Fidelity of the RNA-dependent DNA synthesis exhibited by the reverse transcriptases of human immunodeficiency virus types 1 and 2 and of murine leukemia virus: mispair extension frequencies. Human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2, respectively) exhibit extensive genetic variations. It was postulated that much of this genetic variability stems from the low fidelity of the reverse transcription step. Both HIV reverse transcriptases (RTs) were shown to be particularly error-prone during the in vitro DNA-dependent DNA synthesis relative to other retroviral RTs. Extension of mismatched 3'-termini of the primer DNA was shown to be a major determinant in the infidelity of HIV RTs. However, reverse transcriptases generally exhibit dual template specificities. Therefore, we determined in the current study the fidelity of RNA-dependent DNA synthesis catalyzed in vitro by the RTs of HIV-1 and HIV-2 in comparison with that of murine leukemia virus (MLV) RT. Consequently, we examined the ability of these enzymes to extend preformed 3'-terminal A.A, A.C, and A.G mispairs by quantitating the amount and length of extended primers in a primer extension assay using ribosomal RNA as a template. The results demonstrate that the three RTs studied exhibited efficient extensions from 3'-terminal mispairs with a specificity of A.C greater than A.A greater than A.G. Nevertheless, the HIV RTs are qualitatively as well as quantitatively more error-prone than MLV RT. The mispair extension efficiency appears to be affected mainly by the increase of apparent Km values, rather than by the change in Vmax values.(ABSTRACT TRUNCATED AT 250 WORDS)


Long-range changes in a protein antigen due to antigen-antibody interaction. Amide exchange kinetics were used to probe the conformation of hen egg-white lysozyme complexed with the anti-lysozyme monoclonal antibody HyHEL-5. Following the technique developed by Paterson et al. [(1990) Science 249, 755-759] we used two-dimensional NMR to measure amide exchange kinetics of the lysozyme amide protons in the lysozyme-antibody complex. A total of 15 amide protons showed altered exchange kinetics in the presence of the complex. Five of these 15 protons reside on residues that are found within the epitope as defined by X-ray crystallography. Five residues are located at the perimeter of the epitope. The remaining five residues are removed from the epitope. The perturbation of amide exchange rates at sites distant from the epitope indicates that the formation of antigen-antibody complexes can produce changes in the antigen at sites that are quite distant from the structural epitope.


A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing. Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Multiple cysteins have been postulated to play a key role in this process. To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S. Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity). An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1. The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis. C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant. However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant. The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction. Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759. The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin. C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction. Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor. C462SR1 upon incubation with R2 and [3'-3H,U-14C]UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2. The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7. This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol. The C462AR1 has properties similar to C462SR1. Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.


Ascorbate both activates and inactivates bleomycin by free radical generation. The chemotherapeutic agent bleomycin (BLM) is activated by reducing agents to break isolated DNA. Paradoxically, these same reducing agents protect cellular DNA from BLM damage. To resolve this paradox, we have examined the reaction of FeIIIBLM with DNA in the presence of ascorbate. As expected, ascorbate augments FeIIIBLM-induced DNA damage. However, when ascorbate is added to FeIIIBLM prior to exposure to DNA, a redox-inactive BLM is produced in a reaction that generates the ascorbyl radical. This reaction occurs in both ascorbate-supplemented buffer and unsupplemented plasma. In buffered solution, this reaction was found to be stoichiometric; for each mole of BLM present, 6.9 mol of ascorbate was oxidized and 4.7 mol of oxygen was consumed. Iron was found to serve only as a catalyst for the reaction. These data suggest that both activation of BLM and the generation of redox-inactive BLM occur via the same reaction and that BLM-induced DNA damage depends upon BLM reaching DNA prior to its interaction with reducing agents.


Molecular dynamics simulation of solvated protein at high pressure. We have completed a molecular dynamics simulation of protein (bovine pancreatic trypsin inhibitor, BPTI) in solution at high pressure (10 kbar). The structural and energetic effects of the application of high pressure to solvated protein are analyzed by comparing the results of the high-pressure simulation with a corresponding simulation at low pressure. The volume of the simulation cell containing one protein molecule plus 2943 water molecules decreases by 24.7% at high pressure. This corresponds to a compressibility for the protein solution of beta = 1.8 x 10(-2) kbar-1. The compressibility of the protein is estimated to be about one-tenth that of bulk water, while the protein hydration layer water is found to have a greater compressibility as compared to the bulk, especially for water associated with hydrophobic groups. The radius of gyration of BPTI decreases by 2% and there is a one third decrease in the protein backbone atomic fluctuations at high pressure. We have analyzed pressure effects on the hydration energy of the protein. The total hydration energy is slightly (4%) more favorable at high pressure even though the surface accessibility of the protein has decreased by a corresponding amount. Large pressure-induced changes in the structure of the hydration shell are observed. Overall, the solvation shell waters appear more ordered at high pressure; the pressure-induced ordering is greatest for nonpolar surface groups. We do not observe evidence of pressure-induced unfolding of the protein over the 100-ps duration of the high-pressure simulation. This is consistent with the results of high-pressure optical experiments on BPTI.(ABSTRACT TRUNCATED AT 250 WORDS)


Inhibition of SH2 domain/phosphoprotein association by a nonhydrolyzable phosphonopeptide. Using the association between the pp60c-src/polyoma virus middle T antigen (mT) complex and phosphatidylinositol 3'-kinase (PI 3-kinase) as a prototype for phosphoprotein-SH2 domain interactions, we tested whether a nonhydrolyzable phosphonopeptide would inhibit association. (Phosphonomethyl)-phenylalanine (Pmp) is a nonnatural analogue of phosphotyrosine in which the > C-O-PO3H2 moiety is replaced by > C-CH2-PO3H2. We synthesized a 13 amino acid phosphonopeptide (mT-Pmp315), a related phosphopeptide (mT-pY315), and an unmodified sequence (mT-Y315), all corresponding to the pp60c-src-phosphorylated site of the mT which is within a YMXM motif common to proteins that bind to and activate PI 3-kinase. Only the phosphonopeptide persistently blocked the in vitro association of the baculovirus-expressed pp60c-src/mT complex with cytosolic PI 3-kinase activity. Sustained inhibition of association by the phosphopeptide required the additional presence of vanadate, a potent protein tyrosine phosphatase (PTPase) inhibitor. The phosphopeptide and L-phosphonopeptide bound tightly (KD approximately 10-20 nM) and specifically to isolated SH2 domains of PI 3-kinase p85, demonstrating that the mechanism of inhibited association is competitive binding to PI 3-kinase SH2 domains. We conclude that the appropriate phosphonopeptide sequence inhibits the interaction between a tyrosine-phosphorylated protein and a cognate SH2 domain-containing protein and is resistant to the actions of PTPases. Proteolytically stable phosphonopeptide derivatives should be useful inhibitors of protein-protein interactions when introduced into cells and may provide a basis for the rational design of a new class of chemotherapeutic agent.


Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli. Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.


Determination of rate constants for nucleotide dissociation from Na,K-ATPase. A method for determining individual rate constants for nucleotide binding to and dissociation from membrane bound pig kidney Na,K-ATPase is presented. The method involves determination of the rate of relaxation when Na,K-ATPase in the presence of eosin is mixed with ADP or ATP in a stopped-flow fluorescence apparatus. It is shown that the nucleotide dependence of this rate of relaxation--taken together with measured equilibrium binding values for eosin and ADP--makes possible a reasonably reliable determination of the rate constant for dissociation of nucleotide, i.e., determination of the rate constant k-1 in the following model (where E denotes Na,K-ATPase): [formula: see text] All experiments are carried out at about 4 degrees C in a buffer containing 200 mM sucrose, 10 mM EDTA, 25 mM Tris and 73 mM NaCl (pH 7.4). Values obtained for the rate constants for dissociation are about 6 s-1 for ADP and 2-3 s-1 for ATP.


