Conformation ability test of human, rabbit and bovine plasminogens and their specific interaction with streptokinase. Human, rabbit and bovine plasminogens, having different sensitivity to streptokinase-activating action, differ, according to spectrophotometric titration, tryptophan fluorescence and circular dichroism spectroscopy, in the state of tyrosine and tryptophan residues, and in secondary and tertiary structures. Human plasminogen-streptokinase equimolar complex formation (according to gel chromatography) is accompanied by a differential ultraviolet spectrum. Difference spectroscopy is a convenient and adequate means of studying the formation of the said complexes. Streptokinase-human plasminogen complex formation is not hindered by partial substitution of water (20%) with ethanol or dimethylsulphoxide or by addition of 0.001 M sodium dodecylsulphate. The complex is not formed in 6 M urea, in solution, at pH less than 2.0 or approximately 12.0-13.0, or with bovine plasminogen. Circular dichroism and tryptophan fluorescence spectral pattern changes during streptokinase-plasminogen complex formation enable us to conclude that streptokinase secondary and tertiary structures undergo certain rearrangements in the framework of the complex, while tryptophan-containing sites of the molecule are not drastically changed. The data obtained enable us to presuppose formation of streptokinase-rabbit plasminogen complexes which differ from human plasminogen complexes with streptokinase.


Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei. Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.


GCI: a network server for interactive 3D graphics. The Graphics Command Interpreter (GCI) is an independent server module that can be interfaced to any program that needs interactive three-dimensional (3D) graphics capabilities. The principal advantage of GCI is its simplicity. Only a limited set of powerful features have been implemented, including object management, global and local transformations, rotation, translation, clipping, scaling, viewport operations, window management, menu handling and picking. GCI and the master (client) program it serves run concurrently, communicating over a local or remote TCP/IP network. GCI sets up socket communication and provides a 3D graphics window and a terminal emulator for the master program. Communication between the two programs is via ASCII strings over standard I/O channels. The implied language for messages is very simple. GCI interprets messages from the master program and implements them as changes of graphical objects or as text messages to the user. GCI provides the user with facilities to manipulate the view of the displayed 3D objects interactively, independently of the master program, and to communicate mouse-controlled selection of menu items or 3D points as well as keyboard strings to the master program. The program is written in C and initially implemented using the Silicon Graphics GL graphics library. As the need to link special libraries to the master program is completely avoided, GCI can very easily be interfaced to existing programs written in any language and running on any operating system capable of TCP/IP communication. The program is freely available.


DNA and protein tetragrams: biological sequences as tetrahedral movements. A graphical approach is introduced for representing information-containing sequences in biology. In particular, the procedure takes DNA or protein sequences containing n bases or amino acids, respectively, and computes n three-dimensional real vectors. When displayed on connected tetrahedra these characteristic patterns appear as DNA or amino acid tetragrams T(n). Experiments indicate that these tetragrams are sensitive to certain important patterns in the sequence of bases and allow the human observer to visually detect various properties of biological sequences. The system presented is special in its focus on the fast characterization of the progression of sequence data using a graphics supercomputer with several controlling parameters.


VisiCoor: a simple program for visualization of proteins. A well-drawn picture acts as an excellent metaphor for something real, and human vision provides instant, random access to any part of which the picture represents. It is in this sense that pictures can convey information more effectively than words alone. The power of the graphics work-stations available today makes visual presentation of scientific results a reality. A molecular graphics program for investigating protein structures, as well as several sample plots that show the power of the program, are presented.


A crystallographic molecular lattice builder applied to model lipid bilayers. It is often desirable for noncrystallographers to generate graphical models of three-dimensional crystal structures based on published coordinates of the atoms that make up the crystallographic unit cells. This type of visualization is particularly important for small-molecule crystals, such as lipid crystals, where one may be interested in investigating interactions between the individual molecules in addition to their conformations. BILAYER BUILDER is a program that generates a portion of the entire crystal structure from the coordinates of the molecules in a single unit cell. It gives users of small desktop computers, such as the Apple Macintosh, the capability to generate and examine model crystal structures with a molecular graphics display program. BILAYER BUILDER stores the crystal coordinates in a Brookhaven Protein Data Bank file format for possible use in a variety of applications on many different computers. Initially, it was written for use with lipid crystals and bilayers but may be used for building an assortment of molecular crystals.


A graphics program for the analysis and display of molecular dynamics trajectories. The program SCARECROW has been developed to help the molecular modeler to analyze and display the very big and complex data files produced by molecular dynamics programs. The molecular graphics program SCARECROW is written to support the display, animation, and extensive analysis of molecular dynamics trajectories. Using the macro language it is easy to make scripts for video animation and for the automated display and analysis of time series. Extensive coloring and atom selection commands are included to help the user to focus on relevant regions of the molecule. Time series can be produced and viewed on the screen or transferred to other programs.


A phase II study of recombinant interleukin-2 with or without recombinant interferon-beta in non-Hodgkin's lymphoma. A study of the Cancer and Leukemia Group B. Interleukin-2 (IL-2) and interferon-beta (IFN-beta) have demonstrated activity against lymphoid malignancies, presumably mediated by the augmentation of lymphokine-activated killer (LAK) cell and natural killer (NK) cell activity. There is in vitro and in vivo evidence to suggest that the combination of IL-2 and IFN-beta is synergistic. The Cancer and Leukemia Group B (CALGB) conducted a randomized phase II trial of IL-2 with or without IFN-beta in 49 patients with relapsed or refractory non-Hodgkin's lymphoma (NHL). Overall toxicity was severe, with 17 patients experiencing life-threatening toxicity. Three patients had treatment-related deaths. Responses were noted in seven patients (17%). There were no meaningful differences between treatment arms in toxicity profile, response rate, or modulation of in vivo NK and LAK activity. We conclude that IL-2 with or without IFN-beta is not effective therapy for NHL in the doses and schedule used in this study.


Rapid cytokine release in cancer patients treated with interleukin-2. Serum concentrations of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-6 (IL-6), interleukin-1 (IL-1) and interferon-alpha (IFN-alpha) were determined by commercially available enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) in cancer patients treated with recombinant IL-2 (rIL-2) either as 1-h infusion (3 or 5 x 10(6)/m2) or continuous intravenous infusion for 5 days (3 x 10(6)/m2/day). A significant increase of TNF-alpha and IL-6 serum levels was observed in each patient. One-hour infusion of IL-2 induced a very rapid secretion of TNF-alpha, IL-6 and IFN-gamma with considerably higher peak levels than during IL-2 continuous intravenous infusion. IFN-gamma was released into the blood of all patients receiving IL-2 1-h infusion, but only occasionally during or after IL-2 continuous intravenous infusion. Neither IFN-alpha nor IL-1 were detectable in the serum before, during, or following IL-2 treatment in all patients studied. The kinetics of IL-2 after 1-h infusion fitted to a two-compartment model, suggesting the synthesis of considerable amounts of endogenous IL-2. Following IL-2 1-h infusion, rising TNF-alpha serum levels preceded the increase of serum IFN-gamma or IL-6. The serum peak levels of IFN-gamma and IL-6 decreased rapidly with a half-life of 0.29 to 2.5 h. The concentration time profiles of TNF following 1-h infusion of IL-2 demonstrated a considerably longer half-life than that of intravenously administered recombinant TNF as done in other studies.(ABSTRACT TRUNCATED AT 250 WORDS)


In vivo administration of recombinant macrophage colony-stimulating factor induces macrophage-mediated antibody-dependent cytotoxicity of tumor cells. Macrophage colony-stimulating factor (M-CSF) has been previously shown to facilitate the in vitro survival and differentiation of mononuclear phagocytes. We assessed whether M-CSF administration in vivo could induce macrophages capable of killing tumor via an antibody-dependent mechanism. C57BL/6 mice were given intraperitoneal M-CSF, and peritoneal macrophages were assayed for their ability to kill fluorochrome-labeled R1.1 thymoma cells in vitro in the presence or absence of target-specific antibody. Two-color flow cytometry was used in measuring tumor ingestion by macrophages; macrophages from M-CSF-treated mice eliminated greater than 90% of R1.1 thymoma target within 24 hours, while macrophages from saline-treated controls were ineffective. R1.1 tumor elimination by macrophages depended on the presence of target-specific antibody. These are the first studies that demonstrate the in vivo induction, by M-CSF, of macrophages directly capable of ingesting antibody-conjugated tumor cells.


Phase I study of recombinant human interleukin-2 for pediatric malignancies: feasibility of outpatient therapy. A Pediatric Oncology Group Study. Published data indicate that when recombinant interleukin-2 (rIL-2) is administered to children as a 15-min i.v. bolus, doses of 18 x 10(6) IU/m2 are poorly tolerated, requiring intensive care unit (ICU) management of IL-2-induced hypotension. We administered rIL-2 as a 1- or 2-h i.v. infusion to 11 children with malignancies refractory to conventional therapy. IL-2 was given every Monday/Wednesday/Friday for 3 weeks. Four children received 12 x 10(6) IU/m2/dose, four received 18 x 10(6) IU/m2/dose, and three received 24 x 10(6) IU/m2/dose (1 Cetus Unit = 6 IU). Fever, chills, flushing, nausea, vomiting, transient weight gain, and oliguria were observed at all three dose levels (not dose-limiting toxicities). Cardiovascular toxicity was significantly reduced compared to the bolus regimen. Mild hypotension was observed at all three dose levels; however, there was no severe dose-limiting hypotension. Because of reduced cardiovascular toxicity, IL-2 was safely administered on an outpatient basis. This regimen induced marginal transient increases in natural killer cell activity and lymphokine-activated killer cell activity. No measurable clinical tumor response was observed in any of the 11 children. The maximum-tolerated dose has not been reached. This regimen allows for a considerable cost reduction (outpatient care instead of ICU care) and safety, making further clinical trials on the use of IL-2 in children more feasible.


