Tumor shedding and coagulation.

Three syngeneic carcinomas from two species shed plasma membrane vesicles when cultured in vitro or grown in the ascites tumor form in vivo. Shed vesicles carry procoagulant activity that can account for the activation of the clotting system and the fibrin deposition associated with these and many other types of malignancy in animals and man.

Reaction of immune complexes with Hodgkin's disease tissue cultures: radioimmune assay and immunoferritin electron microscopy.

We examined the binding of soluble immune complexes in sera from patients with Hodgkin's disease to established tissue cultures derived from the tumor. Circulating immune complex levels were determined by the Raji cell assay, and the reaction of serum with cultured cells was examined with a radioimmune assay and by immunoferritin electron microscopy. Serum with elevated immune complexes was found to react with cells of Hodgkin's disease monolayers when tested with radioiodine-labeled antisera against human IgG heavy and light chains and the complement 3 (C3) component. When examined with the electron microscope, monolayers incubated with Hodgkin's disease serum containing immune complex and labeled with ferritin-conjugated antiserum to C3 contained surface-bound ferritin particles with a uniform but discontinuous pattern. Absorption of Hodgkin's disease serum with monolayer cells reduced immune complexes and decreased reactivity of the sample with cultured cells by radioimmune assay. Sera of patients with other disorders and aggregated gamma-globulin with complement, despite markedly elevated immune complex levels, did not react positively with monolayers derived from Hodgkin's disease tumors, and none of the sera reacted with normal cultured spleen. The approximate size of serum components reacting with Hodgkin's disease monolayers was estimated by sucrose density gradient centrifugation. Sedimentation fractions in the 19S region reacted with monolayer cells when tested with 125I-labeled antisera to both IgG and C3 and contained immunoglobulin-complement complexes by gel diffusion and immunoabsorption. A component sedimenting at 7-9S contained immunoglobulin not complexed with complement; this component reacted with monolayer cells when tested with anti-IgG antiserum but did not react when tested with antibody to C3. The reaction of Hodgkin's disease monolayers with serum containing immune complexes differed from that of two suspension culture lines composed of cells with surface complement and IgG Fc receptors. Inasmuch as cells of our long-term Hodgkin's disease monolayers do not contain these surface receptors, possibly the antibody component of the immune complex reacts with antigens on the surface of cultured cells.

A phase I clinical trial with gadodiamide injection, a nonionic magnetic resonance imaging enhancement agent.

Twenty adult male volunteers were studied in an unblinded, ascending-dose study to evaluate the safety, tolerance, and pharmacokinetics of intravenously administered nonionic gadodiamide injection. Dosages administered were 0.05, 0.1, 0.2, and 0.3 mmol/kg. Subjects were monitored from 36 hours before, through 72 hours after administration. There were no clinically relevant changes in vital signs or electrocardiograms. No clinically significant changes occurred in blood or urine laboratory parameters, although a tendency for minor, transient elevations in serum iron levels 8 to 48 hours after administration was noted. These changes were not dose-related. Nine of 20 subjects reported at least one adverse event; all events were transient and of mild intensity, the most common being dizziness/lightheadedness and perversion of taste or smell. One subject reported discomfort consisting of mild stinging at the injection site during administration. Gadodiamide was excreted unmetabolized in the urine with greater than 95% recovery at 72 hours after administration. The serum elimination half-life was approximately 70 minutes.

Effect of manganese (II) bis(glycinate)dichloride on Ca2+ channel function in cultured chick atrial cells.