Regulation of phosphate transport by second messengers in capillaries of the blood-brain barrier. Regulation of phosphate uptake by the blood-brain barrier was studied in isolated bovine capillaries. Dibutyryl cAMP, in the presence of 3-isobutylmethylxanthine, resulted in a dose-dependent inhibition of phosphate uptake. Phosphate influx, with or without 3-isobutylmethylxanthine, was not different. Inhibition of phosphate uptake was also observed when capillaries were preincubated with isoproterenol, parathyroid hormone, insulin and acidic or basic fibroblast growth factors. Treatment of capillaries with vasoactive intestinal peptide, prostaglandin E1, angiotensin II, epidermal growth factor and phorbol esters did not affect phosphate transport. Endothelin I increased phosphate uptake by 15%. Preincubation with cholera toxin also resulted in a dose-dependent decrease in phosphate uptake. In addition, pertussis toxin inhibited phosphate transport by 29%, but only in the presence of 3-isobutylmethylxanthine. These results demonstrate that generation of second messengers, following receptor stimulation, can induce physiological effects on capillary phosphate influx and suggest that G proteins may modulate this transport.


Interaction of sulfhydryl reagents with A-type channels of Lymnaea neurons. The effect of sulfhydryl reagents on macroscopic inactivation of A-current in internally perfused Lymnaea neurons under voltage-clamp conditions was investigated. It was found that the binding of Hg2+ rather than PHMB with channel proteins resulted in a strong decrease of the peak current and the inactivation rate. Hg2+ markedly influenced the steady-state inactivation but did not change the rate of recovery from inactivation. It was found that both reagents reacted with the same groups of the channel protein and that those are most likely sulfhydryl groups. These groups seemed not to be involved in the gating charge movement. Hg2+ ions can immobilize some part of the gating charge thereby resulting in strong changes of the steady-state inactivation.


Inhibitors of the plasma membrane redox system of Zea mays L. roots. The vitamin K antagonists dicumarol and warfarin. The action of the 4-hydroxycoumarins dicumarol and warfarin, antagonists of probable vitamin K type components of the plasma membrane electron-transport system, on plasma membrane redox activity of intact maize roots was compared. Both effectors inhibited electron transfer to extracellular hexacyanoferrate III. While the effect of the strongly lipophilic dicumarol on the electron-transport system was irreversible by rinsing, the inhibition caused by the hydrophilic warfarin could be reverted completely by exchange of the incubation medium. We take these results as possible evidence for the integration of dicumarol into the plasma membrane. The action of warfarin may be confined to enzymic sites freely accessible from the aqueous apoplasmic solution.


The mode of action of Vibrio cholerae cytolysin. The influences on both erythrocytes and planar lipid bilayers. The interaction with erythrocytes of cholera cytolysin (CC) obtained from a non-01 Vibrio cholerae strain results in the osmotic rupture of target cells upon formation by CC of the waterfilled pores in their membranes. The aggregation of several toxin monomers is required for the formation of one CC channel with a radius of 0.9-1.0 nm. The investigations using planar bilayer lipid membranes suggest that the CC-induced pore is an interprotein anion selective channel carrying a fixed positive charge. The role of the charge was supported by the influence of pH on the selectivity, single conductance and voltage gating of the CC channels. The ability of the CC to modify both model and natural membranes has a maximum at pH 6.0-7.0. It was found that CC channels insert into the membrane asymmetrically. The effect of proteolytic treatment of the channel by papain also indicates that the two entrances of the channel protrude from the plane of the membrane into the solution for different distances. It is proposed that the biological effects of the non-01 V. cholera cytolysin are based on its channel-forming activity.


A channel model with fluctuating barrier structures. Following the theory 'Fluctuations of barrier structure in ionic channels' (Läuger, P., Stephan, W. and Frehland, E. (1980) Biochim. Biophys. Acta 602, 167-180), we constructed a model of a channels with several conformational states. The origin of these conformational states and the source for the transitions from one to the other are given explicitly for the presented model. In this work the effect of multiple conformational states on the ion transport process is analyzed. We considered a channel protein with two main barriers and one binding site. The site is surrounded by dipolar groups. The dipole moment of these groups can be reoriented by thermal activity and also by electrical interaction with the transported ions. Differently polarized states generate different activation energy barriers for the ions. The set of conformational states of the channel is constituted by all the possible polarized states of the binding site. Using the rate-theory analysis of ion transport (Glässtone, S., Laider, K.J. and Eyring, H. (1941) The theory of rate processes, McGraw-Hill, New York), the possible coupling between ion flux and the channel conformational transitions has been incorporated into the model by considering the dependence of the rate constants on the heights of the energy barriers. The resulting multistate kinetic equations have been solved numerically. It was shown that the simple saturation characteristic of the flux-concentration curve was obtained. For certain values of the model parameters, the channel shows a strongly different conductance for anions compared to cations. In fact, the model contains an interesting mechanism that exhibits selectivity with respect to the charge of the ions.


Mapping of the epitope on lipoprotein lipase recognized by a monoclonal antibody (5D2) which inhibits lipase activity. A monoclonal antibody, 5D2, which inhibits human lipoprotein lipase (hLPL) activity has been widely used for assessment of LPL immunoreactive mass in the clinical evaluation of patients [1] and for analysis of structure-function relationships of LPL [2,3]. We have mapped the epitope on LPL, recognized by the 5D2 antibody, within residues 396-405. Ala400 is the critical amino acid residue conferring epitope specificity. This knowledge confirms that the C-terminal domain of LPL plays a critical role in LPL activity and also provides important information for studies exploring the structure-function relationship of LPL using this antibody.


Downregulation of hepatic albumin mRNA in response to induced hypercholesterolemia in rabbits. Albumin gene expression was studied in rabbits fed on a cholesterol-rich diet for up to 16 weeks. Livers from experimental animals showed extensive lipid deposition. Hepatic albumin mRNA abundance decreased along the treatment to very low levels after 16 weeks. An 8-fold decrease in the rate of transcription of this gene was also detected. This downregulation of albumin gene expression cannot be attributed to a general impairment of RNA synthesis, as expression of other liver-specific and housekeeping genes did not vary significantly. There was a decrease in ascitic fluid albumin levels after 10 weeks, although serum albumin levels remained unchanged throughout the treatment. Our results are discussed in view of the relationship of albumin levels with hypercholesterolemia.


Sequence and expression of human protein kinase C-epsilon. Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies.


Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans. Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.


Failure of tachykinins including substance P and its fragments to influence proteoglycan and protein synthesis in bovine chondrocytes in vitro. Adult bovine articular chondrocytes were exposed to substance P, neurokinins A and B or substance P fragments, SP1-4, SP1-6 and SP7-11 in vitro. Proteoglycan synthesis was assessed by measuring proteoglycans which were released into the culture medium or incorporated into the cell layer. The intact tachykinins or substance P fragments had no direct effect on proteoglycan synthesis. Nor was total protein production affected. Gel chromatography, under dissociative conditions, revealed that sulphated proteoglycans detected in the medium or cell layer following treatment of chondrocytes with substance P, contained proteoglycans of similar molecular weight to those produced by cells exposed only to diluent controls. Therefore, we conclude that the acceleration of arthritis by substance P does not appear to be mediated through an effect on chondrocyte synthetic function.