Bryostatin 1 activates T cells that have antitumor activity. Several strategies have been used to stimulate the growth of tumor-specific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U/ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 microM ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 microM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominantly CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.


Modulation of tumor-infiltrating lymphocytes derived from human renal cell carcinoma by interleukin-4. Current methods of expanding tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma bulk cultures result in a heterogeneous population of cells with low tumor-killing specificity. To improve the yield of cells with higher autologous and lower nonspecific cytotoxicity, interleukin-4 (IL-4) was added to high (1,000 U/ml)- and low (20 U/ml)-dose IL-2 and compared to cultures grown without IL-4 for proliferation, phenotype, and cytotoxicity against targets including autologous and allogeneic tumors. When compared to culture in IL-2 alone, the addition of IL-4 improved overall expansion in both high-dose (mean fold expansion of 2,061 vs. 1,087) and low-dose (mean fold expansion of 1,904 vs. 262) IL-2. Enhancement of TIL proliferation was dependent on the timing of IL-4 addition to the culture; augmented growth occurred only when IL-4 was added with or following activation by IL-2. The phenotype consisted primarily of CD3+/CD4+ lymphocytes with a reciprocal reduction in CD56+/CD16+ cells. Finally, there was a significant reduction in nonspecific cytotoxicity against K-562, M-14, and allogeneic tumor targets, but no significant change against autologous tumor. We conclude that IL-4 has an important regulatory effect on the expansion of renal cell carcinoma TILs in IL-2 by the promoting growth of CD3+/CD4+ lymphocytes and inhibiting the growth and nonspecific cytotoxicity associated with LAK-like CD16+/CD56+ cells. These findings may be beneficial in extracting more potent effector cells from bulk TIL culture for use in clinical trials.


Activation of human monocytes by interleukin-2 and various cytokines. We previously reported that human macrophages cultured with IL-2 for a long period (lymphokine-activated macrophages, LAMs) showed high tumoricidal activity against human and murine leukemic cell lines through a different mechanism from lymphokine-activated killer (LAK) cells. In this report, we investigated the effects of various cytokines on the tumoricidal activity of IL-2-induced LAMs against HeLa cells. CSF-1 and IL-1 were found to enhance the tumoricidal activity of LAM in a dose-dependent manner, whereas IFN-gamma and TNF had inhibitory effects. CSF-1 in combination with a low dose of IL-2 synergistically induced LAMs with highly tumoricidal activity. We also found that monocytes from some donors that did not respond to IL-2 were differentiated to tumoricidal macrophages by treatment with a combination of CSF-1 and IL-2. Furthermore, IL-2-induced LAMs were found to produce cytotoxic factors in the culture medium when they were cocultured with tumor cells, and the cytotoxic activity in the culture supernatant of LAMs was also increased by the incubation of LAMs with CSF-1. The cytotoxicity of the supernatants from macrophages with different tumoricidal activity correlated with their cell-mediated cytotoxicity. It is suggested from these results that the cytotoxicity of LAMs is regulated by CSF-1, IL-1, IFN-gamma, and TNF, and that the production of cytotoxic molecules is involved in cell-mediated killing by LAMs.


The concentration dependence of the depolarization of yeast by monovalent cations. Monovalent cations decrease the initial rate of uptake of the membrane potential probe 2-(dimethylaminostyryl)-1-ethyl-pyridinium (DMP) into metabolizing cells, showing that the cells are depolarized. A steep decrease in this rate was found even at low cation concentrations, reaching 62%, 42%, 58%, 40% and 40% at high concentrations of K+, Rb+, Cs+, Na+ and Li+, respectively. The corresponding concentrations at which half-maximum decrease was found were 0.22, 0.36, 1.2, 17 and 17 mM. These values are of the same order of magnitude as the half-saturation concentrations for monovalent cation uptake by the yeast.


Amitriptyline attenuates the febrile response to a pyrogen in rabbits. We investigated the effect of a tricyclic antidepressant (TCA) agent on fever in rabbits. In the first series of experiments, the TCA agent amitriptyline hydrochloride (AMI) was administered intraperitoneally (i.p.) at a dose of 1 mg/kg or 10 mg/kg daily for three weeks. The rabbit's response to intravenous (i.v.) injection of 0.1 micrograms/kg of the lipopolysaccharide (LPS) of Salmonella typhosa was tested on a weekly basis. In the second series of experiments, rabbits were given simultaneous i.v. injections of either 1 mg/kg or 10 mg/kg of AMI together with 0.1 microgram/kg of LPS. After two weeks of chronic i.p. administration of 10 mg/kg AMI, and for the remainder of the experimental period, the rabbit's response to the LPS was significantly attenuated when compared to control animals. When AMI was administered simultaneously with the LPS, a significant dose-dependent antipyresis was observed from 50 minutes after injection, compared to the response of control animals. AMI had no effect on the body temperature of afebrile animals.


Lithium fluxes in Paramecium and their relationship to chemoresponse. Paramecia respond to environmental stimuli by altering swimming behavior to disperse from or accumulate in the vicinity of the stimulus. We have found, using the T-maze assay, that treatment of paramecia with LiCl in a time- and concentration-dependent manner modifies the normal response to folate, acetate, and lactate from attraction to no response or even repulsion. Responses to NH4Cl were unaffected and to cAMP were variably affected by LiCl. Cells incubated in the presence of K+, or both Na+ and K+, but not Na+ alone reliably recovered attraction to folate. Treatment of cells with 4 mM LiCl for 1 h dramatically slowed swimming speed from about 1 mm/s in NaCl or KCl (control) to 0.18 mm/s in LiCl. Li-treated cells subsequently incubated in 4 mM NaCl, KCl or sequentially in KCl and NaCl for a total of 20 min increased their swimming speed to 0.35, 0.45 and 0.67 mm/s, respectively. Paramecia readily took up Li+ in Na(+)- and K(+)-free media reaching intracellular concentrations of 5-10 mM in 10 mM extracellular Li+. Efflux of intracellular Li+ was stimulated 35% by extracellular 10 mM NaCl and 185% by 10 mM KCl over 10 mM choline chloride. Incubation of cells in 10 mM LiCl for 1 h inhibited the rate of Ca2+ efflux by 44% compared to cells in 10 mM NaCl. This may relate to the mechanism by which Li+ perturbs chemoresponse. A mutant with defects in Ca homeostasis responds normally to NH4Cl, but not to any of the stimuli that are affected by LiCl.


Transbilayer incorporation of 1-pyrenebutyltrimethylammonium by blood platelets and its application for analyzing changes in physico-chemical properties of the membrane lipid bilayer induced by platelet activation. The binding of cationic butyltrimethylammonium derivative of pyrene to bovine platelets was initially rapid and then increased gradually, unlike the bindings of other anionic and neutral derivatives of pyrene tested. The rate of increase in binding of the cationic probe depended on temperature and was due to its incorporation into the cytoplasmic side of the platelet membranes, as shown quantitatively by monitoring decrease in its extractability with albumin. The penetration into the inner membrane compartment did not reach equilibrium even after 4 h at 37 degrees C. Slow penetration of a fluorescent probe such as this is useful in studies on the physico-chemical properties of the outer layer and cytoplasmic side of the platelet membranes and their changes. Initial rapid binding of the cationic probe to platelets, representing the binding of the probe to the outer layer of the plasma membrane, was increased by ionomycin-induced platelet activation. Fluorescence spectra in the presence of a relatively high concentration of the cationic probe showed increase of the excimer of the cationic probe accompanied with the incorporation of the probe to the cytoplasmic side. On ionomycin-induced activation, the excimer-to-monomer intensity ratio of the probe in the cytoplasmic side of the platelet membranes decreased, possibly due to decrease in fluidity of the lipid layer near the probe or change in distribution of the probe.


Peripheral-type mitochondrial binding sites for benzodiazepines in GH3 pituitary cells. Benzodiazepines (BZs) interact with two classes of high affinity binding sites, equilibrium dissociation constants in the nanomolar range, a neuronal or central-type and a non-neuronal or peripheral-type. The peripheral-type binding site has been shown to be present on the outer mitochondrial membrane and appears to be involved in regulation of cholesterol transport in steroid hormone-producing endocrine cells. In rat pituitary GH3 cells, BZs bind to receptors for thyrotropin-releasing hormone (TRH) and via interaction at a different site block Ca2+ influx through voltage-sensitive channels. These, however, are low affinity interactions occurring at micromolar BZ concentrations. Here, using [3H]Ro 5-4864, we report that GH3 cells also have high affinity peripheral-type BZ binding sites. Apparent equilibrium dissociation constants of 7.8 +/- 1.7 nM and 9.3 +/- 4.5 nM for [3H]Ro 5-4864 were measured with intact cells and isolated mitochondria, respectively. As predicted from studies of these sites in other cells, the order of potencies of BZs to displace [3H]Ro 5-4864 was Ro 5-4864 greater than diazepam (DZP) much greater than clonazepam (CIZP); chlordiazepoxide (CDE) did not affect binding. Nifedipine, a dihydropyridine antagonist of Ca2+ channels that has been shown to displace BZs from peripheral-type sites in other cells, was shown to be a competitive inhibitor of [3H]Ro 5-4864 binding with a half-effective concentration in the micromolar range. Ro 5-4864, however, had no effect on Ca2+ influx or efflux in mitochondria isolated from GH3 cells. Hence, GH3 cells exhibit mitochondrial, peripheral-type BZ binding sites but the role of these putative receptors in these neuroendocrine cells, which do not produce steroid hormones, is unclear.