Manganese (II) bis(glycinate)dichloride (Mn(glycinate)2) is a coordination complex of manganese with application as a contrast enhancement agent for magnetic resonance imaging in the heart. To determine the cardioactivity of the manganese ion in this chelation cage, the effects of Mn(glycinate)2 on Ca channel function in the cultured chick atrial cell was studied. Mn(glycinate)2 decreased amplitude of contraction in chick atrial cells from embryos 14 days in ovo with complete inhibition of beating at 1 mM and half-maximal effect at 0.1 mM. Under control conditions, Bay K 8644, a Ca channel activator increased amplitude of contraction by 86% with a half maximal effect at 3.2 x 10(-7) M. In the presence of 0.025 mM Mn(glycinate)2, a concentration which had no effect on the amplitude of contraction, the maximum response to Bay K 8644 was decreased to 31%. Mn(glycinate)2 had no effect on the EC50 for the response to Bay K 8644, 1.7 +/- 0.1 x 10(-9) M (S.E.M., n = 4) in control cells compared to 2.2 +/- 0.4 x 10(-9) M (S.E.M., n = 4) in cells incubated with Mn(glycinate)2. 45Ca2+ uptake over 5 min in cultured chick atrial cells decreased from 2.0 nmol/mg protein in control cells to 1.5 nmol/mg protein in the presence of 10(-5) M PN200-110, a Ca2+ channel blocker, a decrease of 28%. 45Ca2+ uptake decreased to 0.94 nmol/mg protein (53%) in the presence of 1 nmol Mn(glycinate)2. Effects of Mn(glycinate)2 and PN200 were not additive. These data demonstrate that Mn(glycinate)2 exerts its negative inotropic effect, at least partially, by interfering with the function of the L-type Ca channels at high concentrations.

Renin synthesis by canine aortic smooth muscle cells in culture.

Angiotensin-I generating activity has been detected in homogenates of arterial tissue but it remains unclear whether this enzymatic activity results from the presence of renin itself or from the action of other proteases such as cathepsin D. In an assay system employing anephric dog plasma as substrate and buffered to pH 7.4, we detected angiotensin-I generating activity in homogenates of canine aortic smooth muscle cells. This enzymatic activity was in large part inhibitable by renin-specific antisera raised to pure canine renal renin. Immunofluorescent study of cultured arterial smooth muscle cells was also performed using renin specific antiserum. Granular cytoplasmic immunofluorescence was detected when specific antirenin serum was used but not when preimmune serum was employed. The addition of pure canine renin to the renin antiserum during staining suppressed the granular immunofluorescence confirming the specificity of staining. Finally, biosynthetic radiolabelling studies were performed. Immunoprecipitation of newly synthesized proteins with antirenin serum and staphylococcal protein A followed by gel electrophoresis and autoradiography demonstrated the synthesis of an immunoreactive protein with the molecular weight of renin. Pretreatment of the antirenin serum with pure canine renin resulted in the disappearance of this immunoreactive protein band. Thus these studies provide multiple lines of evidence to indicate the in situ synthesis of renin by vascular smooth muscle cells.

The relationship between thermodynamics and the toxicity of gadolinium complexes.

The suitability of gadolinium complexes as magnetic resonance imaging contrast agents depends on a number of factors. A thermodynamic relationship to toxicity exists if one assumes that the chemotoxicity of the intact complex is minimal but that the toxicity of the components of the complex (free metal and uncomplexed ligands) is substantial. Release of Gd3+ from the complex is responsible for the toxicity associated with gadolinium complexes; this release appears to be a consequence of Zn2+, Cu2+, and Ca2+ transmetallation in vivo. This hypothesis is supported by acute toxicity experiments, which demonstrate that despite a 50-fold range of LD50 values for four Gd complexes, all become lethally toxic when they release precisely the same quantity of Gd3+, and by subchronic rodent toxicity experiments, which demonstrate a set of gross and microscopic findings similar to those known to be caused by Zn2+ deficiency. Finally, this hypothesis predicts that subtle changes in formulation can further enhance the intrinsic safety of these complexes.

Human pharmacokinetics of a perfluorocarbon ultrasound contrast agent evaluated with gas chromatography.

The purpose of this study was to prospectively study the human pharmacokinetics of an ultrasound (US) contrast agent through its active ingredient, dodecafluoropentane (DDFP). Expired air and blood samples were collected from 24 volunteers after IV administration from 0.01 to 0.1 mL/kg. They were analyzed by a gas chromatographic method specially adapted to the study of DDFP. Blood data fitted to an open one-compartment model. Elimination half-life range was 1.8 to 2.5 min. The area under the curve was correlated to the dose (r(2) = 0.99). Mean blood clearance ranged from 30 to 49 mL/min kg. Blood apparent distribution volume ranged from 0.09 to 0.15 L/kg. In expired air, DDFP concentration exhibited a biexponential decay. The percentage of recovery was 98 +/- 19% at 2 h. No extraneous peaks were observed, indicating no detectable DDFP metabolites. It was concluded that DDFP pharmacokinetics in blood fitted to an open one-compartment model with a fast elimination half-life. Recovery in expired air was almost complete 2 h after administration.

