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This paper develops and implements a new constraint formulation for treating nonlinear beam-to-shell, beam-to-solid, and shell-to-solid connections in explicit finite element analyses. The traditional methods commonly adopted to treat element couplings, namely transition elements or multi-point constraints, are investigated and their limitations are outlined and discussed. The newly proposed generalised constraint formulation enforces the kinematical and dynamic consistency across the element coupling interface simultaneously, and a method for implementing them in the explicit central difference integration algorithm is devised. Particular attention is devoted to the treatment of problems involving large rotations and deformations. To validate our constraint method, a number of finite element test cases are examined. Comparison of results of the new method and existing commercial codes against analytical results reveals good accuracy and robustness of the new algorithms in the treatment of both static and dynamic problems encountering large deformations and rotations.

Objectives: To examine the efficacy and mechanism of δ- or 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on a human hepatocellular carcinoma cell line. Materials and methods: The optimal uptake of photosensitizer ALA in HepG2 (p53 wild) cells was investigated by means of spectrometric measurement. Cell viability was determined by trypan blue exclusion assay. Morphological apoptotic changes in HepG2 cells before and after ALA-mediated PDT were determined by microscopic examination. Detection of apoptotic bodies was examined by DAPI staining. The changes in p53 expression were revealed by the immunostaining method. Results: ALA/protoporphyrin IX (PpIX) was mainly located in the cytoplasm of HepG2 cells. The maximal cellular uptake occurred after 18 h in vitro incubation. The photocytotoxic assay showed that ALA PDT induced 80% killing at 2 mM drug dose and 2 J/cm2 light intensity. Up to 70% of cells showed membrane blebbing and positive DAPI staining, indicating that ALA–PDT-mediated cell death was predominantly via apoptosis. In addition, p53 was upregulated after treatment, implying that p53 might evoke apoptotic cell death. Conclusions: HepG2 cell line is sensitive to ALA-mediated PDT. ALA–PDT induces apoptosis in the HepG2 cell line that may be mediated by a p53-dependent pathway.

This paper presents a unified novel technique for enforcing nonlinear beam-to-shell, beam-to-solid, and shell-to-solid constraints in explicit finite element formulations. The limitations of classical multi-point constraint approaches are examined at length, particularly in the context of explicit solution schemes. Novel formulation of a generalized constraint method that ensures proper element coupling is then presented, and its computer implementation in explicit integration algorithms is discussed. Crucial in this regard is the accurate and efficient representation of finite rotations, accomplished using an incremental rotation tensor. The results of some illustrative test cases show the accuracy and robustness of the newly developed algorithm for a wide range of deformation, including that in which large rotations are encountered. When compared to existing works, the salient features of the current method are in evidence.

Using frequency-modulation atomic force microscopy (FM-AFM) at sub-nanometer vibration amplitudes, we find in the system $n$-dodecanol/graphite that solvation layers may extend for several nanometers into the bulk liquid. These layers maintain crystalline order which can be imaged using FM-AFM. The energy dissipation of the vibrating tip can peak sharply upon penetration of molecular layers. The tip shape appears critical for this effect.

The antiviral activity of recombinant human macrophage CSF (M-CSF) against genital herpes simplex virus type-2 (HSV-2) infection in guinea pigs was investigated. M-CSF stimulates proliferation of human and guinea pig peripheral blood monocytes, specifically the plastic adherent esterase-positive mononuclear cells. When anti-HSV-2 activity of M-CSF was evaluated in guinea pigs by 6 daily injection (s.c.) of M-CSF at various doses (5 x 10(5) to 7 x 10(7) U/kg), we found 2 x 10(6) U/kg to be the optimum dose for protective efficacy against primary HSV-2 infection. Either at a lethal, 5 x 10(5) pfu, or sublethal 5 x 10(4) pfu of virus challenge, animals treated with the optimum regimen of M-CSF exhibited lower herpetic lesion scores (p less than 0.005), and lower mortality (p less than 0.025) than animals in placebo group. M-CSF treatment increased the HSV-infected cell killing activities of plastic-adherent mononuclear cells, indicating that in vivo administration of M-CSF may activate the antiviral effects of guinea pig macrophages that may play a role in protection against severity and mortality of herpetic disease.