Involvement of histidine residues and sulfhydryl groups in the function of the biotin transport carrier of rabbit intestinal brush-border membrane. Possible involvement of histidine residues and sulfhydryl groups in the function of the intestinal brush-border membrane (BBM) transporter of biotin was investigated. This was done by examining the effects of pretreatment of BBM vesicle (BBMV) isolated from rabbit intestine with the histidine-specific reagent diethyl pyrocarbonate (DEPC) and the sulfhydryl group-specific reagents p-chloromercuribenzenesulfonic acid (p-CMBS) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) on carrier-mediated biotin transport. Pretreatment of BBMV with DEPC caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with DEPC provided significant protection to biotin transport. Treatment of DEPC-pretreated vesicles with the reducing agents dithiothreitol and 2,3-dimercaptopropanol failed to reverse the inhibitory effect of DEPC on biotin transport. The inhibitory effect of DEPC was found to be mediated through a marked decrease in the number of the functional biotin transport carriers with no change in their affinity, as indicated by the severe inhibition in the Vmax but not the apparent Km of the biotin transport process, respectively. Pretreatment of BBMV with p-CMBS and NBD-Cl also caused significant inhibition in the initial rate of biotin transport without affecting the substrate uptake at equilibrium. Addition of biotin plus Na+ to vesicle suspensions prior to treatment with p-CMBS (or NBD-Cl) failed to protect biotin transport from inhibition. On the other hand, treatment of vesicles pretreated with p-CMBS (or NBD-Cl) with the reducing agents dithiothreitol and mercaptoethanol caused significant reversal in the inhibition of biotin transport. The inhibitory effects of p-CMBS (and NBD-Cl) on biotin transport was also found to be mediated through inhibition in the Vmax, but not the apparent Km, of biotin transport process. These results indicate the involvement of histidine residues and sulfhydryl groups in the normal function of the biotin transport system of rabbit intestinal BBM. Furthermore, the results also suggest that the histidine residues are probably located at (or near) the substrate-binding site while the sulfhydryl groups are located at a site other than the substrate binding region.


Pentazocine reduces cholinergic responses in the guinea-pig extrahepatic biliary tract by a non-opiate mechanism. The mechanical responses of the guinea-pig gallbladder and common bile duct to pentazocine were measured in vitro. At concentrations of 1 microM or higher, pentazocine reduced the responses of the gallbladder and common bile duct to electrical stimulation and to carbachol. This antagonism was not blocked by naloxone and appears to be due to an atropine-like action of pentazocine. At concentrations of 5 microM or higher pentazocine induced gallbladder contraction which was naloxone-insensitive. Both effects were not affected by tetrodotoxin and appear to be due to direct action on the muscle. The antimuscarinic activity of pentazocine should be taken into consideration when this opioid is administered experimentally or clinically.


Antidepressant activity of S-adenosyl-L-methionine in mice and rats. S-Adenosyl-L-methionine (SAM), main methyl donor, was tested in mice and rats in several models which are predictive of possible antidepressant activity. In the forced swimming test in rats the effect of SAM was compared with that of the tricyclic antidepressant amitriptyline. SAM decreased dose-dependently immobility time in the forced swimming test in mice and rats, these effects being antagonized by haloperidol and prazosin (the latter only in rats). Locomotor or exploratory activity in mice and rats was not increased by SAM. D-Amphetamine-induced locomotor hyper-activity in rats was increased by repeated (14 days, twice daily) treatment with SAM. Behavioural stimulation induced by D-amphetamine or L-dopa (given with benserazide) in mice was not changed by a single dose of SAM. The drug reduced hypothermia induced by apomorphine in mice. Hypothermia induced by reserpine or clonidine in mice was not antagonized. SAM increased the amplitude of the acoustic startle reflex. The above results indicate that the psychopharmacological profile of SAM resembles that of antidepressants in only some tests. The mechanism by which SAM produces its antidepressant effect needs further investigation.


Merocyanine 540 as a probe to monitor the molecular packing of phosphatidylcholine: a monolayer epifluorescence microscopy and spectroscopy study. The characteristics of the fluorescent dye, merocyanine 540 (MC-540), incorporated in monolayers of 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were studied in different states of molecular packing. Conditions for phase separation in these monolayers were defined by their pressure/area (pi-A) isotherms. Within the liquid expanded (LE) and the liquid condensed (LC) coexisting phases of DPPC monolayers, low light level epifluorescence microscopy revealed 'dark' discoid domains embedded in a 'bright' matrix. Under the same conditions, and at temperatures as low as 12 degrees C, the pi-A isotherms of POPC demonstrate the existence of a single phase, and no fluorescent domains were observed. Fluorescence spectra of MC-540 labelled monolayers, recorded in different structural states, reveal three distinct emission peaks: a 572 nm peak, present for monolayer packing conditions at low surface pressures; a 585 nm peak, similar to that obtained from dye molecules in fluid phase lipid bilayers, and observed here within the respective area/molecule ranges of 54-62 A2 and 62-69 A2 for monolayers of DPPC and POPC with diminishing intensity at increasing surface pressure; and finally, a peak at 560 nm, which predominates in densely packed POPC monolayers. Our results are interpreted on the basis of dye partitioning between monolayer and subphase, and different orientations of the dye with respect to the monolayer in various structural states. The usefulness of MC-540 to differentiate lipid packing in cell membranes is discussed.


Melatonin levels are decreased in rheumatoid arthritis. The hormone, melatonin, is a product of the pineal gland. This methoxy-indole, also known as N-acetyl-5-methoxy-tryptamine, is structurally related to indomethacin, a derivative of methylated indole. The fact that indomethacin has been used successfully in patients suffering from certain chronic inflammatory conditions (such as rheumatoid- and osteoarthritis) gives rise to the question as to whether melatonin also possesses anti-inflammatory virtues. This hypothesis has been tested by determining melatonin concentration levels by means of a radioimmunoassay in patients with rheumatoid arthritis. The daytime melatonin levels of untreated patients were significantly lower (mean concentration = 5.76 pg/ml) than the normal value (mean concentration = 15-33 pg/ml). The second part of this study showed that the administration of indomethacin (100 mg/day) to normal healthy subjects led to a 14.7 pg/ml decrease in plasma melatonin levels. This may indicate that melatonin and indomethacin act synergistically.


A role for the transient increase of cytoplasmic free calcium in cell rescue after photodynamic treatment. Chinese hamster ovary (CHO) cells and T24 human bladder transitional carcinoma cells were treated with the photosensitizers aluminum phthalocyanine (AlPc) and hematoporphyrin derivative (HPD), respectively. Exposure of both sensitized cell lines to red light caused an immediate increase of cytoplasmic free calcium, [Ca2+]i, reaching a peak within 5-15 min after exposure and then returning to basal level (approximately 200 nM). The level of the peak [Ca2+]i depended on the light fluence, reaching a maximum of 800-1000 nM at light doses that kill about 90% of the cells. Loading the cells with the intracellular calcium chelators quin2 or BAPTA prior to light exposure enhanced cell killing. This indicates that increased [Ca2+]i after photodynamic therapy (PDT) contributed to survivability of the treated cells by triggering a cellular rescue response. The results of experiments with calcium-free buffer and calcium chelators indicate that both in CHO cells treated with AlPc and with HPD-PDT of T24 cells extracellular Ca2+ influx is mainly responsible for elevated [Ca2+]i. PDT is unique in triggering a cell rescue process via elevated [Ca2+]i. Other cytotoxic agents, e.g., H2O2, produce sustained increase of [Ca2+]i that is involved in the pathological processes leading to cell death.


The permeability and the effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory and experiment. The model of Cruzeiro-Hansson et al. (Biochim. Biophys. Acta (1989) 979, 166-1176) for lipid-cholesterol bilayers at low cholesterol concentrations is used to predict the thermodynamic properties and the passive ion permeability of lipid bilayers as a function of acyl-chain length and cholesterol concentration. Numerical simulations based on the Monte Carlo method are used to determine the equilibrium state of the system near the main gel-fluid phase transition. The permeability is calculated using an ansatz which relates the passive permeability to the amount of interfaces formed in the bilayer when cholesterol is present. The model predicts at low cholesterol contents an increase in the membrane permeability in the transition region both for increasing cholesterol concentration and for decreasing chain length at a given value of the reduced temperature. This is in contrast to the case of lipid bilayers containing high cholesterol concentrations where the cholesterol strongly suppresses the permeability. Experimental results for the Na+ permeability of C15PC and DPPC (C16PC) bilayers containing cholesterol are presented which confirm the theoretical predictions at low cholesterol concentrations.