Evidence for the existence of RNA of Ca(2+)-channel alpha 2/delta subunit in Xenopus oocytes. Ba(2+)-currents (IBa) through voltage-dependent Ca(2+)-channels were studied in Xenopus oocytes injected with RNA from several excitable tissues, using the two-electrode voltage-clamp technique. Previous studies have shown that the expression of cardiac Ca(2+)-channels can be suppressed by an hybrid-arrest procedure that includes co-injection of the tissue-derived RNA with an 'antisense' oligonucleotide complementary to a part of RNA coding for the Ca(2+)-channel alpha 1 subunit. In this study, this method was used to investigate the role of the alpha 2/delta subunit. Co-injection of RNA extracted from either rabbit heart, rat brain or rat skeletal muscle (SkM) with 'antisense' oligonucleotides complementary to the alpha 2/delta subunit RNA did not substantially affect the expression of IBa in the oocytes. Using the Northern blot hybridization method, it was shown that native oocytes contain large amounts of alpha 2/delta subunit RNA of Ca(2+)-channel. It is proposed that te oligonucleotide treatment fails to eliminate the alpha 2/delta RNA because of the vast excess of endogenous alpha 2/delta RNA. These results impose a limit on the use of the hybrid-arrest method.


Pulsed static magnetic field effects on in-vitro pineal indoleamine metabolism. In-vitro rat pineal glands stimulated with the beta-adrenergic receptor agonist isoproterenol to induce melatonin synthesis and exposed for 1 h to a pulsed 0.4-G static magnetic field demonstrated significant inhibition of serotonin-N-acetyltransferase activity and melatonin content. 2-h exposure to pulsed magnetic field also resulted in a significant reduction in isoproterenol-induced serotonin-N-acetyltransferase activity. These results support the idea that the cultured pineal gland can be affected directly by artificially generated weak magnetic fields.


Molecular weight determination and compositional analysis of dextran-protein conjugates using low-angle laser light scattering technique combined with high-performance gel chromatography. The low-angle laser light scattering technique combined with high-performance gel chromatography was applied for characterization of the dextran-ovalbumin and dextran-lysozyme conjugates obtained from the mild heating in dry state, which is attracting interest as a way leading to stabilization of proteins and to production of proteins with excellent emulsifying or antimicrobial ability (Nakamura, S., Kato, A. and Kobayashi, K. (1991) J. Agric. Food Chem. 39, 647-650). According to the above technique, providing the information about the molecular weight distribution and the composition of the conjugates, one or two dextran molecules were found to be linked to one molecule of the proteins. In addition, each of the conjugates was shown to exist as an oligomeric assembly of which formation is promoted by an increase in the salt concentration of buffer. The observations suggest that the increase in the hydrophobicity of the protein moiety as a result of partial denaturation and the introduction of the hydrophilic dextran chain affords the conjugate an amphiphilic property.


Membrane proteins from lymphoblastoid cells showing cross-affinity for alpha-fetoprotein and albumin. Isolation and characterization. AFP or SA immobilized on nitrocellulose membranes (AFP-NC or SA-NC) were used as affinity matrices to purify cell membrane proteins with affinity for AFP (AFP-BP) and for SA (SA-BP) from membrane-enriched extracts of Raji cells (a B-lymphoma cell line), as well as for normal resting and activated peripheral blood lymphocytes (PBMC). SDS-PAGE and ligand blotting assays showed that AFP-BP and SA-BP isolated from Raji cells are probably identical molecules. They consisted of two sets of polypeptides of 31 kDa and 18 kDa. The glycoprotein nature of isolated 31 kDa and 18 kDa peptides was suggested by positive staining with Schiff's reagent, and amino-acid analysis revealed similar amino-acid composition for the two glycoproteins. In human PHA-activated PBMC, only the 18 kDa polypeptide was identified and isolated as AFP-BP or SA-BP. As in Raji cells, this 18 kDa polypeptide, isolated by affinity for AFP or for SA, appeared to be the same molecule. Contrary to Raji cells and activated PBMC, no proteins with an affinity for AFP or for SA were identified or isolated in resting PBMC. These observations strongly suggest that the isolated 31 kDa and 18 kDa glycoproteins are probably AFP receptors previously demonstrated in several neoplastic and normal cells undergoing growth and/or differentiation; indeed, they were identical to albumin-binding proteins described by others.


Immunochemical study of equine chorionic gonadotropin (eCG/PMSG): antigenic determinants on alpha- and beta-subunits. In the present study we have established an immunochemical mapping of equine Chorionic Gonadotropin (eCG/PMSG) using three monoclonal antibodies (mAbs), namely the antibodies ECG01, E10 and D7, raised against the native hormone. These antibodies do not bind to reduced, alkylated hormone, suggesting that they recognize discontinuous rather than continuous epitopes. We have also assessed the reactivity of mAbs towards human CG, and ovine, porcine, equine and bovine LH and FSH. The antigenic determinant recognized by ECG01 is localized on the alpha-subunit of equine gonadotropins and of human CG and LH. The epitopes recognized by E10 and D7 mAbs appear to be very similar and are present on the beta-subunit of eCG and of LHs from all species tested, except hLH, as well as on porcine and equine FSHs. Attempts to specify the amino-acid residues involved in these epitopes suggest that ECG01 mAb might preferentially bind to residues around position 70 whereas the region around disulfide bridges Cys-88-Cys-90 might be involved in the epitopes recognized by D7 and E10 mAbs. Topographical relationships of epitopes show that ECG01 mAb never binds to eCG simultaneously with either D7 or E10 mAbs. Furthermore, simultaneous binding of D7 and E10 mAbs on eCG could not be achieved. Thus, these three epitopes appear to be closely located on the surface of eCG. Finally, ECG01 mAb inhibits eCG binding to LH and FSH receptors, suggesting that its antigenic site is closely related to hormone-receptor interaction site(s).


Characterization of the interaction of yeast enolase with polynucleotides. Yeast enolase is inhibited under certain conditions by DNA. The enzyme binds to single-stranded DNA-cellulose. Inhibition was used for routine characterization of the interaction. The presence of the substrate 2-phospho-D-glycerate reduces inhibition and binding. Both yeast enolase isozymes behave similarly. Impure yeast enolase was purified by adsorption onto a single-stranded DNA-cellulose column followed by elution with substrate. Interaction with RNA, double-stranded DNA, or degraded DNA results in less inhibition, suggesting that yeast enolase preferentially binds single-stranded DNA. However, yeast enolase is not a DNA-unwinding protein. The enzyme is inhibited by the short synthetic oligodeoxynucleotides G6, G8 and G10 but not T8 or T6, suggesting some base specificity in the interaction. The interaction is stronger at more acid pH values, with an apparent pK of 5.6. The interaction is prevented by 0.3 M KCl, suggesting that electrostatic factors are important. Histidine or lysine reverse the inhibition at lower concentrations, while phosphate is still more effective. Binding of single-stranded DNA to enolase reduces the reaction of protein histidyl residues with diethylpyrocarbonate. The inhibition of yeast enolase by single-stranded DNA is not total, and suggests the active site is not directly involved in the interaction. Binding of substrate may induce a conformational change in the enzyme that interferes with DNA binding and vice versa.


Rapid purification and N-terminal sequencing of a potato tuber cyclic nucleotide binding phosphatase. A high affinity cyclic nucleotide binding phosphatase was purified to homogeneity from potato tubers by a rapid procedure involving batchwise elution from carboxymethylcellulose and gel filtration. The phosphatase has a molecular weight of 28,000 as estimated from both SDS-PAGE and gel filtration. The phosphatase binds to Con A-agarose and is eluted by 0.5 M alpha-methylglucoside. The phosphatase catalyses the hydrolysis of nucleoside monophosphates, p-nitrophenylphosphate and O-phospho-L-tyrosine, but not of O-phospho-L-serine or O-phospho-L-threonine. N-terminal sequencing of the phosphatase has revealed significant homology with two similar-size soybean leaf and stem storage glycoproteins.