Focus on energy and environmental sustainability policy has put automotive research & development directed to developing high efficiency and low pollutant power train. Diffused flame controlled diesel combustion has reach its limitation and has driven R&D to explore other modes of combustions. Known effective mode of combustion to reduce emission are Low temperature combustion (LTC) and homogeneous charge combustion ignition by suppressing Nitrogen Oxide(NOx) and Particulate Matter (PM) formation. The key control to meet this requirement are chemical composition and distribution of fuel and gas during a combustion process. Most research to accomplish this goal is done by manipulating injected mass flow rate and varying indirect EGR through intake manifold. This research paper shows viable alternative direct combustion control via co-axial direct EGR injection with fuel injection process. A simulation study with OpenFOAM is conducted by varying EGR injection velocity and direct EGR injector diameter performed with under two conditions with non-combustion and combustion. n-heptane (C7H16) is used as surrogate fuel together with 57 species 290 semi-detailed chemical kinetic model developed by Chalmers University is used for combustion simulation. Simulation result indicates viability of co-axial EGR injection as a method for low temperature combustion control.

This work presents a novel technique for enforcing the beam-to-shell, beam-to-solid, and shell-to-solid family of constraints in explicit finite element formulations. The limitations of classical multipoint constraint approaches are examined at length, with robustness and accuracy examined through numerical examples. Novel formulation of the new constraint method is presented, and its implementation in the explicit finite element computational cycle discussed. The results of some illustrative test cases employing the newly proposed method are given, showing excellent accuracy even in the presence of large rotations. When compared to existing works, the salient features of the current method are in evidence.Copyright © 2004 by ASME

The therapeutic efficacy and tumor accumulation of a liposome formulation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), an effective agent used in the treatment of malignant brain tumors, was examined in an animal tumor model. Pharmacokinetic studies in normal and tumor-bearing rats indicated that a 2-fold greater plasma exposure was achieved with liposome-formulated CCNU compared with the free drug. In Fisher rats bearing s.c. tumors 36B-10, tumor growth was delayed substantially when liposomal CCNU was delivered compared with free-drug treatment. In single-dose treatments of 20, 35, and 50 mg/kg, tumor progression after each dose was reduced approximately 2-fold with liposomal compared with free CCNU (four animals in each treatment group). Multiple-dose treatments (given as three weekly doses with eight animals in each treatment group) with cumulative doses of 80 and 100 mg/kg of free and liposomal CCNU also resulted in a 2-fold reduction in tumor progression when compared with free-drug treatment. When drug levels in tumors relative to plasma were examined, it was observed that tumor drug concentrations did not exceed those found in plasma after administration of free CCNU; after administration of liposomal CCNU, however, tumor concentrations exceeded those in plasma by nearly 10-fold. These results suggest that the increased efficacy of liposome-formulated CCNU may be attributable to enhanced drug accumulation in tumor tissues.