Amphotericin B-phospholipid interactions responsible for reduced mammalian cell toxicity. When interacting with phospholipid in an aqueous environment, amphotericin B forms unusual structures of markedly reduced toxicity (Janoff et al. (1988) Proc. Natl. Acad. Sci. USA 85, 6122-6126). These structures, which appear ribbon-like by freeze-fracture electron microscopy (EM), are found exclusively at amphotericin B to lipid mole ratios of 1:3 to 1:1. At lower mole ratios they occur in combination with liposomes. Circular dichroism (CD) spectra revealed two distinct modes of lipid-amphotericin B interaction, one for liposomes and one for the ribbon-like structures. In isolated liposomes, amphotericin B which comprised 3-4 mole percent of the bulk lipid was monomeric and exhibited a hemolytic activity comparable to amphotericin B suspended in deoxycholate. Above 3-4 mole percent amphotericin B, ribbon-like structures emerged and CD spectra indicated drug-lipid complexation. Minimal inhibitory concentrations for Candida albicans of liposomal and complexed amphotericin B were comparable and could be attributed to amphotericin a release as a result of lipid breakdown within the ribbon-like material by a heat labile extracellular yeast product (lipase). Negative stain EM of the ribbon-like structures indicated that the ribbon-like appearance seen by freeze-fracture EM arises as a consequence of the cross-fracturing of what are aggregated, collapsed single lamellar, presumably interdigitated, membranes. Studies examining complexation of amphotericin B with either DMPC or DMPG demonstrated that headgroup interactions played little role in the formation of the ribbon-like structures. With these results we propose that ribbon-like structures result from phase separation of amphotericin B-phospholipid complexes within the phospholipid matrix such that amphotericin B release, and thus acute toxicity, is curtailed. Formation of amphotericin B-lipid structures such as those described here indicates a possible new role for lipid as a stabilizing matrix for drug delivery of lipophilic substances, specifically where a highly ordered packing arrangement between lipid and compound can be achieved.


A second phenazine methosulphate-linked formate dehydrogenase isoenzyme in Escherichia coli. A biochemical and immunological study has revealed a new formate dehydrogenase isoenzyme in Escherichia coli. The enzyme is an isoenzyme of the respiratory formate dehydrogenase (FDH-N) which forms part of the formate to nitrate respiratory pathway found in the organisms when it is grown anaerobically in the presence of nitrate. The new enzyme, termed FDH-Z, cross reacts with antibodies raised to FDH-N and possesses a similar polypeptide composition to FDH-N. FDH-Z catalyses the phenazine methosulphate-linked formate dehydrogenase activity present in the aerobically-grown bacterium. FDH-Z and FDH-N exhibit distinct regulation. Like formate dehydrogenase N, formate dehydrogenase Z is a membrane-bound molybdoenzyme. With nitrate reductase it can catalyse electron transfer between formate and nitrate. Quinones are required for the physiological electron transfer to nitrate. It seems likely that like FDH-N, FDH-Z functions physiologically as a formate: quinone oxidoreductase.


Induction of a Ca(2+)-activated K+ channel in human erythrocytes by mechanical stress. Mechanical deformation of normal ATP-replete human erythrocytes increased their permeability to Ca2+ sufficiently to turn on the Ca(2+)-activated K+ channel (the Gardos channel). When Ca2+ is absent, mechanical deformation of normal erythrocytes induces an equivalent increase the permeability of both Na+ and K+, In the presence of 0.1 to 1 mM Ca2+, a further increase in the K+ efflux rate was seen. There was no increase in Na+ flux above that induced by deformation itself. The involvement of the Ca(2+)-activated H channel was verified by showing the specific inhibitors of the channel, quinine and charybdotoxin, prevent the Ca(2+)-induced increase in K+ efflux. These results are consistent with a model of sickle cell dehydration proposed by Bookchin et al. ((1987) Prog. Clin. Biol. Res. 240, 193-200). The estimated rate of Ca2+ entry under these conditions (37 degrees C, 1000 dyne/cm2, and laminar shear) was about 1 mmol/loc per h.


Temperature-dependent effects of cholesterol on sodium transport through lipid membranes by an ionizable mobile carrier. Temperature-jump relaxation experiments on Na+ transport by (221)C10-cryptand were carried out in order to study the influence of cholesterol and its temperature-dependence on ion transport through thin lipid membranes. The experiments were performed on large, negatively charged unilamellar vesicles (LUV) prepared from mixtures of dioleoylphosphatidylcholine, phosphatidic acid and cholesterol (mole fractions 0-0.43), at various temperatures and carrier concentrations. The initial rates of Na+ transport and the apparent rate constants of its translocation by (221)C10 increased with the carrier concentration and the temperature. The incorporation of cholesterol into the membranes significantly reduced the carrier concentration- and temperature-dependence of these two parameters. The apparent energy required to activate the transport decreased significantly with increasing carrier concentrations at any given cholesterol molar fraction, and increased significantly with the cholesterol molar fraction at any given carrier concentration. Our interpretation of the action of cholesterol on this transport system is based on the assumption that the binding cavity of cryptands is likely to be located towards the aqueous side of the dipole layer. The results are discussed in terms of the structural, physico-chemical and electrical characteristics of carriers and complexes, and of the interactions occurring between an ionizable mobile carrier and the membrane.


Freeze-fracture analysis of the effects of intermediates of the phosphatidylinositol cycle on fusion of rough endoplasmic reticulum membranes. While searching for the identity of the effector of the putative GTP-binding protein involved in fusion of rough endoplasmic reticulum (RER) cell-free incubation conditions were found permitting fusion in a GTP-independent manner. Membrane fusion was obtained using medium required to study synthesis of phosphatidylinositol (PI). We now report on the effects of various co-factors and intermediates of the PI cycle on the interaction of rough microsomes. By freeze-fracture, fusion of rough microsomes was defined as the appearance of fracture-planes of membrane larger than those of unincubated membrane. Cytosine triphosphate (CTP, 3 mM) in the presence of 2 mM MnCl2 was most effective in stimulating fusion. Guanosine triphosphate (GTP) at the same concentration, could substitute for CTP to stimulate fusion, ATP, ITP, UTP and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) could not. When combined together in the same medium CTP potentiated the effect of GTP. Arachidonic acid (20 micrograms/ml) also stimulated fusion in the presence of MnCl2. This led to the appearance of large fracture-planes of membrane with a heterogeneous distribution of intramembranous particles. Other saturated fatty acids at the same concentration did not stimulate fusion. Phosphatidylinositol (PI, 50 micrograms) and 2 mM MnCl2 had a similar effect as arachidonic acid and MnCl2 in stimulating fusion. The PI effect was largely augmented in the presence of CTP. Our results are consistent with the concept that metabolism of phospholipids may modulate GTP-dependent fusion of RER membranes.


The relaxation effect as observed on lipid suspensions of low polydispersity. Lipid suspensions with a low polydispersity (delta = 0.15 +/- 0.05, as given by photon correlation spectroscopy (PCS)) were used to elucidate the origin of the disagreement between the experimental zeta potential values (zeta sm), obtained from the electrophoretic mobilities through the Smoluchowski equation, and double-layer theory prediction (zeta potential) at low salt concentrations. The values of zeta sm, measured for cardiolipin and phosphatidylserine suspensions in monovalent electrolytes, were compared with the correspondent theoretical values of the zeta potentials correlated for the relaxation effect; the correction was made according to the S.S. Dukhin theory of electrophoresis. It was found, that this correction eliminates the disagreement for cardiolipin in NaCl entirely; it partly solves the problem for cardiolipin in KCl but fails to improve the situation for phosphatidylserine in NaCl.


Triggered release of hydrophilic agents from plasmalogen liposomes using visible light or acid. Triggered release from liposomes composed of semi-synthetic 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphocholine (plasmalogen) lipids has been demonstrated using either aerobic visible illumination or low pH to induce leakage. The photodynamic release system consists of three functional components: (1) small (less than 1000 A) unilamellar plasmalogen vesicles (SUVs) containing encapsulated glucose, (2) oxygen and (3) zinc phthalocyanine (ZnPc) incorporated within the hydrophobic region of the SUV membrane. Irradiation (lambda greater than 640 nm) at 37 degrees C of air-saturated 1-alk-1'-enyl-2-palmitoyl-sn-glycero-3-phosphocholine (PlasPPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (8:1, mol/mol) liposomes at physiologically relevant temperatures results in glucose release rates that are twice those of the corresponding dark control. Photolysis of argon-saturated PlasPPC/DPPC liposomes or of identical vesicles lacking either ZnPc or the plasmalogen vinyl ether bond exhibit glucose release curves which are indistinguishable from the dark control. Irradiation under identical conditions, but in the presence of 100 mM sodium azide, also results in no increased rate of glucose release above that of the dark control. TLC analysis indicates that oxidized lipid species are produced only in air-saturated, irradiated plasmalogen liposomes. The acid lability of the plasmalogen vinyl ether linkage has also been used to trigger release of entrapped calcein. At pH 4.2, the release rate at 37 degrees C is increased 4-fold over rates observed at pH 8. TLC analysis indicates formation of a lysoplasmalogen product. Taken together, these results indicate that both photodynamic and acid triggering can be used to increase plasmalogen liposome permeability and suggest that these liposomes are potentially useful for drug delivery applications.