Heat and autoclave resistance of cell-spreading activity of vitronectin. We have investigated the heat- and autoclave-resistant properties of the cell-spreading activity of vitronectin, a cell-spreading glycoprotein in animal blood plasma. Vitronectin heated at 100 degrees C for 10 min or autoclaved at 121 degrees C at 1.2 kg/cm2 for 20 min retained the same cell-spreading activity as native vitronectin. In contrast, fibronectin and type-I collagen treated in the same way lost their activity almost completely. GRGDSP remarkably inhibited the cell-spreading activity of native, heated and autoclaved vitronectins. GRGESP did not inhibit the activity of native vitronectin, but, unexpectedly, partially inhibited the activity of both heated and autoclaved vitronectins. In SDS-polyacrylamide gel analysis under reducing conditions, vitronectin heated at 100 degrees C migrated mainly as a monomer, but autoclaved vitronectin migrated at both the top and front of the gel instead of at the position of the monomer. The change in molecular size during the heat- and autoclave treatments was partially prevented by adding 10 mM dithiothreitol or 2% 2-mercaptoethanol to the protein solution.


The effect of vanadate upon the expression of phenylalanine hydroxylase in streptozotocin-diabetic rat liver. Induction of diabetes in rats is associated with a significant elevation in the phenylalanine hydroxylating capacity of the liver. This phenomenon reflects an increase in the abundance of both phenylalanine hydroxylase protein and phenylalanine hydroxylase-specific mRNA. These changes can be abolished by insulin-dependent control of diabetes. We show here that the control of diabetes by oral administration of sodium orthovanadate will also nullify the diabetes-related alterations in phenylalanine hydroxylase expression. In addition, diabetes-induced changes in the extent of phosphorylation of phenylalanine hydroxylase are reversed by either insulin or vanadate treatment in vivo. These treatments also abolished the diabetes-related, approx. 30-fold, decrease in glucagon sensitivity of phenylalanine hydroxylation in isolated liver cells.


Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells. An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated MCAD cDNA, containing the entire coding region, was placed between the SV40 early promoter and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.


In vivo antitumor activity of a panel of four monoclonal antibody-vinca alkaloid immunoconjugates which bind to three distinct epitopes of carcinoembryonic antigen. A panel of four murine monoclonal antibodies apparently directed against three distinct epitopes of carcinoembryonic antigen (CEA) was conjugated via oxidized carbohydrate groups to 4-desacetylvinblastine-3-carboxyhydrazide. The resulting antibody-vinca conjugates were evaluated for antitumor activity against 2-9-day-established LS174T human colorectal carcinoma xenografts. The antibodies (immunoglobulin G, IgG) employed in this study were 11.285.14 (IgG1), 14.95.55 (IgG2a), CEM231 (IgG1), ZCE025 (IgG1). Additive immunofluorescence studies indicated that CEM231 and ZCE025 recognized the same or a closely related epitope(s) on CEA which was distinct from the two epitopes bound by 11.285.14 and 14.95.55. The in vivo antitumor efficacy studies demonstrated that chemoimmunoconjugates prepared from 14.95.55 and ZCE025 were more active than the conjugates constructed from the 11.285.14 and CEM231 antibodies. The 14.95.55 and ZCE025 immunoconjugates were also more efficacious than free drug or drug conjugated to irrelevant murine IgG. The presence of increased carbohydrate content on the light chain of ZCE025 may have been responsible for the ability to construct ZCE025-vinca conjugates with about twice the drug content (approximately 10 mol of vinca/mol of IgG) than was achieved with the other antibodies. The highly conjugated form of ZCE025 demonstrated similar efficacy but was much less toxic than a ZCE025 conjugate containing 5 mol of vinca/mol of IgG. These data indicated that significant differences existed in the ability of monoclonal antibodies to target a cytotoxic agent for effective antitumor activity even when the immunoconjugates recognized the same antigen or even the same or closely related antigen epitope(s). Furthermore, these differences could not have been identified without extensive in vivo evaluation for antitumor efficacy.


Activity of ribosomal and tRNA promoters of Bacillus subtilis during sporulation. The rrnB operon in Bacillus subtilis includes 21 tRNA genes downstream of the ribosomal genes. In addition to the dual promoters upstream of these ribosomal genes, a second promoter is found within the tRNA gene region. In this study, these two promoter regions were inserted before the lacZ gene and each was integrated as a single-copy into the B subtilis chromosome to examine their activity during sporulation. Both promoters exhibited similar strength and temporal downregulation from vegetative growth through t3 of sporulation. At t3, transcription from both promoters was approximately 20% of that in logarithmic growth. No obvious function of the second promoter is evident except to boost transcription of downstream genes. This function may be important because the ribosomal promoters of rrnB are weak relative to other ribosomal promoters. For instance, the rrnO promoters were much stronger than the rrnB ribosomal promoters and considerably more active at t3. Thus, all ribosomal promoters are not of the same strength nor are they transcriptionally downregulated to the same extent during sporulation. However, both the rRNA and tRNA promoters in the rrnB operon are similar in strength and downregulation.


Surgical treatment of acute malignant large bowel obstruction. To evaluate the morbidity and mortality in all patients operated on urgently for acute large bowel obstruction caused by carcinoma of the colon or rectum during a 10 year period. Retrospective study. Aalborg Hospital, Denmark. 156 consecutive patients operated on for obstructing primary colorectal cancers. Operations done, morbidity and mortality. 95 patients (61%) had advanced disease (Dukes' stage C or "D") and their median age was 73 years (range 38-93). 97 had the obstructing lesion resected with a 30 day mortality of 5%. 43 patients underwent primary resection and 4 died (9%), and 54 underwent staged resection with one death (2%). Complications were common, particularly after staged resections, median hospital stay being 19 days after primary, compared with 30 days after staged resection. 59 patients (38%) had palliative operations with 29 deaths (49%); in 39 the tumor was completely unresectable. Patients with obstructing primary colorectal cancers are a high risk group who are characterised by advanced disease and old age. Only prospective trials comparing different operations can assess whether it is possible to achieve a reduction in mortality.


Identification of the cross-reacting antigen among Actinobacillus pleuropneumoniae strains of serotype 1, 9 and 11 by use of monoclonal antibodies. Two monoclonal antibodies (MAbs), lMAb-1 and lMAb-5, against Actinobacillus pleuropneumoniae serotype 1 were obtained. In enzyme-linked immunosorbent assay-inhibition tests with whole cell antigens obtained from serotype 1 to 12 strains of A. pleuropneumoniae, lMAb-1 reacted to only a serotype 1, strain 4074. The epitope recognized by lMAb-1 was a carbohydrate sensitive to periodate oxidation and resided on capsular polysaccharide (CP) of A. pleuropneumoniae serotype 1. On the other hand, lMAb-5 reacted with serotype 1, 9 and 11 strains at the same degree and its epitope was found to be located on O-polysaccharide of serotype 1, 9 or 11 lipopolysaccharide (LPS). These results showed that CP was one of the serotype-specific antigens of A. pleuropneumoniae, and that O-polysaccharide of LPS obtained from serotype 1, 9 or 11 strain was the cross-reacting antigen among these strains.


The DNA-binding protein HBsu is essential for normal growth and development in Bacillus subtilis. The hbs operon of Bacillus subtilis comprises a single gene which is localized between the spoIVA and mtrA open reading frames, and is situated at 204 degrees of the standard map of B subtilis. Expression of hbs is initiated from two distinct promoters called P1 and P2. The transcription initiation sites have been mapped by primer extension analysis. Sequences upstream from P1 show a -35 and -10 region which may be recognized by the vegetative form of RNA polymerase E sigma A, whereas sequences upstream from P2 may be recognized by either E sigma C or E sigma H minor forms of RNA polymerase. In vegetative cells, hbs is highly and equally transcribed from both promoters, P1 and P2. In contrast, in sporulating cells, hbs is expressed predominantly from P2. In order to study the physiological role of HBsu, we must overcome our failure to interrupt the hbs gene within the B subtilis chromosome by using a previously constructed strain, BM19, bearing hbs under the control of the IPTG-inducible spac-1 promoter. In this strain, growth was found to depend highly on hbs expression. In the absence of IPTG, growth was strongly affected culminating in a filamentous cell morphology. Although sporulation in IPTG-uninduced BM19 cells was poor, due to the limited cell growth, the outgrowth of those spores was delayed by 1 h. In contrast, in the presence of IPTG, a condition that induces hbs expression, normal outgrowth of spores was observed. The proposed essentiality of the hbs gene product for growth and development in B subtilis may be attributed to its interaction with replication and transcription as a consequence of its facility to wrap DNA and to condense the chromosome into nucleosomelike structures. A comparative sequence analysis of HBsu with 18 homologous histonelike proteins of diverse origin demonstrated their high conservation throughout evolution.