To reduce the systemic toxicity and prolong the systemic presence of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), a lipid-based drug carrier was designed and characterized.The degree of CCNU association with lipid vesicles composed of 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) (1:1, m/m) was characterized and the drug decomposition rates of lipid-drug complexes were monitored. Effects of lipid association on drug potency against medulloblastoma cells and total systemic drug exposure in rats were determined.At a CCNU:lipid molar ratio greater than 1:5, more than 90% of the drug was associated with the lipid vesicles. In aqueous suspensions, lipid association significantly reduced the first-order drug decomposition rate. In addition, lipid-associated CCNU exhibited a 4-fold increase in drug sensitivity with medulloblastoma cells. IC50 values for CCNU admixed and encapsulated with lipid vesicles were 18+/-4.9 and 14.0+/-2.2 microM, respectively, compared to 83+/-11.0 microM for free CCNU. When administered to rats, lipid-associated CCNU increased the AUC (area under the concentration-time curve) of CCNU by approximately 2-fold (20.46+/-2.15 compared to 39.59+/-1.87 microg x min/ml), and the terminal half-life (t1/2beta) by almost 9-fold (17+/-9 compared to 147+/-48 min) over free CCNU. Despite the increase in total systemic drug exposure, rats treated with lipid-associated CCNU exhibited a significantly lower frequency of acute neurotoxicity.These data indicate that CCNU associated with lipid vesicles may increase drug stability, potency, and systemic exposure in rats.

To determine whether the non-toxic pentameric B subunit of Cholera toxin (CTB) binding to ganglioside GM1 on both the lipid vesicles and epithelial cells may provide a means to target lipid vesicles to mucosal cells expressing surface GM1.Sonicated lipid vesicles containing ganglioside GM1 were prepared. Inter-vesicle cross-linking due to pentameric CTB binding to these GM1 vesicles was determined with a sub-micron particle analyzer. Association of CTB to GM1 vesicles was analyzed with continuous sucrose gradient centrifugation. CTB-mediated binding of GM1 vesicles to human mucosal epithelial cells (Caco-2 and HT-29), mucous membranes of mouse trachea, and nasal tissues were detected with fluorescent labeled vesicles.An increase in lipid particle size due to binding of CTB to lipid vesicles and inter-vesicles cross-linking was detected. At a 30-to-1 mole ratio of membrane-bound GM1-to-CTB, optimum increase in GM1 vesicle aggregation, was detected. Under such conditions, all the added CTB molecules were associated with GM1 vesicles. Time course analysis showed that inter-vesicles cross linking by CTB was detectable within 10 min. and reached a maximum value at 60 min. CTB associated GM1-vesicles bind to mucosal epithelial cells HT-29 and Caco-2 with similar affinity [Kd = 7.8 x 10(-4) M lipid (Caco-2) and 7.6 x 10(-4) M lipid (HT-29)]. GM1 mediated binding specificity was demonstrated by blocking with anti-GM1 antibody and the insignificant degree of CTB-associated GM1 vesicle binding to GM1 deficient C6 cells.The CTB-mediated GM1 binding to multiple membrane surfaces provides selective localization of GM1 vesicles to GM1 expressing mucosal cells and tissues. The strategy may be useful in localizing drugs and proteins to gut and respiratory tract mucosa.

Stringent emission policy has put automotive research & development on developing high efficiency and low pollutant power train. Conventional direct injection diesel engine with diffused flame has reached its limitation and has driven R&D to explore other field of combustion. Low temperature combustion (LTC) and homogeneous charge combustion ignition has been proven to be effective methods in decreasing combustion pollutant emission. Nitrogen Oxide (NOx) and Particulate Matter (PM) formation from combustion can be greatly suppressed. A review on each of method is covered to identify the condition and processes that result in these reductions. The critical parameters that allow such combustion to take place will be highlighted and serves as emphasis to the direction of developing future diesel engine system. This paper is written to explore potential of present numerical and experimental methods in optimizing diesel engine design through adoption of the new combustion technology.

The present study was designed to investigate the effects of 13-cis-retinoic acid (13-cis-RA) (100 micrograms/mouse/day) and androgen ablation (castration) alone and in combination on growth of a human prostatic carcinoma line (LNCaP) transplanted to athymic nude mice as an experimental model. The results of these studies suggest that; (1) androgen ablation (castration) significantly decreased the size of LNCaP xenograft as compared to untreated animals; (2) when 13-cis-RA was administered to nude mice carrying established tumors (0.51 +/- 0.04 cm3), the tumor size was significantly reduced as compared to untreated controls (0.65 +/- 0.06 cm3 versus 1.63 +/- 0.12 cm3). About 50% of the animals in this group showed xenografts necrosis followed by complete regression of tumors by five months; (3) the combination of androgen ablation and 13-cis-RA treatment to nude mice carrying tumors showed synergistic effect in decreasing the tumor size. These results indicate that combination therapies based on androgen ablation and retinoid administration may be a useful approach for the treatment of prostate cancer.