Effect of oligomers of ethylene glycol on thermotropic phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles. The effect of oligomers of ethylene glycol (EG) on thermotropic phase transitions of dipalmitoylglycerophosphatidylcholine multilamellar vesicles (DPPC-MLV) were investigated. Diethylene glycol (di-EG) had a biphasic effect on transition temperature, reducing pre-transition temperature (Tp) at low concentrations but increasing main transition temperature (Tm) and extinguishing pre-transition at high concentration. Results of the X-ray diffraction method and the excimer method indicated that di-EG induced interdigitated gel phase (L beta 1 phase) in the DPPC membranes at high concentration. Phase diagram of temperature-di-EG concentration for DPPC-MLV was determined by use of X-ray diffraction and differential scanning calorimetry, which was similar to that of temperature-EG concentration. The minimum concentration of di-EG where L beta 1 phase was induced was 42%(w/v), which was larger than that of EG (30%(w/v)). On the other hand, in the presence of triethylene glycol (tri-EG), Tm and Tp increased with an increased in tri-EG concentration, as well as poly(ethylene glycol). These differences, between the effects of di-EG and those of tri-EG, might be due to the differences of their sizes.


Presence of small GTP-binding proteins in the peroxisomal membrane. Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.


Subcellular distribution of carbonic anhydrase in the enterocyte of the rabbit ileum and the Caco-2 cell. Evidence for the presence of two isozymes bound to brush-border membranes. The activity of CA has been determined in the membranes of enterocytes from rabbit ileum and of Caco-2 cells. No CA activity was detected in the BLM, but the activity in the BBM (43 and 7 WAU/mg protein for rabbit and Caco-2, respectively) was doubled by the addition of Triton. These two types of activity could be distinguished in rabbit ileum by their different IC50 in the presence of acetazolamide (10(-5) and 5 x 10(-7) M) and their different sensitivities to heat. They were not modified by inhibitors of cytoplasmic isozymes and seem to correspond to two forms of CA, one situated in the extracellular leaflet of the BBM and the other one in the intracellular leaflet.


Protein flux across the membrane of single secretion granules. We have applied, for the first time to our knowledge, X-ray microscopy to measure the mass of protein contained in single sub-cellular membrane-bound structures and to make high resolution, time-resolved observations on them. Using this method we have been able to follow the flux of protein out of secretion (zymogen) granules isolated from the acinar cells of the exocrine pancreas. The results provide direct visual and quantitative confirmation of the hypothesis that the membrane enclosing this object is permeable to its various contained proteins, although the mechanism remains unknown.


Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes. We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.


Tellurite-induced damage of the erythrocyte membrane. Manifestations and mechanisms. Chemical and biophysical mechanisms underlying the thiol-dependent lytic action of tellurite (and selenite) on human erythrocytes were investigated using native and GSH-depleted cells. Exposure of GSH-depleted cells to tellurite alone produces oxidative cross-linking of membrane thiols paralleled by a moderate membrane leakiness comparable in its extent to that induced by other SH-oxidizing agents (diamide, periodate). Exposure to tellurite in presence of endogenous or exogenous GSH produces marked leakiness which stems from the formation of aqueous leaks permeant to ions and nonelectrolytes and sensitive to inhibition by phloretin. Apparent pore radii, derived from exclusion limits for polar non-electrolytes, range from 0.3 to at least 1.3 nm. Leak size increases with increasing exposure time and concentration of the modifier. Leak formation is paralleled by membrane rigidification based on the cross-linking of spectrin. Thiol-dependent leak formation by tellurite in GSH-depleted cells can be sustained not only by exogenous GSH but also by other thiols. Progress of leak formation by tellurite/thiol can not be reliably quenched by procedures such as removal of tellurite from the medium, inhibition of anion transport via band-3 protein, washing of the cells or low temperature. The reaction can, however, be terminated, even in the presence of tellurite, by addition of N-ethylmaleimide, presumably due to the blockage of thiols or thiol-analogous tellurium compounds. N-ethylmaleimide even brings about a partial reversal of leakiness, suggesting the contribution of a reversible and an irreversible component of tellurite damage. Membrane perturbation by tellurite/thiol involves the formation of a membrane permeant tellurium species, possibly HTe-, which is likely to induce progressive damage of membrane proteins by a redox shuttle going along with a formation of elemental tellurium and its reduction by thiols.


Denaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride; identification of inactive, partially unfolded, dimeric intermediates. The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.


Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast. The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14)DM (lanosterol 14 alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14)DM from Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of 26-hydroxylanosterol and 25-hydroxy-24,25-dihydrolanosterol with a lower activity than lanosterol and 24,25-dihydrolanosterol. This enzyme demethylated the (Z)-24-ethylidene-24,25-dihydrolanosterol with a low rate, but did not metabolize the E-isomer. The apparent Km of 26-hydroxylanosterol was 10.8 microM, which was higher than that of lanosterol, but lower than that of 24,25-dihydrolanosterol. On the other hand, competition experiments suggested that the affinity of 25-hydroxy-24,25-dihydrolanosterol and (Z)-24-ethylidene-24,25-dihydrolanosterol for P-450(14)DM was significantly lower than that of 24,25-dihydrolanosterol. Integration of the present results with the preceding ones (Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1991) Biochim. Biophys. Acta, 1081, 262-266 and Aoyama, Y. and Yoshida, Y. (1991) Biochem. Biophys. Res. Commun., 178, 1064-1071) suggests that yeast P-450(14)DM recognizes two parts of the side-chain, the structure around C-24 and the terminal fork consisting of C-25, C-26 and C-27.


Localization and characterization of hemoglobin-degrading aspartic proteinases from the malarial parasite Plasmodium falciparum. Three hemoglobin-degrading proteinases were partially purified from food vacuoles isolated from trophozoite-stage forms of the malarial parasite Plasmodium falciparum. Two of the proteinases (M1 and M2) were solubilized by repeated sonication. The remaining proteinase (M3) was solubilized by treatment of the particulate fraction with taurocholic acid, suggesting that proteinase M3 is a membrane-bound proteinase whereas proteinases M1 and M2 are weakly associated with parasite membrane. The location of these proteinases suggests that they may participate in the digestion of host cytosolic protein. After partial purification, but not before, proteinases M1, M2 and M3 are highly sensitive to pepstatin, supporting their designation as aspartic proteinases. These aspartic proteinases show broad specificity for protein substrates. Native hemoglobin, acid denatured hemoglobin and oxidatively damaged hemoglobin are comparable substrates. Hemoglobin within the food vacuole was shown to be primarily native hemoglobin. Chemical modification studies indicate that these three aspartic proteinases have similar properties. The peptide maps from degradation of hemoglobin, however, suggest that aspartic proteinases M1, M2 and M3 are distinct proteinases.


NADPH activates a decarboxylation reaction catalysed by lamb liver 6-phosphogluconate dehydrogenase. NADP-dependent lamb liver 6-phosphogluconate dehydrogenase catalyses the oxidative decarboxylation of 2-deoxy-6-phosphogluconate, an analogue of the natural substrate. The first products of the reaction are NADPH and 3-keto-2-deoxy-6-phosphogluconate. The NADPH, released from the enzyme, binds to the coenzyme site of the same or the other subunit, activating the decarboxylation reaction in which has not a redox role, since it can be substituted by an analogue devoid of enzymatic redox power. These findings are compared to those obtained with other NADP-dependent decarboxylating dehydrogenases.


A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus. A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.


Conversion of non-functional to functional iron following reconstitution of hemerythrin. A recent report from this laboratory (Zhang, J.-H., Kurtz, D.M., Jr., Xia, Y.-M. and Debrunner, P.G. (1991) Biochemistry 30, 583-589) described a procedure for reconstitution of a functional di-iron site in the octameric, non-heme iron O2-carrying protein, hemerythrin by addition of ferrous salts to apoprotein, followed by slow dilution of the denaturant. Although the resulting protein contained its full complement of iron, i.e., 2 Fe per subunit, about 30% of the iron was found to remain ferrous under ambient O2, i.e., this iron was incapable of forming an O2 adduct. In this report a method is described for obtaining essentially fully functional hemerythrin by passage of the freshly reconstituted protein through an [oxy/30% non-functional----met----deoxy----oxy redox cycle. UV/vis absorption and 57Fe Mössbauer spectroscopies show that little or no non-functional iron remains in the reconstituted oxyhemerythrin after the redox cycle. Quantitations of protein and diiron sites show that, during the first step of the redox cycle, the non-functional iron is converted to a form that is spectroscopically indistinguishable from that of native methemerythrin. Far-UV circular dichroism shows that the secondary structure of this reconstituted methemerythrin is essentially identical to that of native protein. Non-denaturing polyacrylamide gel electrophoresis shows that the size and charge of the native and reconstituted proteins before and after redox cycling are essentially identical. These results indicate that the non-functional iron is converted to a functional form by the redox cycling, and that the key step in this conversion is the [oxy/30% non-functional]----met transformation.


Effect of NaCl addition on nanosecond O2 escaping reaction of myoglobin: evidences for the transition of myoglobin dynamic structure at 20 degrees C. We studied the nanosecond (ns) geminate O2 escape reaction from the protein interior of myoglobin (Mb) to the solvent phase in the temperature range of 5-40 degrees C containing 0-0.1 M NaCl. In the flash photolysis experiments, we found that both the rate constant, kout, and its Arrhenius plot changed upon the variation of the NaCl concentration. In particular, it was noteworthy that the Arrhenius plot of kout dramatically changed in its slope, keeping the break at 20 degrees C, upon the addition of NaCl, indicating that the thermodynamic parameters such as an enthalpy of activation (delta H not equal to) and an entropy of activation (delta S not equal to) are different between above and below 20 degrees C, and that they are further altered upon the NaCl addition to the sample solution. From these results, we suggested that the Mb dynamic structure in the ns geminate O2 escape reaction is sensitively regulated by the interaction of the protein surface and the salt. The present study also showed that an inconsistency of the Arrhenius plot of kout between Chatfield et al. ((1990) J. Am. Chem. Soc. 112, 4680-4687) and us ((1990) J. Biol. Chem. 265, 18823-18828) is probably due to the difference in the solution condition.


