleoside triphosphates were required for transfer, and transfer was facilitated by a cytosol fraction of Mr greater than 10,000. In the presence of both nucleoside triphosphate and cytosol, radiolabeled proteins were transferred in a manner dependent upon both time and temperature. Transfer appeared to be both vectorial and specific in that, with Golgi apparatus (or endoplasmic reticulum) as both donor and acceptor, only negligible time and temperature-dependent transfer was observed. The test system described is expected to facilitate further investigation of the transfer process and to provide a convenient assay to guide transition vesicle isolation and characterization.

Is the drug-responsive NADH oxidase of the cancer cell plasma membrane a molecular target for adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC50 for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.