[Hemobilia following biliary drainage: percutaneous treatment with arterial embolization and biliary fibrinolysis]. The Authors report a case of haemobilia following the performance of biliary drainage. Angiography of the celiac tripod and superior mesenteric artery was carried out in order to evidence the lesion and to perform, during the same session, embolization of the leaking vessel by means of fibrin sponge particles (Spongostan). Subsequent cholangiographic controls demonstrated the presence of bile ducts clots then treated by loco-regional infusion of urokinase. On the basis of the Authors' personal experience, the combination of loco-regional biliary fibrinolytic treatment and embolisation leads to the resolution of the cause of hematobilia and of retention jaundice.


Use of a modified Transwell culture to quantitate the contribution of non-IL-7 substances to growth of an IL-7-dependent pre-B cell clone. Clone 3 is a mouse pre-B cell line that cannot grow in standard tissue culture media but is immortalized by coculture with a bone marrow-derived feeder layer. In addition, clone 3 cells can be passaged indefinitely in recombinant interleukin-7 (IL-7) in the absence of a feeder layer if maintained at high cell density. Using monoclonal antibody to IL-7 and Transwells ligated to dialysis membranes, we have examined the relative contributions of IL-7, both endogenous and exogenous, and of "non-IL-7" feeder layer factors to growth promotion of clone 3 cells. There is synergy between feeder layers and exogenous IL-7 that is most marked when the latter is present in suboptimal concentrations. The synergizing activity is not neutralized by antibody to IL-7 and appears to be freely dialyzable. This "non-IL-7" effect is common to two different feeder layers, the one derived from bone marrow (3E) being an IL-7 producer, and the other, 3T3 fibroblasts, making no detectable IL-7. These experiments reveal a substantial contribution of the dialyzable moiety to the total feeder layer effect, and are the first to demonstrate cytokine-dependent low-molecular-weight synergy in a coculture. This demonstration is possible because the synergizing cofactor(s) can cross a semipermeable membrane whereas the cytokine and its neutralizing antibody cannot.


Acute phase reactants and interleukin 6 after total hip replacement. Effects of high dose corticosteroids. A randomised study was performed to evaluate the association between some commonly measured acute phase proteins and interleukin-6 after a standard musculoskeletal operation, and to investigate the effect of high doses of corticosteroids on these proteins. Eight men and four women with osteoarthrosis but who were otherwise healthy and who were each to have an uncemented hip prosthesis inserted by the porous coated anatomical technique, were included. Patients were randomised to receive methylprednisolone 30 mg/kg body weight 1 1/2 hours before, and four and 12 hours after, operation (n = 6) and compared to a control group (n = 6). Plasma concentrations of C reactive protein, haptoglobin, orosomucoid and alpha 1-antitrypsin; serum concentration of albumin; packed cell volume; white cell count; and plasma concentration of interleukin-6 were measured. The increases in concentrations of acute phase proteins in plasma were significantly less in the group given steroids, but this did not have any obvious clinical consequences. Increase in the concentration of interleukin-6 preceded the increases in acute phase proteins in both groups, reflecting the role of interleukin-6 in the regulation of expression of acute phase protein genes in hepatic cells. The increase of interleukin-6 in the group receiving steroids was less pronounced than that in the control group, indicating that corticosteroids inhibit the generation of interleukin-6 in vivo.


Serotonergic measures in suicide brain: the concentration of 5-HIAA, HVA, and tryptophan in frontal cortex of suicide victims. Concentrations of 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and tryptophan (TRP) were determined in the frontal cortex of individuals who died by suicide, homicide, accident, or the result of physical diseases. Females had significantly higher tryptophan concentrations than males. There was a significant correlation HVA and the interval between death to refrigeration of the body. Mean HVA levels were higher from noon to 5 PM. Suicide and homicide victims had significantly higher cortical HVA concentrations than those who died of physical disease but not accident victims. This was not accounted for by gender, age, postmortem interval from death to refrigeration of the body or to autopsy, specimen storage time, or drug effects. The ratio of HVA/5-HIAA was also significantly higher in suicides compared with those who died of physical disease. No differences in cortical 5-HIAA or tryptophan concentrations between the four groups were found. There were no differences in the levels of the three substances in violent and nonviolent suicides. There were no significant correlations between 5-HIAA, HVA and TRP concentrations in all subjects or any of the four subgroups. The implications of these findings for the role of serotonin and dopamine in suicide and violence are discussed.


Relationships among concentrations of steroids, inhibin, insulin-like growth factor-1 (IGF-1), and IGF-binding proteins during follicular development in weaned sows. The experimental objective was to determine how insulin-like growth factor binding proteins (IGFBP), as examined by Western ligand blot procedures, related to porcine follicular steroidogenesis. Weaned sows were ovariectomized at various times after litter removal in three experiments. In experiments 1 and 2, sows were ovariectomized at 48-120 h after weaning. In experiment 1, pools of all small (1-3 mm), medium (greater than 3-6 mm), or large (greater than 6-9 mm) follicles were made for each sow; in experiment 2, fluid was collected individually from the 10 largest follicles per ovary. A third experiment was conducted to examine changes after an ovulatory dose of hCG, but prior to ovulation. In this experiment, sows were treated with eCG at weaning, given hCG 72 h later, and ovariectomized 0-36 h after the ovulatory dose of hCG. Follicular fluid was collected from the 10 largest follicles per sow. In experiments 1 and 2, IGFBP-3 in follicular fluid remained constant over follicle diameters and stage sof development, and IGFBP-2 decreased with advancing follicular development as concentrations of estradiol, androstenedione, and progesterone increased. In experiment 1, after the presumed LH surge when the concentration of all steroids was low, there was a sharp increase in band intensity for IGFBP-2. Similarly, estradiol and androstenedione were low in preovulatory sows in experiment 2, though progesterone increased and IGFBP-2 decreased with follicle diameter. In experiment 3, progesterone remained elevated from 0 to 36 h after hCG, even though IGFBP-2 did not increase until after 24 h post-hCG.(ABSTRACT TRUNCATED AT 250 WORDS)


Uterine uptake of alpha 2-macroglobulin and alpha 1-proteinase inhibitor from the blood during early implantation in the mouse. The objective of this study was to investigate the uterine uptake of plasma alpha 1-proteinase inhibitor (53,000 Da) and alpha 2-macroglobulin (725,000 Da) from the blood during implantation in the mouse using isotopic methods. The uterine uptake of albumin (67,000 Da) and immunoglobulin G (150,000 Da) were also measured for comparison. Rates of uptake were assessed from permeability-surface area products estimated from the rate at which the tissue volume of distribution approaches its steady-state value. The permeability-surface area product estimates at implantation sites were 13.3 and 54.8 ml/100 g.h for alpha 2-macroglobulin and alpha 1-proteinase inhibitor, respectively. Given the circulating levels of these proteins in mice, these results demonstrate that considerable amounts of plasma proteinase inhibitors are extravasated into the interstitium in the vicinity of the implanting blastocyst. The permeability-surface area products of all the proteins studied, except immunoglobulin G, were greater at implantation compared to non-implantation sites, confirming greater vascular permeability to plasma proteins at implantation sites compared to non-implantation sites. Estimates of the permeability-surface area products of the studied proteins showed that the uterine vasculature was generally more permeable to proteins with a small than with a large molecular size. Nevertheless, the ratio of the permeability-surface area product between implantation and non-implantation sites for the proteins ranged from 1.1 to 2.9 with no obvious relationship to molecular size.(ABSTRACT TRUNCATED AT 250 WORDS)