Hydrophobic interaction of lysozyme and alpha-lactalbumin from equine milk whey. From fluorescence measurements on mixtures of bis-ANS and equine lysozyme and from Ca(2+)-dependent hydrophobic interaction chromatography of equine lysozyme, it is demonstrated that Ca2+ binding induces a conformational change upon which hydrophobic regions in the protein become less accessible. Bis-ANS fluorescence titrations in the absence of Ca2+ and in 2 mM Ca2+ are also performed with equine alpha-lactalbumin variants B and C. These variants differ by an amino-acid exchange Asp----Ile at residue 95. The fluorescence titration curves indicate that the accessibility of the probe to the Ca2+ conformers is clearly influenced by the mutation. The Ca(2+)-dependent exclusion of a hydrophobic domain is used in a new and simplified method for preparing lysozyme and alpha-lactalbumins simultaneously from equine milk whey.


Adenosine deaminase from Saccharomyces cerevisiae: kinetics and interaction with transition and ground state inhibitors. Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme. The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate. The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme. The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported. The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.


Promotion of beta-structure by interaction of diabetes associated polypeptide (amylin) with phosphatidylcholine. The interaction of the diabetes associated polypeptide (amylin) with dimyristoylphosphatidylcholine (DMPC) was assessed by measurements of turbidity (absorbance at 400 nm) and secondary structure by CD spectroscopy. In trifluoroethanol, human amylin adopts a highly alpha-helical conformation while the rat peptide is less structured. In water, the rat peptide is largely disordered and the human peptide exhibits a combination of alpha- and beta-structures. Mixtures of DMPC and the rat peptide have no effect on either the turbidity of the DMPC or the CD spectrum of the peptide. By contrast, mixtures of the human peptide with DMPC form relatively clear mixtures similar to those observed with amphipathic alpha-helical peptides, but the structure adopted, based on the CD spectrum, is largely beta. These data demonstrate that fundamental differences in the structures adopted by amylins from human and rat species exist in mixtures with DMPC and suggest that these differences may be related to the formation of amyloid fibrils in the human amylin peptide which are not observed in the rat peptide.


Limited proteolysis of chicken gizzard 5'-nucleotidase. Chicken gizzard 5'-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5'-nucleotidase by limited proteolysis. A hydrophobic proteolytic fragment carrying the intact C-terminus, as well as two major hydrophilic products, were identified. We developed procedures for specific radiolabelling of the active center of 5'-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5'-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5'-Nucleotidase represents a binding protein for components of the extracellular matrix. The interaction between 5'-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5'-nucleotidase. In summary, we have further characterized chicken gizzard 5'-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5'-nucleotidase.


Structure and metabolism of multiple heparan sulphate proteoglycans synthesized by the isolated rat glomerulus. Metabolism of biosynthetically [35S]sulphate-labelled heparan sulphate proteoglycan (HSPG) was studied in the isolated glomerulus. Chromatography and electrophoresis resolved HS into 5 components, designated HS1a, HS1b, and HS2 to HS4 in order of increasing Kd. Both HS1a (250 kDa) and HS1b (130 kDa) are present in the glomerular basement membrane and have glycosaminoglycan chains of 25-45 kDa. Chemical analysis of glycosaminoglycan chains indicated a similar content of 50% N-sulphation and 30% 6-O-sulphation on the hexosamine residues of all HSs, with the remaining 20% of sulphate likely at the 2-O-position of uronic acid residues. By pulse-chase analysis, the basement-membrane fraction was found to have a half-life of residency in the glomerulus of 37 h. Both HS1a and HS1b are mainly released intact into the medium and are not further broken down in that compartment. In contrast, HS2 is almost completely released into the medium immediately after synthesis and is not normally recovered from the tissue. It is a 90-kDa HSPG with a hydrophobic core protein and glycosaminoglycan chains similar in size to those of HS1. In addition to these larger PGs, HS3 and HS4 represent glycosaminoglycan chains with little or no core protein. HS1a, HS1b and HS2 were iodinated and deglycosylated. Each has a 30-kDa core protein in addition to 18 kDa of chondroitinase ABC- and nitrous-acid-resistant O-linked carbohydrate. This suggests the possibility of a single core protein with variable glycosylation and destination. HS1a has 5-6 glycosaminoglycan chains, HS1b 2-3 and HS2 1-2. We propose that basement-membrane HSPG (HS1a and HS1b) and a related, underglycosylated secreted HSPG (HS2) are the major HSPGs synthesized by the isolated glomerulus. Other molecular species may represent discrete steps in the turnover of basement-membrane HSPG.


Ammonium chloride-induced acidification in renal TALH SVE.1 cells monitored by 31P-NMR. The aim of this study was to investigate the effect of NH4+ on the intracellular pH in TALH SVE.1 cells derived from the medullary thick ascending limb of Henle's loop (TALH) of rabbit kidney. These cells are specialized to perform NH4+ transport in vivo. Intracellular pH was monitored by 31P-NMR. The steady state intracellular pH (pHi) under standard conditions was 7.24 +/- 0.04 (n = 46). Exposure to NH4Cl resulted in an initial intracellular acidification of the TALH SVE.1 cells, followed by a recovery to the initial steady-state pHi value. The NH4(+)-induced acidification followed saturation kinetics up to 20 mM NH4Cl (delta pHmax = 0.2 pHunits). Half-maximal acidification was observed at 0.6 mmol/l. The intracellular acidification due to NH4Cl exposure was completely inhibited by 0.1 mM of the diuretic bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter. The effect of bumetanide was dose-dependent and a Ki value of 8.10(-7) M was calculated. NH4+ influx via K+ channels or the (Na+ + K+)ATPase could not be detected. pHi recovery to the initial value was caused mainly by amiloride-sensitive Na+/H+ exchange and to a lesser extent by an amiloride-insensitive system, which was not studied in detail. In the presence of bumetanide, pulses of high concentrations of NH4Cl induced small intracellular alkalinizations. From these experiments, an intrinsic buffer capacity (beta i) in TALH SVE.1 cells of 26 +/- 3 mM x pH-1 (pHi = 7.65) was determined. It could also be shown that the TALH SVE.1 cells exhibit maximal 'functional buffer capability' between pHout 6.9 and 7.3. Within these limits the cells can maintain their intracellular pH at a constant level, even though the extracellular pH changes. These data strongly suggest that the Na+/K+/2Cl- cotransporter is the main site of NH4+ entry into rabbit thick ascending limb cells in culture. A high intracellular buffer capacity and potent acid extrusion mechanism cooperate in counteracting the intracellular acidification caused by NH4+ influx into the cell.


Abnormal accumulation and toxicity of polyamines in a difluoromethylornithine-resistant HTC cell variant. Mammalian cells possess an inducible, active polyamine transport system that is stringently regulated by feedback controls. This study provides evidence that DH23b cells, which were initially selected from the rat hepatoma HTC line for overproduction of ornithine decarboxylase, demonstrate an abnormality in the regulation of polyamine transport. Exposure of these cells to micromolar levels of spermidine or spermine resulted in inhibition of protein synthesis and eventual cell lysis. These effects were not due to by-products of polyamine oxidation by serum oxidases as neither inhibition of protein synthesis nor cell lysis was mitigated by aminoguanidine, reduced glutathione, dithiothreitol, or catalase. Although the polyamine transport system in the DH23b cells has the same Km and Vmax as that in the parental HTC line, the variant cells accumulated abnormally high levels of both spermidine (8-times normal) and spermine (4-times normal). In the HTC line, however, transport of both polyamines as well as putrescine was feedback inhibited within approx. 3 h, while in the variant cells uptake was not diminished by 12 h and terminated only with cell lysis. The DH23b cells appear to lack the normal mechanism responsible for feedback control of active polyamine incorporation. This defect provided the opportunity to manipulate intracellular levels of spermidine from 30 to approx. 800% of normal, allowing the demonstration that cellular protein synthesis is as sensitive to spermidine levels as previous in-vitro studies had suggested.


Guanine nucleotides stimulate carboxyl methylation of kidney cytosolic proteins. We studied the effect of guanine nucleotides on the carboxyl methylation catalyzed by class II protein carboxylmethyltransferases (PCMT). Addition of guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) promoted a time- and concentration-dependent enhancement of protein methylation in the cytosolic fraction isolated from kidney cortex. GTP gamma S affected the kinetics of the methylation reaction, as reflected by alterations of both apparent Km and Vmax of the methyltransferase. This effect was specific for guanine nucleotides and was completely abolished by addition of S-adenosyl-L-homocysteine, a well-known inhibitor of methyltransferase-catalyzed reactions. No GTP gamma S stimulation of methylation was found in cytosolic extracts from any of the other tissues studied, including brain, testis, spleen, and liver, nor in brush-border membranes isolated from the kidney cortex. The methylated proteins were highly sensitive to moderately alkaline conditions, suggesting that the methyl esters were formed on L-isoaspartyl residues and thus methylated by a class II PCMT. These results suggest that class-II-associated protein methylation activity from the soluble fraction of the kidney can be regulated by guanine nucleotides.