We investigated the ability of human recombinant interleukin-7 (IL-7) to enhance the immune responses of mice vaccinated with either the alum-associated or liposome-formulated recombinant human immunodeficiency virus (HIV)-envelope protein, env-2-3SF2 (a nonglycosylated denatured gp 120 of HIV-1SF2 produced in genetically engineered yeast). Pathogen-free (C3H) mice were vaccinated on days 0, 14, and 28 with 10 micrograms of either the alum-associated env-2-3SF2 or liposome-formulated env-2-3SF2, both containing a lipophylic muramyl tripeptide, MTP-PE. Liposome-formulated IL-7 (5 micrograms/mouse) or empty liposomes were given on days 7, 14, 21, and 28. Antibody response against the immunized antigen, evaluated on day 21 and day 35 or 42, showed that liposome-formulated antigen induced higher antibody titer than did alum-associated antigen, and these antibody responses can be enhanced by concurrent administration of IL-7 liposomes. Spleen cells were harvested on day 21 and day 35 or 42 to evaluate cytotoxic T lymphocyte responses directed against autologous cells infected with vaccinia virus-expressing HIV-envelope protein. Mice treated with liposome-formulated antigen expressed the highest cytotoxic t-lymphocyte (CTL) activity, regardless of whether IL-7 liposome was given as an immune potentiator. In contrast, spleen cells from mice vaccinated with alum-associated antigen exhibited minimal CTL response, which was enhanced by concurrent IL-7 liposome treatment. Collectively, IL-7 liposome treatment enhanced the antibody production of the alum-associated or liposome-formulated env-2-3SF2, whereas its enhancement of CTL activity was detected only in mice vaccinated with alum-associated antigen.

The effects of liposome formulation on interleukin-7 (IL-7)-dependent lymphopoietic activity was investigated based on the pharmacokinetics and tissue distribution profile of soluble and liposome-formulated recombinant human IL-7. Using 125I-IL-7, we determined the role of liposome formulation on in vivo IL-7 disposition by analyzing injection site, blood, tissue, and urinary kinetics. Following a 30- to 40-microgram subcutaneous dose of soluble IL-7, most of the IL-7 was eliminated through urinary excretion within 24 hr. An equivalent subcutaneous dose of liposome-encapsulated IL-7 resulted in a peak level less than one-tenth that seen with soluble drug but produced sustained blood and urinary levels for 5 days. The bioavailability of liposome-encapsulated IL-7 was comparable to that of soluble IL-7, as determined by both blood and urinary data. Kinetic analysis of IL-7 at the subcutaneous injection site indicated that liposome encapsulation significantly reduced the rate of disappearance at the injection site. Studies with a mixture of 40% liposome-encapsulated and 60% soluble IL-7 gave an intermediate response between that of soluble IL-7 and that of liposome-encapsulated IL-7. Characterization of blood cells from IL-7-treated animals indicated that treatment with two weekly doses of mixed IL-7 liposomes (40% liposome encapsulated IL-7) significantly increased the total numbers of lymphocytes by day 14.(ABSTRACT TRUNCATED AT 250 WORDS)