Expression of c-kit protein during placental development. The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor and is shown to be allelic with the white-spotting locus (W) of the mouse. In order to elucidate the role of c-kit protein during placental development, we have examined the expression of c-kit protein in the uterus and placenta of mice at pre- and post-implantation stages by the avidin-biotin-peroxidase (ABC) method using rat anti-mouse c-kit monoclonal antibody. At Days 3 and 5 of pregnancy and pseudo-pregnancy, c-kit protein was detected in the glandular epithelium, but little expression was observed in the luminal epithelium. At Day 7 of pregnancy, expression was detected in the stromal cells around the uterine crypts of the mesometrial portion, but not in the vigorously proliferating decidual cells around the developing embryo. At Days 9 and 10 of pregnancy, the decidua basalis facing invading trophoblasts gradually expressed c-kit protein. In the mature placenta, c-kit protein was detected in the labyrinthine and decidual layers, but in neither the giant trophoblastic nor the spongiotrophoblastic layer. By Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR), c-kit mRNA was detected at the stages of periimplantation and placental development. These results suggested that the c-kit protein might be involved in the proliferation and differentiation of placenta.


Mammary gland differentiation in hypophysectomized, pregnant mice treated with corticosterone and thyroxine. Swiss Webster mice were hypophysectomized or sham-operated on Day 11 of pregnancy. The animals were fitted s.c. with osmotic minipumps containing either corticosterone (B) dissolved in Molecusol (Pharmatec, Alachua, FL) or the vehicle alone immediately after they were hypophysectomized. Animals in some of the experimental groups also received thyroxine (T4) in their drinking water. The mice were killed on Day 18 of gestation, and mammary tissue was homogenized and extracted for assessment of DNA, RNA, alpha-lactalbumin, and alpha-casein. Serum was assayed for placental lactogen-I (PL-I), and placental lactogen-II (PL-II), B, and T4. The concentration of PL-II in serum was elevated in the hypophysectomized mice, whereas the PL-I concentration did not differ among experimental groups. Hypophysectomy decreased both T4 and B concentrations in serum, and administration of these hormones restored their serum concentrations to normal or, in some cases, somewhat higher than normal levels. Hypophysectomy reduced the total RNA content and RNA/DNA ratio of the mammary gland, but treatment with B alone or with B and T4 restored RNA levels to those of sham-operated animals. T4 alone was ineffective in restoring RNA levels. Sham-operated animals that received hormonal treatment (B and T4) had the highest levels of RNA in the mammary tissue. Hypophysectomized animals had reduced content and concentration of alpha-lactalbumin in the mammary gland as compared to all other experimental groups.(ABSTRACT TRUNCATED AT 250 WORDS)


[Cholinergic receptors and anesthesia].

[Cholinergic receptors and anesthesia]. Postsynaptic chemically controlled ion channels are a possible location for the action of anaesthetics. The nicotinic acetylcholine receptor (AcChoR) is a member of a superfamily of chemically controlled ion channels, which have a large number of structural similarities in common. This receptor provides an excellent model for studying the effects of anaesthetics on excitable membrane proteins since it is well characterised and available in large quantities. The AcChoR consists of five subunits that surround a central ion channel. Anaesthetics can inhibit the function of the AcChoR by several different mechanisms: 1. By directly blocking the ion channel, 2. by an allosteric inhibition of the ion channel, 3. by changing the properties of the cell membrane surrounding the receptor. In high concentrations, anaesthetics stabilize the receptor in a desensitized state. Investigation of the mode of action of anaesthetics on the AcChoR provides important information as to the mechanisms of anaesthesia.


[Molecular and cellular actions of inhalation anesthetics]. The cellular and molecular basis of inhalational anaesthesia is still unknown. Recent biochemical, neurophysiological and pharmacological interest has focussed on membrane proteins in the neuron, the smallest functional entity in the brain. In this review, the effects on ion transport and ion channels potentially relevant to anaesthesia are analysed with reference to the most extensively investigated volatile anaesthetics ether, chloroform, halothane, methoxyflurane, isoflurane, and enflurane. Synaptic transmission proved a major target of all the volatile substances tested so far. Their inhibitory effect seems to be due to an alteration in the Ca(2+)-dependent stimulus-secretion coupling within the presynaptic nerve endings. In contrast, the phosphatidylinositol pathway proved relatively insensitive. In the postsynaptic membrane, receptor-operated ion channels, K+ and Cl- channels are considered possible targets for anaesthetic vapours. Likewise, guanine nucleotide-binding proteins, so-called G proteins, are promising candidates for future research. So far, only limited data exist on the effects of anaesthetics on non-neuronal cellular components of the CNS, e.g. glial cells. In view of the rapidly increasing number of potential target mechanisms, the future challenge of biologically oriented anaesthesia research will be to puzzle out those mechanisms most relevant to the still mysterious state of anaesthesia.


Cell growth on immobilized cell-growth factor. II. Adhesion and growth of fibroblast cells on poly(methyl methacrylate) membrane immobilized with proteins of various kinds. The surface of poly(methyl methacrylate) membrane was partially hydrolysed and the carboxyl groups produced were coupled with various protein molecules with water-soluble carbodiimide. The immobilized proteins were a cell-growth factor insulin, cell adhesion factors fibrinogen and fibronectin, and serum proteins albumin and gamma-globulin. The insulin-immobilized poly(methyl methacrylate) membrane strongly accelerated the growth and slightly accelerated the adhesion of fibroblast cells. The immobilized fibronectin and fibrinogen enhanced the cell adhesion, and the former also accelerated the cell growth. The immobilized albumin and gamma-globulin influenced the adhesion and growth of cells very little. It was found that various proteins specifically influence the adhesion and growth of cells in an immobilized state.


[Exocrine pancreas involvement in chronic enteropathies in infants and children]. On a number of 135 cases of chronic enteropathies in patients aged 6 months--15 years, the trypsin, amilasis and lipasis levels in duodenal aspirate were investigated. In a high number, the decrease of pancreatic enzymes activity was noted and the majority of cases were accompanied by a marked dystrophy (grades II or III). Thus, it was not possible to indicate whether the low levels were primary or secondary determined.


Complement activation and adsorption of protein fragments by functionalized polymer surfaces in human serum. The interactions between blood and polymer surfaces used in extracorporeal circulations result in variable activations of the immune system of complement. Measuring concentrations of C3a or C5a in supernatant blood or serum after contact with the surface has been the most usual way of assessing this activation. Most polymer surfaces bearing various chemical groups were found to adsorb C3a and sometimes C5a. After taking into account adsorption, a good correlation was found between total C3a generated and CH50 units consumed by most of the polymer samples tested. Measuring only C3a remaining in the fluid phase should not be considered sufficient to conclude that a material surface is not an activator of complement.