Safety assessment of the use of perflenapent emulsion for contrast enhancement of echocardiography and diagnostic radiology ultrasound studies.

PURPOSE: The studies were undertaken to assess the safety of the use of perflenapent emulsion (EchoGen, SONUS Pharmaceuticals, Bothell, Wash.) for contrast enhancement of echocardiography and radiology ultrasound studies. MATERIALS AND METHODS: In all, 1,001 patients or subjects were enrolled in 21 clinical studies. Clinical laboratory test, pulse oximetry, vital signs, and electrocardiograms (ECGs) were obtained in 818 patients before and after administration of perflenapent emulsion and active control [5% sonicated human albumin, (Albunex, Molecular Biosystems, Inc., San Diego, Calif.)] or placebo (saline) to determine mean changes from baseline. Adverse event rates were monitored following administration of perflenapent emulsion in 743 patients and placebo in 151 patients. RESULTS: No clinically significant abnormalities in clinical laboratory, pulse oximetry, vital signs, or ECG evaluations were observed. Values following administration of perflenapent emulsion were comparable with those following placebo or active control. Perflenapent emulsion (50/743; 6.7%) was comparable with placebo (4/151; 2.6%) in the overall incidence of adverse events considered related to the test article. Adverse events that occurred with a frequency of > or = 1% within 30 min after perflenapent emulsion administration were vasodilation and taste aberration. Adverse events which were mostly mild to moderate in intensity, began within 10 to 20 min after administration, and resolved spontaneously within 10 to 20 min. CONCLUSION: Perflenapent emulsion is generally well tolerated in patients undergoing echocardiography and ultrasound of other target organs.

Lower mean weight after 14 days intravenous administration peptide YY3-36 (PYY3-36) in rabbits.

OBJECTIVE: Endogenous peptide YY(3-36) (PYY(3-36)) is associated with postprandial regulation of appetite. We investigated the safety and effectiveness of peripherally administered synthetic human PYY(3-36) for 14 days in New Zealand white rabbits. Weight gain and food consumption were assessed and pharmacokinetics and toxicity characterized. RESEARCH METHODS AND PROCEDURES: In all, 24 animals were randomized to one of four intravenous treatment groups - control (0.9% saline) or PYY(3-36) bolus at 4.1, 41.0, or 205 microg/kg/day. Body weight and consumption of fixed food allotment were measured daily. Hematology and serum chemistries were profiled at baseline and Day 15, and pharmacokinetics measured following dose 14. Histopathologic examination of designated tissues and organs in control and PYY(3-36) 205 mug/kg animals was conducted. All animals were subject to clinical and macroscopic observation. RESULTS: The trend effect of higher dose PYY(3-36) on lower average weight was significant (P = 0.01; Day 14 compared to baseline) and its effect on reduced food consumption was suggested (P = 0.065; number of days < or =75% food eaten, compared with control). Hematology and clinical chemistries were within normal limits pretest and at Day 15. No clinical, macroscopic, histologic, or microscopic changes related to the test article were observed over the course of study. DISCUSSION: Lower average weight occurs in rabbits treated once daily with intravenous injection PYY(3-36) (205 microg/kg/day) over 14 days. No clinical or histologic signs of toxicity were observed. Further research is warranted to describe alternate routes of peripheral administration for optimizing weight control.

The relA locus specifies a positive effector in branched-chain amino acid transport regulation.

The regulation of branched-chain amino acid transport and periplasmic binding proteins was studied in Escherichia coli strains which were isogenic except for the relA locus, the gene for the "stringent factor," which is responsible for guanosine tetraphosphate synthesis. The strain containing the relA mutation could not be derepressed for the synthesis of leucine transport or binding proteins when shifted from a medium containing all 20 amino acids in excess to one in which leucine was limiting. The relA+ strain showed normal derepression under these conditions.

Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur.

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.

Conformational studies of aqueous melittin: thermodynamic parameters of the monomer-tetramer self-association reaction.

The self-association reaction in which four melittin molecules associate to form an aqueously soluble tetramer was studied by fluorescent spectroscopy. At 23 degrees C, pH 7.15, gamma/2 0.50, the dissociation constant, Kd, is 3.20 x 10(-16) M3. At 23 degrees C, gamma/2 0.60, melittin has an amino acyl group with a proton ionization constant at ca. 10(-6) M, which must be un-ionized for tetramer formation to occur. The change in Kd with temperature indicates the forward reaction (tetramer formation) proceeds primarily by entropic changes, with delta H degrees = -20.3 kJ/mol of monomer and delta S degrees = 211 J/(K . mol of monomer). The observed enthalpic and entropic values for the tetramerization reaction are consistent with the expected contributions of both nascent hydrogen bonds and hydrophobic stabilization to the reaction. The ionic strength dependence of the tetramerization reaction was found to be consistent with an Edsall-Wyman treatment of activity coefficients. Specifically, the calculated charge of melittin varied from 2.5 (pH 10.53, gamma/2 less than 0.08) to ca. 6 (pH 7.15, gamma/2 greater than 0.3) and showed a strong dependence on gamma/2.

Carcinosarcoma of the prostate: case report and review of the literature.

We report the third case of carcinosarcoma of the prostate. The epithelial element consisted of a highly malignant adenocarcinoma. The mesenchymal component was composed of malignant osteosarcoma and chondrosarcoma.

Conformational studies of aqueous melittin. Characteristics of a fluorescent probe binding site.

The structural basis of the interaction of melittin with amphipathic molecular assemblies, i.e. membranes, was investigated by studying the binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to melittin by ultraviolet and fluorescence spectroscopy. Monomeric melittin did not significantly bind TNS as judged by UV and fluorescent spectroscopy. Tetrameric melittin bound two TNS molecules per protomer with dissociation constants (Kd) of 4.2 X 10(-6) M. TNS binding to tetrameric melittin led to an increase in fluorescence quantum yield of 180-fold over the value for TNS alone in aqueous buffer (phi H2O = 0.004). Five independent experimental findings suggest that the arginine residues of melittin provide one portion of the TNS binding site (presumably by formation of an especially stable "ringed-structure" salt bridge between the tetrahedral sulfonyl anion of TNS and the argininyl residues of melittin): 1) the Kd for binding is independent of pH from 6.0 to 10.8, the range in which the alpha-aminoglycine and epsilon-aminolysines titrate; 2) TNS binding fails to perturb the kinetics of the reaction of 2,4,6-trinitrobenzenesulfonate with Lys-21 or Lys-23; 3) 1,7,21,23-acetyl-melittin, in which the NH2 terminus and all lysines are acetylated, binds TNS with a Kd similar to that for normal melittin; 4) guanidination of the NH2-terminal glycines and lysines of melittin (forming N-guanidoglycine and homoargininyl residues, respectively) increases the number of TNS molecules bound per protomer to approximately 5; 5) conversion of Arg-22 and Arg-24 to the anionic N7, N8-(2,3-dihydro(7,7-dimethyl))bicyclo[2.2.1] heptane - 1 - methanesulfonylborate complex abolishes TNS binding, as judged by fluorescence.

Biochemical studies of immune complexes. I. Isolation of circulating immune complexes using a bifunctional exclusion chromatography matrix.

A simple and rapid method of isolating immune complexes from serum is described. The method utilizes the binding properties of a bifunctional chromatography matrix consisting of diethylaminoethyl and Cibacron blue F3GA functional groups linked to agarose beads. This column matrix binds all normal serum proteins except IgG and transferrin, excluding these proteins, and permitting them to be collected in the wash-through, void volume. Immune complexes prepared in vitro with human serum albumin were rapidly isolated from starting serum by this method. This method may have general application for the purification of immune complexes from serum in pathological conditions and should foster subsequent biochemical and immunological studies, including identification and characterization of immune complex antigens.

Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.

The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.