We have optimized a procedure to isolate placental trophoblasts and Hofbauer cells simultaneously in a quantity sufficient for short-term cultures and then used these placental cells to investigate the effects of zidovudine (ZDV) on trophoblast and Hofbauer cell functions. Of more than 10 term placentas tested, ZDV inhibits DNA synthesis of trophoblasts in a concentration-dependent manner with half the maximal inhibitory concentration (IC50) of 9.88 +/- 1.35 microM. Of the hormones evaluated, production of progesterone by trophoblasts is most sensitive to ZDV (IC50 = 3.65 +/- 0.29 microM). The inhibitory effect of ZDV on the secretion of placental lactogen and choriogonadotropin by the trophoblasts was detected only at a much higher concentration (or = 60 microM). ZDV does not affect trophoblast or Hofbauer cell protein synthesis. Collectively, our results indicate that at clinically relevant concentrations (or = 10 microM), ZDV significantly inhibits both the DNA synthesis of placental trophoblasts and their production of progesterone, while having a minimal effect on protein synthesis of both types of placental cells.

Dioleoyl phosphatidylethanolamine (DOPE) does not form stable bilayer liposomes at room temperature and neutral pH. However, stable unilamellar liposomes could be prepared by mixing DOPE with a minimum of 12% of a haptenated lipid, N-(dinitrophenylaminocaproyl)-phosphatidylethanolamine (DNP-cap-PE). When the liposomes bound to rabbit anti-DNP IgG that had been adsorbed on a glass surface, lysis of the liposome occurred with the release of the contents into the medium as judged by the fluorescence enhancement of an entrapped self-quenching dye, calcein. On the other hand, incubation of the same liposomes with glass surfaces coated with normal rabbit IgG had little effect. In addition, free anti-DNP IgG induced aggregation of the liposomes but did not cause any dye release. Liposomes composed of dioleoyl phosphatidylcholine (DOPC) and DNP-cap-PE did not lyse when added to the glass surfaces coated with either anti-DNP IgG or normal IgG. A likely mechanism for liposome lysis is that the DNP-cap-PE laterally diffuse to the contact area between the liposome and the glass. Binding of the haptenated lipid with the immobilized and multivalent antibody trap the haptenated lipids in the contact area. As a result of lateral phase separation, lipids may undergo the bilayer to hexagonal phase transition, leading to the leakage of the entrapped dye. Because both the free hapten and the free antibody inhibited the liposome leakage, this process could be used to assay for the free hapten or antibody. We have shown that inhibition assays performed by using this principle can easily detect 10 pmol of free DNP-glycine in 40 microliter. Furthermore, by substituting human glycophorin A, a transmembrane glycoprotein, for the lipid hapten, we have demonstrated that this assay system is also applicable to detect protein antigen with a sensitivity of sub-nanogram level.

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.

What appears to be a mediator of insulin action has been successfully produced in rat adipocytes plasma membrane upon its treatment with insulin at concentrations of 50-200 microunits/ml. This apparent mediator, when isolated and presented to adipocyte cells, mimics insulin action in the lowering of hormonally stimulated cAMP levels as well as in stimulating lipogenesis and antilipolysis. The cAMP-lowering activity of such a mediator can be quantitated as insulin-activity equivalents. Insulin at 200 microunits/ml causes, in terms of insulin-activity equivalents, a generation of as much as 50 times more of insulin mediator. The magnitude of amplification is even greater when the amount of insulin bound to its receptor is taken into account in this calculation. The action of insulin in the generation of cAMP-lowering mediator is abolished by the insulin antibody. Inactive insulin analogs do not effectively generate such a mediator activity. On the other hand, while the cAMP-lowering action of insulin shown in the bioassay system is completely inhibited by the insulin antibody, the action of such a mediator is only slightly inhibited by the same antibody. The mediator has a low molecular weight and attributes that resemble a peptide. It can be separated from insulin in a Sephadex G-25 column and has a molecular size smaller than the insulin A-chain but larger than ATP. The molecular weight of this mediator is similar to the insulin mediators isolated by other investigators. In view of the fact that it is small in size and mimics several actions of insulin when used in extracellular situations, its theoretical, as well as practical, implications are substantial.