Inhibition of endotoxin-mediated activation of endothelial cells by a perfluorocarbon emulsion. Endothelial cell (EC) activation plays a key role in the inflammatory response by promoting the margination of leukocytes in inflamed loci. Augmented leukocyte margination to activated EC is mediated by the increased display of leukocyte adhesion molecules on EC surface membranes. The biocompatibility of synthetic oxygen-transport fluids is intimately linked to EC function, since one of the first tissues encountered by such fluids is the vascular endothelium. We investigated the effect of one such agent, a phospholipid-based perfluorocarbon emulsion containing 90% w/v perflurooctyl bromide (perflubron, PFOB) on EC activation. Human umbilical vein EC (HUVEC) were activated by 5 U/ml interleukin-1 (IL-1), 20 U/ml tumor necrosis factor (TNF), or 50 ng/ml E coli endotoxin (LPS) in the presence or absence of up to 20%, w/v perflubron. HUVEC activation was monitored by the extent of up-regulation of expression of intercellular adhesion molecule-1 (ICAM) and endothelial-leukocyte adhesion molecule-1 (ELAM). Exposure of HUVEC to perflubron did not alter the up-regulation of ICAM or ELAM in response to IL-1 or TNF (n = 20). However, at 10% perflubron ICAM up-regulation in response to LPS was inhibited by 95 +/- 6% (n = 9; p less than .05). ELAM expression was similarly affected. The concentration of perflubron required to diminish LPS-induced up-regulation by 50% was 6.0 +/- 0.6% (n = 3). The inhibitory effect of 10% perflubron was overcome by greater than 1 ug/ml LPS (n = 3) and the inhibitory effect was attenuated by adding perflubron to the cultures after LPS.(ABSTRACT TRUNCATED AT 250 WORDS)


Hemoglobin linked to polyanionic polymers as potential red blood cell substitutes. A variety of chemical modifications of hemoglobin (Hb) have been proposed in order to transform it into cell-free oxygen carriers. These modifications are intended to increase the plasmatic half-life of the protein and to lower its affinity for oxygen. We have designed and prepared derivatives of dextran and polyoxyethylene functionalized so that, after their covalent fixation onto Hb, they can act as permanent effectors and thus lower the oxygen affinity of the protein while decreasing its renal excretion. The main characteristic of these functionalized polymers is that they possess a relatively high density of anionic groups. Benzene hexacarboxylate (BHC) and tetracarboxylate (BTC) linked to water-soluble polymers such as polyoxyethylene or dextran, yielded polymeric derivatives which, even after reaction with oxyHb, gave rise to conjugates with a lower oxygen affinity than the native protein. We showed on a conjugate obtained by the fixation of a BHC-monosubstituted polyoxyethylene onto oxyHb, that the low oxygen affinity was due to the preferential binding of the polymer-linked BHC to the beta-terminal valine residues. In the reaction of dextran-linked benzene tetracarboxylate (dex-BTC) with oxyHb, a lot of parameters had to be optimized in order to obtain conjugates well fitted to the purpose of blood substitute. Thus the polymer content in BTC and the amounts of reagents used for the reaction determined the oxygen-binding properties, the molecular weights and the biochemical characteristics of the conjugates, as well as the viscosity and oncotic pressure of the solutions. This optimization resulted in products which are now studied in-vivo.


A potential blood substitute from carboxylic dextran and oxyhemoglobin. I. Preparation, purification and characterization. Human adult hemoglobin (Hb) from peripheral venous blood was covalently linked, under its oxygenated form, to a dextran polymeric effector to yield conjugates with low oxygen affinity. The polymeric effector was synthesized by directly coupling benzene tetracarboxylic anhydride to dextran (MW approximately 10,000) under mild conditions. The reaction parameters were chosen so that the resulting polymeric derivative was only very little cross-linked and possessed benzene tricarboxylate site concentrations lesser than 1 site for 10 glucose units, as it was found that for higher concentrations the polymer exhibited anticlotting properties. The reaction between oxyHb and the polymer (dex-BTC) was carried out in water in the presence of a water-soluble carbodiimide (EDCI). By optimization of the reaction conditions, a dextran-Hb conjugate with a P50 of 21 torr (37 degrees C, 50mM Bis-tris buffer pH 7.4, 0.14M NaCl, 40 mM glucose) and a potential O2 release of 0.30 ml/g Hb between oxygen partial pressures of 100 and 40 torr, was obtained. The viscosity and oncotic pressure of a solution containing about 6% of conjugated Hb were respectively 1.8 cSt (37 degrees C) and 28 torr (25 degrees C). This solution could be stored frozen at -20 degrees C for a long time, without modification of its properties.


Effect of dextran benzene polycarboxylates on the functional properties of human hemoglobin. The present study deals with the fixation of benzene polycarboxylates such as benzene hexacarboxylate (BHC) or benzene tetracarboxylate (BTC) onto dextran, with the aim of obtaining polyanionic polymers capable of decreasing the oxygen affinity of Hb by interacting with its phosphate binding site. The synthesis was carried out according to several reaction schemes. The polyanionic polymers presented the strongest effector properties, when the polyanionic molecule (BHC or BTC) were directly fixed onto the hydroxyl functions of dextran in the presence of a carbodiimide or by means of the benzene tetracarboxylic anhydride. The introduction of a spacer arm between the polymer and the polyanionic molecule led to the formation of crosslinking between chains of dextran. Among the synthesized polymers those which possess effector properties similar to those of 2,3-diphosphoglycerate (2,3-DPG) were bound onto oxyHb. The resulting covalent conjugates exhibited a low oxygen affinity and a molecular size quite fitted to a potential use as blood substitutes.


A potential blood substitute from carboxylic dextran and oxyhemoglobin. II. Physicochemical and physiological assessments. Preliminary results on guinea pig. The hemoglobin solution covalently linked to dextran benzene tetracarboxylate (dex-BTC) described in Part I, offered the following characteristics: [Hb] = 70 g/l, non modified Hb less than 15%, metHb less than 5%, P50 close to blood value, viscosity and oncotic pressure near to physiological values (37 degrees C). It was biocompatible, stable in plasma, non hypotensive in rat model, non pyrogenic and did not present abnormal toxicity (on mice, according to the french Pharmacopea). Nevertheless, on account of the presence of dextran in the conjugate and due to the literature, we observed an anaphylactoïd reaction in rats consecutive to the injections: absence of hypotension but presence of a massive oedema resulting from an important increase in the capillary permeability with harmful consequences to the vascular retention and survival time. As guinea pigs are insensitive to dextran, we are beginning to assess this conjugate solution in this animal. The first results are very promising: a lack of mortality after large injection, an almost complete vascular retention, a weak urinary loss (only non conjugated Hb), and a plasmatic half-disappearance time close to 7h. Results from haemorrhagic shocks to a final hematocrit less than 0.10 l/l and total and isovolemic exchange transfusions seem to prove a real oxygen-carrying capacity of this new Hb solution.


A potential blood substitute from carboxylic dextran and oxyhemoglobin. III. Evaluations by perfusion of normal and ischemic guinea-pig heart. Hemodynamic parameters of six groups of guinea-pig hearts were studied by the working heart technic of Neely. Three groups were respectively perfused with: Krebs-Henseleit, purified native hemoglobin 1gm/dl and hemoglobin conjugated to dextran benzene tetracarboxylate 1 gm/dl. Three other groups were perfused under the same conditions except that after 30 mn of perfusion, 10 mn of total ischemia were produced followed by 30 mn of reperfusion with the previous solutions. All solutions contained 10 g/l of BSA. Hearts perfused with Hb solutions without ischemia or after ischemia show better parameters than with Krebs-Henseleit. These observations suggest that contrary to previously published results, purified Hb and more, dextran-BTC-Hb appear to be perfusable and are less deleterious for heart than saline without hemoglobin.