Measurement of intracellular Ca2+ in young and old human erythrocytes using 19F-NMR spectroscopy. Elevated cell calcium has been implicated in functional changes with human erythrocyte aging. However, until recently it has been difficult to measure free ionic intracellular calcium in red cells. We have made use of a fluorinated calcium chelator probe (5,5'-difluoroBAPTA) and fluorine nuclear magnetic resonance (19F-NMR) techniques to measure changes of intracellular Ca2+ concentrations ([Ca2+]i) with cell aging. We have demonstrated in these studies that human erythrocyte [Ca2+]i is significantly elevated as a function of in-vivo aging. Young cells, the least dense fraction of density-separated erythrocytes, contained an average of 62 (+/- 4) nM Ca2+ (+/- S.E.), whereas the oldest, most dense cell fraction contained 221 nM Ca2+ (+/- 25). Mechanisms by which intracellular [Ca2+] increases with in-vivo aging are currently under investigation.


Glucocorticoid stimulation of amnion cell prostaglandin synthesis: suppression by protein kinase C inhibitors and independence of phorbol ester-sensitive protein kinase C. Glucocorticoids stimulate the prostaglandin E2 production of confluent amnion cell cultures, but have no stimulatory effect on the PGE2 output of freshly isolated human amnion cells. Since protein phosphorylation may modify the responsiveness of target cells to steroids, and activators of protein kinase C (PKC), as well as corticosteroids, promote amnion cell PGE2 output by stimulating the synthesis of prostaglandin endoperoxide H synthase (PGHS), we investigated the possibility that PKC is involved in the glucocorticoid-induction of PGE2 synthesis in cultured amnion cells. The dexamethasone-induced PGE2 output of arachidonate-stimulated cells was blocked by the protein kinase inhibitors staurosporine, K-252a, H7, HA1004, and sphinganine, in a manner consistent with their effect on PKC. However, dexamethasone increased the PGE2 production of cultures treated with maximally effective concentrations of the PKC-activator compound TPA. Moreover, dexamethasone stimulated PGE2 synthesis in cultures which were desensitized to TPA-stimulation by prolonged phorbol ester treatment. Concentration-dependence studies showed that staurosporine completely (greater than 95%) blocked glucocorticoid-provoked PGE2 synthesis at concentrations which did not inhibit TPA-stimulated prostaglandin output, and that K-252a inhibited the effect of TPA by more than 95% at concentrations which decreased the effect of dexamethasone only moderately (approximately 40%). Dibutyryl cyclic AMP had no influence on the basal- or dexamethasone-stimulated PGE2 production, and on the staurosporine inhibition of the steroid effect. These results show that glucocorticoids and phorbol esters control amnion PGE2 production by separate regulatory mechanisms. It is suggested that the response of human amnion cells to glucocorticoids is modulated by protein kinase(s) other than phorbol ester-sensitive PKC and cyclic AMP-dependent protein kinase.


Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine. Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A2 and D. Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using [1-14C]oleate-labelled autoclaved E. coli (20 microM) as substrate. Inhibition is comparable using the same concentrations (20 microM) of [1-14C]oleate-labelled C. albicans or E. coli, or aqueous dispersions of 1-acyl-2-[1-14C]linoleoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. Inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory. Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.


Exo-protein kinase release from intact cultured aortic endothelial cells. Extracellular protein kinase activity is demonstrated in intact cultured porcine aortic endothelial cells and is characterised. When cells were incubated with [gamma-32P]ATP (1 microM) a major cell surface protein, corresponding to 115 kDa, and at least four serum proteins (19, 21, 55 and 126 kDa) became phosphorylated. Protein kinase activity is released by intact endothelial cells, which is not due to cell damage, as judged by various cell viability parameters (e.g., release of marker enzymes, trypan blue exclusion). The activity of the protein kinase released amounted to 170 fmol/min per mg endothelial cell protein with phosvitin as substrate, which represents 9% of the total cellular phosvitin protein kinase activity. Repetitive incubation of endothelial cells substantially decreased phosvitin-kinase release. Exo-protein kinase is not influenced by cAMP and cGMP but is effectively inhibited by heparin (EC50, 0.3 microgram/ml). The findings clearly demonstrate: (1) exo-protein kinase is released by intact porcine aortic endothelial cells; (2) substrates of this enzyme are endothelial surface proteins and serum proteins.


Poly(ADPR)polymerase expression and activity during proliferation and differentiation of rat astrocyte and neuronal cultures. Poly(ADPR)polymerase (poly(ADPR)P) mRNA and enzymatic activity levels were investigated in primary cultures of rat astrocytes and neurons in the absence or presence of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF), respectively. In cultured rat astrocytes, a biphasic increase in poly(ADPR)P mRNA, associated with enhanced nuclear poly(ADPR)P enzymatic activity, were observed. The first rise in poly(ADPR)P mRNA and enzymatic activity is at the beginning of cell proliferation and the second with the occurrence of cell differentiation. In the presence of bFGF (5 ng/ml) the mRNA peaks and the differentiation-associated poly(ADPR)P enzymatic activity undergoes a 2-fold increase. In neuronal cultures an initial high level of poly(ADPR)P mRNA is followed by a decrease while differentiation is progressively achieved. A limited increase of poly(ADPR)P activity is observed during this phase. In the presence of NGF (50 ng/ml), similar poly(ADPR)P mRNA expression and enzymatic activity patterns are observed. The results suggest that poly(ADPR)P is involved at the onset of nerve-cell proliferation and differentiation.


Cell-free phosphorylation of the murine small heat-shock protein hsp25 by an endogenous kinase from Ehrlich ascites tumor cells. The small heat-shock protein hsp25 of the Ehrlich ascites tumor exists in one non-phosphorylated (hsp25/1) and two phosphorylated (hsp25/2, hsp25/3) isoforms. In stationary phase tumor cells, a protein kinase activity was detected which phosphorylates hsp25/1, resulting in the formation of several phosphorylated hsp25 isoforms, including those occurring naturally in the tumor. Cell-free phosphorylation of hsp25 required Mg2+ and ATP and was independent of Ca2+, phosphatidylserine, cAMP and cGMP. Polymyxin B inhibited, specifically, hsp25 phosphorylation, whereas trifluoperazine, staurosporine and the protein inhibitor of protein kinase A had no effect. In its properties, the hsp25 phosphorylating kinase differs from other common kinases such as protein kinases A and C, calcium/calmodulin-dependent kinases, and the ribosomal protein S6 kinase.


cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations. Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RI alpha, RI beta, RII alpha, and RII beta, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14-15-day-old rats. Following separation by DEAE-cellulose, R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14-15 day-old rats, showed the presence of a prominent type I holoenzyme containing RI alpha subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RII beta subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominantly RII alpha and, to a lesser extent RII beta subunits (peak 3). The 53 kDa RI beta protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14-15-day-old, but not adult, rats exhibited high levels of C subunit-free RI alpha, a result not predicted by mRNA studies. This latter result may be attributable to direct RI alpha regulation or to indirect RII beta regulation at a time during testis development prior to germ cell maturation.


Amylin inhibits insulin-stimulated glucose uptake in C2C12 muscle cell line through a cholera-toxin-sensitive mechanism. Rat amylin inhibits insulin-stimulated glucose uptake with an IC50 of 12.1 +/- 4.1 pM in C2C12 myotubes. The maximal inhibition is 64 +/- 5.4% observed at a 100-pM dose of the peptide. Consistently, presence of 100 pM amylin shifted the dose-response curve of insulin to the right, increasing the ED50 from 0.71 to 16 nM. No effect of amylin is observed on basal glucose uptake in these cells. Cholera-toxin treatment of the cells did not affect the insulin-stimulated glucose uptake, while the inhibitory effect is completely lost in toxin-treated cells. These findings strongly suggest that rat amylin is active at a physiological concentration and the amylin inhibition of glucose uptake is mediated through a cholera-toxin-sensitive mechanism.


Hypofrontality on topographic EEG in schizophrenia. Correlations with neuropsychological and psychopathological parameters. Topographic EEG was performed in 17 DSM-III-R schizophrenic patients and in 15 sex- and age-matched healthy controls. Eleven patients were first-onset (neuroleptic naive) schizophrenics. EEG band power was compared with psychopathology, neuropsychology and neurological soft signs. The EEG was recorded at 14 topographic locations monopolarly and movements of the eye and of the lid were monitored by two bipolar electro-oculogram (EOG) derivations, one vertical and one horizontal. A multivariate correction of EOG artefacts was performed based on regression analysis with respect to EOG channels. Schizophrenic patients showed higher mean and median power in most bands. These differences were marked in the delta band, in the fast alpha and beta bands, in particular at left frontal sites. Delta power at F7 was by far the best separating variable between schizophrenics and controls in a discriminant analysis. Significant positive correlations were found between the Brief Psychiatric Rating Scale scores "Anxiety-depression" and "Activation" and power in the fast bands and negative ones between "Anergia" and the beta bands. Positive significant correlations emerged between the total score in the Negative Symptoms Rating Scale and the amount of delta power, predominantly over the temporal region. Impairment in the Luria-Nebraska neuropsychological scores "Rhythm" and "Memory" correlated highly significantly with EEG band power. No correlations were found between neurological soft signs and EEG band power. Our results are in line with the hypothesis of a hypofrontality in schizophrenia. It is unlikely that these findings are an artefact of prior psychiatric treatment, as they were also observed in first-onset, neuroleptic naive schizophrenics.(ABSTRACT TRUNCATED AT 250 WORDS)


Smooth pursuit eye movements and neuropsychological tests in schizophrenic patients: possible involvement of attentional components. The relationships among different components of frontal lobe dysfunction and voluntary attention were studied. Drug-free schizophrenic patients and matched normal controls were recruited and assessed for smooth pursuit eye movements, voluntary saccadic eye movements and by means of neuropsychological tests (Toluse-Pieron test and Wisconsin Card Sorting Test). No clear-cut relationship was found between eye movement performance and neuropsychological impairment.