We have shown previously that target-sensitive immunoliposomes composed of palmitoyl antibody stabilized phosphatidylethanolamine bilayers could be destabilized by binding to the target cells (Ho, R. J. Y., Rouse, B. T., and Huang, L., Biochemistry (1986) 25, 5500-5506). Target-sensitive immunoliposome-encapsulated and free cytotoxic drugs of nucleoside analogs cytosine-beta-D-arabinoside (AraC) or acycloguanosine (acyclovir, ACV) were compared for their antiviral efficacy and cell cytotoxicity. Target-insensitive immunoliposomes and nontargeted liposomes were also investigated. When the mouse fibroblast L929 cells were infected at low multiplicity with herpes simplex virus, AraC encapsulated in target-sensitive immunoliposomes composed of transphosphatidylated egg phosphatidylethanolamine effectively inhibited virus replication and had far less cell cytotoxicity than free drug. As a measure of cytotoxicity, the drug concentration required to inhibit 50% of [3H]thymidine incorporation from 6 to 42 h (CD50) was determined. For free AraC, this value was 0.3 ng/ml, whereas for target-sensitive immunoliposome-encapsulated AraC, the CD50 exceeded 1 microgram/ml. However, target-sensitive immunoliposome-encapsulated AraC was virus inhibitory (50% effective dose = ED50) at 1.8 ng/ml. A free drug concentration of at least 1000-fold greater was required for comparable antiviral activity. A similar phenomenon was observed when ACV was administered via target-sensitive immunoliposomes. The CD50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 12.5 ng/ml and 1.4 micrograms/ml, respectively, whereas the ED50 values of the free and target-sensitive immunoliposome-encapsulated ACV were 1.1 and 125 ng/ml, respectively. Consequently, our results indicated the superiority of target-sensitive immunoliposomes at drug delivery, especially when drugs were cytotoxic to cells. The use of liposomes of the target-insensitive variety provided some enhancement of activity, but this was several-fold less than that observed with target-sensitive immunoliposomes. In addition, the nucleoside transport inhibitors, p-nitrothiobenzylinosine and dipyridamole, were shown to inhibit the liposome-mediated antiviral activity of AraC. This finding indicated that site-specific cytosolic delivery of nucleoside analogs by target-sensitive immunoliposomes involved a cellular nucleoside transport system. A mechanism of action is proposed.

Cyclic nucleotide phosphodiesterase activity of several tissues of rat is inhibited by an endogenous factor isolated from rat adipocytes following exposure of these cells to agents that raise intracellular cyclic AMP levels. The inhibitory action was demonstrated with varying cAMP concentrations from 0.1-400 muM. Enzyme from 10,000 X g supernatant of epididymal adipose tissue was inhibited approximately 2-3 fold more than the plasma membrane of adipocytes by a given concentration of the feedback regulator. Kinetic analysis of cAMP phosphodiesterase of plasma membrane showed that feedback regulator (8.8 U/ml) inhibited the Vmax 48%. The maximum inhibition of phosphodiesterase by feedback regulator (20 U/ml) was about 80%. The apparent Km for cAMP was increased. The ability of phosphodiesterase from several tissues of rat (10,000 X g supernatant) to hydrolyze cAMP and cGMP was tested. Feedback regulator inhibited cGMP hydrolysis in cardiac muscle and 5 other tissues 23-92% more than it inhibited the hydrolysis of cAMP. The physiological significance of this inhibitory effect can begin to be clarified when the feedback regulator is purified to homogeneity and characterized.