Anxiogenic properties of yohimbine. I. Behavioral, physiological and biochemical measures. The anxiogenic effects of yohimbine, a specific alpha-2-receptor antagonist were examined by administering 20 mg yohimbine orally to 8 panic patients on placebo treatment, 7 panic patients on alprazolam treatment and 12 controls using a double-blind randomized design, instructions that minimized the expectancy of experiencing a panic attack and two additional structured situations. Yohimbine induced more pronounced increases in anxiety and panicky ratings, norepinephrine secretion, maximum heart rate and high heart rate variability and decreases in skin temperature in panic patients compared with controls. However, possibly owing to an instructional set and experimental design that distracted patients from unpleasant bodily sensations no panic attacks were observed.


Anxiogenic properties of yohimbine. II. Influence of experimental set and setting. To study the pharmacological induction of stress along with psychological stress and their possible interaction, 20 mg yohimbine and placebo orally were administered to 8 panic patients on placebo treatment, 7 panic patients on alprazolam treatment and 12 controls in a double-blind crossover design. Two structured situations which can be considered as 'neutral' stressors were included: a mental arithmetic task and a continuous performance task. Mental arithmetic induced robust increases in ratings of panicky, anxiety, nervousness, heart rate and electrodermal activity, while the continuous performance task induced increases exclusively in skin conductance reaction. Patients responded to these tasks less than controls with regard to subjective ratings and electrodermal activity. Yohimbine did not potentiate the response to the tasks in the patients. In controls, heart rate during the mental arithmetic task, but not during rest, was increased after yohimbine. In contrast to other yohimbine challenge studies no panic attacks were observed. It is hypothesized that the experimental design together with an instructional set that reduces expectancy factors and the inclusion of structured and time-limited tasks in a challenge paradigm is able to reduce the anxiogenic effects of yohimbine.


The deleterious effect of ocular artefacts on the quantitative EEG, and a remedy. The effect of ocular artefacts on spectral EEG parameters is assessed statistically. These artefacts are caused by movements of the eyeball and/or of the lid. Further, methods for correcting ocular artefacts are presented and evaluated. This methodological study is based on data from an investigation comparing the EEG of schizophrenic patients (n = 17) with healthy controls (n = 15). Ocular artefacts are monitored by the bipolar vertical and the bipolar horizontal electro-oculogram (EOG). It is shown that the influence of ocular artefacts on the measured electrical activity in the frontal region is larger than the cerebral potentials which the EEG is ideally intended to record. The more frequent occurrence of blinks and eye movements in schizophrenic patients may lead to an artificial enhancement of slow frequency EEG power for schizophrenics and eventually "false significances". In contrast to this, we found more significant group differences when correcting for EOG artefacts than without it. This can be attributed to a very much inflated sample variability of the uncorrected EEG, due to the individually varying EOG power. We conclude that it may not be sufficient to select visually epochs for analysis that are considered artefact-free. Rather, one should monitor EOG artefacts and apply an appropriate correction.


Subclinical hyperthyroidism: physical and mental state of patients. We investigated whether subclinical hyperthyroidism [subnormal basal thyroid-stimulating hormone (TSH) level, attenuated TSH response to thyrotropin-releasing hormone (TRH) stimulation, peripheral thyroid hormones within normal range] is accompanied by physical and mental changes. Thirty-five subclinically hyperthyroid patients (27 female, 8 male) were compared with 60 overtly hyperthyroid patients (51 female, 9 male) and with 28 euthyroid control patients (18 female, 10 male) with respect to physical symptoms, affective state, short-term memory, ability to concentrate and psychomotor performance. Patients with subclinical hyperthyroidism ranged between the other two groups. The major difference between controls and subclinically hyperthyroid patients was an increase in frequency of nervous symptoms and symptoms due to an increase of metabolic rate and thermal regulation changes. The major differences between subclinically hyperthyroid and overtly hyperthyroid patients were psychomotor impairment and symptoms of increased metabolic rate. Self-ratings of affective state tended to be similar in patients with subclinical and overt hyperthyroidism. The ability to concentrate and short-term memory were not impaired in any group. Symptoms in patients with subclinical hyperthyroidism probably result from central changes which lead to attenuated TSH responses to TRH, or from elevated but still normal thyroxine levels, which possibly enhance the effect of catecholamines.


[Results of radioiodine therapy in different forms of hyperthyroidism in relation to the planned dosage]. The aim of this study was to assess the efficacy of radioiodine therapy (131J) in a large group (n = 925) of hyperthyroid patients treated at two major departments of nuclear medicine (Freiburg, abbr. FR, and Munich, abbr. M). 761 patients suffered from non-immunogenic hyperthyroidism (Plummer's disease) and the remaining 164 patients from immunogenic hyperthyroidism (Graves' disease). In these cases, radioiodine therapy using doses between 60 and 80 Gy proved ineffective, FR (80 Gy) recording 28% success and M (60 Gy) 54%. A dose of 150 Gy, however, is successful in more than 80% of the cases: FR 81%, M86%. However, the incidence rate of hypothyroidism increases consecutively with 150 Gy: FR 49%, M 62%. In patients suffering from Plummer's disease, the solitary autonomous nodule can be eliminated by radioiodine therapy (400 Gy) with a high rate of success (95%); the same applies to multinodular autonomous adenomas. The therapeutic concept applying a dose of 400 Gy to the total functional autonomous tissue (delineated by ultrasound) yields slightly better results (95%) than 150 Gy applied to thyroid gland (M88%, FR82%). This dosimetric compromise is a practicable alternative which is tolerably successful. In patients suffering from disseminated non-immunogenic hyperthyroidism, a dose of 150 Gy applied to the entire organ succeeds in 85% of the cases. The rate of hypothyroidism resulting from these dose recommendations is the lesser evil compared to residual or recurrent hyperthyroidism, since hypothyroid patients can be treated without any problem with thyroid hormones.


Serum prolactin concentrations and epilepsy. A study which compares healthy subjects with a group of patients in presurgical evaluation and circadian variations with those related to seizures. In 20 healthy subjects (10 female and 10 male) and 17 patients undergoing presurgical epilepsy evaluation with intracranial EEG electrodes, circadian variations of serum prolactin (PRL) were measured. A comparison between the peak values found in normals with the postictal rises in patients, led us to consider 700 microU/ml to be the threshold of diagnostic value and the observed rises above this level to be all induced by seizures. In order to assess the clinical value of this threshold, PRL was measured postictally in a further 30 patients with epilepsy and in 11 patients with psychogenic seizures. In none of the latter group did PRL rises exceed 700 microU/ml, while they did so in 39% of the complex partial seizures and in 80% of the tonic-clonic seizures. There was no significant difference with respect to sex (a rise over 700 microU/ml in 42% in male and in 55% in female patients). Based on the findings in 17 patients investigated by means of intracranial electrodes, we were not able to establish different criteria for different focus localisations: in 66% of both temporal as well as frontal lobe seizures the 700 microU/ml level was exceeded. As a trend, in the period preceding an epileptic seizure we found a slightly decreasing PRL level, whereas in healthy persons the PRL concentrations gradually increased in the 40 minutes before the maximum spontaneous peak was reached.


[Reactive enlargement of cervical lymph nodes and cervical lymph node metastases: sonography (M/Q quotient) and computed tomography]. Ranking of sonographic maximum/transverse diameter quotients was compared with the ranking of CT in respect of identification and exclusion of cervical lymph node metastases. Both sonography and CT are distinguished by a high degree of sensitivity in the imaging of cervical lymph node metastases. Sonography, with the assistance of the maximum/transverse diameter quotient, can differentiate between benign enlarged and non-enlarged lymph nodes on the one hand and cervical lymph node metastases on the other, with a safety of 95 per cent. CT can yield definite information regarding the tumour status only after contrast medium administration under the criteria of central hypodensity and peripheral marginal enhancement. CT is much less specific than the sonographic M/T quotient (66% vs 95%). Accuracy of sonography (94%) is clearly superior to that of CT (79%).


[The mean optical density in x-ray pictures: measurement methodology, results and parameters]. A simple testing method for defining the average density or the integral density of x-ray images is described. In a semiquantitative way the physicians in radiological offices can measure the proper integral density. The amount of scattered x-rays influences the integral density significantly. Proper values for testing the integral density of x-ray images are described.


[FCR AC-1: prototype of a new generation of equipment for digital luminescence radiography (DLR)]. The promising perspective to integrate the project-image-radiography into future picture archiving and communication systems (PACS) failed in medium terms because of high costs. A new generation of compact computed radiography systems (CR) facilitate the entry into storage phosphorus technique for routine application while future PACS-connection is still possible. In August 1990 the Institute of Diagnostic Radiology (University of Düsseldorf) received the first FCR AC-1 in Germany for clinical trials. The acquired experiences expounded by case reports emphasize the diagnostic benefit of this technique. The varying use of expandible FCR AC-1 systems is illustrated.