What appears to be a mediator of insulin action has been successfully produced in rat adipocytes plasma membrane upon its treatment with insulin at concentrations of 50-200 microunits/ml. This apparent mediator, when isolated and presented to adipocyte cells, mimics insulin action in the lowering of hormonally stimulated cAMP levels as well as in stimulating lipogenesis and antilipolysis. The cAMP-lowering activity of such a mediator can be quantitated as insulin-activity equivalents. Insulin at 200 microunits/ml causes, in terms of insulin-activity equivalents, a generation of as much as 50 times more of insulin mediator. The magnitude of amplification is even greater when the amount of insulin bound to its receptor is taken into account in this calculation. The action of insulin in the generation of cAMP-lowering mediator is abolished by the insulin antibody. Inactive insulin analogs do not effectively generate such a mediator activity. On the other hand, while the cAMP-lowering action of insulin shown in the bioassay system is completely inhibited by the insulin antibody, the action of such a mediator is only slightly inhibited by the same antibody. The mediator has a low molecular weight and attributes that resemble a peptide. It can be separated from insulin in a Sephadex G-25 column and has a molecular size smaller than the insulin A-chain but larger than ATP. The molecular weight of this mediator is similar to the insulin mediators isolated by other investigators. In view of the fact that it is small in size and mimics several actions of insulin when used in extracellular situations, its theoretical, as well as practical, implications are substantial.

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.

Forskolin is a unique diterpene activator of adenylate cyclase which has been extensively used in the study of cAMP generating systems. This report describes the production of antibodies to forskolin and the optimization of two sensitive assay methods for such antibodies. 7-0-Hemisuccinyl 7-deacetyl forskolin, coupled to either human serum albumin or goat IgG, was injected into goats to elicit antibodies to the forskolin hapten. Two assay methods, a radioimmunoassay with [12-3H]forskolin as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) with horse radish peroxidase-labelled rabbit anti-goat IgG as an indicator, were optimized to test for the presence of forskolin antibodies in antisera and isolated IgG fractions. The titers for forskolin antisera were 4000-10000. Both assay methods can be adapted to quantify forskolin and its protein conjugates. The availability of antibodies to this diterpene will be useful in accelerating the understanding of the mechanism of adenylate cyclase activation by forskolin.

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

Interactions between target-sensitive (TS) immunoliposomes and herpes simplex virus (HSV) were investigated. Target sensitivity of phosphatidylethanolamine (PE) immunoliposomes is a result of the ability of acylated monoclonal anti-HSV glycoprotein D (gD) to stabilize the bilayer phase of PE, whereas by itself, PE does not form stable liposomes (Ho, R. J. Y., Rouse, B. T., and Huang, L. (1986) Biochemistry 25, 5500-5506). Upon binding of these immunoliposomes to HSV antigen-containing gD, destabilization of PE immunoliposomes was observed. By encapsulating either a self-quenching fluorescent dye, calcein, or alkaline phosphatase inside the liposomal compartment, the HSV-induced destabilization of TS immunoliposomes was shown to be target-specific. Neither Sendai, Semliki Forest, nor Sindbis virus could significantly destabilize the TS immunoliposomes. Moreover, HSV-induced liposome destabilization could be inhibited by free anti-gD (the same antibody used in TS immunoliposomes) but not by monoclonal anti-HSV glycoprotein B, indicating that the interaction was antigen-specific. Destabilization could also be induced by binding to truncated gD (tgD), but only when in a multivalent form immobilized on latex beads. Truncated gD is a cloned, 312-amino acid fragment of HSV-gD that lacks the transmembrane segment. Preincubation of soluble tgD with the TS immunoliposomes failed to induce destabilization and, in addition, abolished the tgD-bead-induced destabilization. This finding strongly indicated that multivalent binding is essential for TS immunoliposome destabilization. Using alkaline phosphatase encapsulated in the liposomes, TS immunoliposomes could be used to detect HSV in fluid phase with 50% signal recorded at 5 microliters of 3.2 x 10(3) pfu/ml; at least 10-fold more sensitive than the standard double-antibody sandwich enzyme-linked immunosorbent assay. The interactions described here may be useful in designing a homogeneous and sensitive immunoliposome assay.