Treatment of cerebral ischemia with Dextran-40 or Fluosol DA 20%. A cat stroke model was used to evaluate the efficacy of Dextran-40 (DEX) or Fluosol-DA 20% (FDA) in the treatment of focal cerebral ischemia. The animals were assigned randomly to one of three treatment groups: control, isovolemic hemodilution with DEX or isovolemic hemodilution with FDA. The oxidation state of cytochrome aa3 was measured in-vivo using near infrared reflectance spectrophotometry. The cerebral edema was measured by magnetic resonance imaging (MRI). The MRI edema indices for the three groups revealed that the FDA group had less edema (p less than 0.05), approaching that of non-stroke controls. The relative oxidation state of aa3 for the DEX group declined both during and after hemodilution. At the ninth hour post stroke the FDA group was better (aa3 more oxidized. p less than 0.025). Changes in blood and plasma components were reflective of the extent of hemodilution. Whole blood viscosity analysis revealed a difference (p less than 0.05) at the lower shear rates comparing DEX to FDA with FDA being higher than DEX. Two animals in each of the groups were allowed to awaken at the end of the procedure for functional assessment. These observations suggest that hemodilution with FDA following stroke significantly reduces early post-ischemic cerebral edema, improves oxidation in the peri-infarct area and appears to minimize functional deficits.


Mechanism of protein folding. IV. Forming and breaking of disulfide bonds in bovine pancreatic tripsin inhibitor. The folding mechanism of bovine pancreatic tripsin inhibitor (BPTI) is explained theoretically on the basis of the island model, where the driving force of folding is hydrophobic interaction. For this purpose, we take a look at the formation and breaking of disulfide bonds during the folding process of BPTI. The intermediate conformations and the native one are successfully obtained, which satisfy the so-called "lampshade" geometrical criterion for the formation of the disulfide bonds. The folding pathway is consistent with the renaturation experiment by Creighton. In addition, an elaborate treatment of side chains of amino acid residues by the software programme CHARMm confirms quantitatively the formation of disulfide bridges.


Spectroscopic study of the interaction between poly-(9-vinyladenine) and single or multistrand RNA. The interaction between poly(9-vinyladenine) (PVAd) and poly[r(U)] was investigated by means of uv, CD, 1H-, and 31P-nmr spectroscopies. The interaction was dependent on the molecular weight of PVAd determined by uv and CD spectroscopies. Based on imino proton nmr, it was clearly found that PVAd formed the complex with poly[r(U)] by complementary hydrogen bonding. The interaction of PVAd with double- and triple-stranded helices of RNA was also investigated by uv melting behavior and 31P-nmr spectroscopy. The results suggested that PVAd could not interact with the double-stranded poly[r(A)].poly[r(U)] but did with the triple-stranded RNA.


Adenocarcinoma-reactive human monoclonal antibody MS2B6 defines an antigen in simple glandular epithelium. A human monoclonal antibody (MAb), MS2B6, produced from splenocytes isolated from a patient with advanced papillary serous cystadenocarcinoma of the ovary, defines a unique human tumor-associated antigen. This antigen, EA2B6 (epithelial antigen 2B6), is expressed in a tissue-restricted manner on cultured and fresh human adenocarcinomas and some normal glandular epithelial tissues. EA2B6 is a 38-48 kD protein antigen that co-fractionates with the nuclear matrix-intermediate filament scaffold of simple glandular epithelial tissues. EA2B6 is a molecule with restricted solubility, and in vitro antigen-antibody binding is dependent on the antigen being presented on a solid support. To determine if EA2B6 is a cytokeratin, competition studies were undertaken with several cytokeratin-specific murine monoclonal antibodies. None of these antibodies inhibited the binding of human MAb MS2B6 to partially purified EA2B6. Less than 1% of HT29 colon adenocarcinoma cells and fresh ovarian adenocarcinoma ascites cells express EA2B6 on their surface. The majority of EA2B6 is intracellular. Because of the restricted tissue distribution of this antigen and stability of the antibody, we believe MS2B6 is a good candidate for MAb-mediated diagnosis and therapy of human adenocarcinomas.


Rapid toxicological model for use in assessing ocular drugs. Nonsteroidal antiinflammatory drugs (NSAIDs) were applied to corneas either by in vitro or in vivo methods. The in vitro method involved excising and mounting corneas in a perfusion system at 37 degrees and exposing drug for 2.5 h. The in vivo methods represent either topical administration to the rabbit eye or topical in vivo infusion using a fixed well which permitted a constant concentration (0.05 per cent) to be applied to the eye of anesthetized rabbits for up to 120 min. An overlay grid procedure using scanning electron microscopy (SEM) showed less per cent endothelial damage with in vivo methods than with the in vitro method of administration, but per cent damage depended on which section was viewed. Damage to the epithelium and endothelium were also assessed by quantitative carboxyfluorescein and Janus green staining and uptake procedures, respectively, following drug exposure by the in vivo infusion method. Results for the epithelium indicated that the more lipophilic NSAIDs damaged the epithelial layer to a greater degree than newly synthesized hydrophilic NSAIDs. Damage to the epithelium correlated to the surface activity of the NSAIDs. Qualitative assessment of epithelial and endothelial toxicity can be performed with SEM and transmission electron microscopy (TEM) while vital staining procedures and the SEM grid procedure can be used to quantitatively assess corneal toxicity. The staining methods, however, possess advantages over SEM and TEM procedures in that they are rapid and do not require laborious preparation. As a result of these characteristics, the vital staining procedures could be used as part of a biopharmaceutical screening technique in evaluating new ophthalmic drugs.


Decreased protein kinase C activity is associated with programmed cell death (apoptosis) in freshly isolated rat hepatocytes. Apoptosis of freshly isolated rat hepatocytes was induced by either the omission of fetal bovine serum in the culture medium or addition of the protein kinase C inhibitors polymyxin B or staurosporine. The time-course of DNA breakdown into oligonucleosome-sized fragments and the activity of protein kinase C was determined. Hepatocytes were found to be sensitive to bleomycin which induced a high degree of DNA breakdown even within 30 min incubation. Both staurosporine and polymyxin B induced DNA degradation in hepatocytes after three hours incubation, an effect that was partially prevented by phorbol myristate acetate (PMA). After eight hours incubation, PMA failed to counteract this action and itself produced the apoptosis of rat hepatocytes. The results suggest the involvement of protein kinase C in hepatocyte survival.


Rat liver metabolism of benzo[b]naphtho[2,1-d]thiophene. Thioarenes, sulfur-containing polycyclic aromatic hydrocarbons, have been detected in a number of environmental sources. The metabolism of one thioarene, benzo[b]naphtho[2,1,d]thiophene ([2,1]BNT), by F344 rat liver 9000g supernatant (S-9) was studied. [2,1]BNT which is structurally analogous and has similar carcinogenic potency to chrysene, was metabolized to six ethyl acetate-extractable metabolites when incubated with S-9 from Aroclor 1254-treated F344 rats. Each metabolite was collected from reverse-phase HPLC, and their identities were determined by analysis of MS and NMR data. In order of elution from HPLC they are as follows: (1) trans-1,2-dihydroxy-1,2-dihydrobenzo[b]naphtho[2,1-d]thiophene, (2) benzo[b]naphtho[2,1-d]-thiophene sulfone, (3) benzo[b]naphtho[2,1-d]thiophene sulfoxide, (4) trans-3,4-dihydroxy-3,4-dihydrobenzo[b]naphtho[2,1-d]thiophene, (5) 8- or 9-hydroxybenzo[b]naphtho[2,1-d]thiophene, and (6) 7-hydroxybenzo[b]naphtho[2,1-d]thiophene. In addition, the identities of metabolites 1, 2, 3, and 4 were confirmed by comparison to standards. The syntheses of the sulfone and sulfoxide of [2,1]BNT are reported here. The syntheses of the dihydrodiols were reported previously. Metabolite 5, a hydroxy[2,1]BNT, was the major metabolite formed by liver S-9 from untreated F344 rats. Microsomal preparations from these rats also produced significant amounts of the dihydrodiols, 1 and 4, and the sulfoxide, 3. Microsomes prepared from Wistar rats produced dihydrodiols and the sulfone and sulfoxide of [2,1]BNT. Therefore, [2,1]BNT is metabolized by both ring oxidation and sulfur oxidation in these two strains of rats.


Remnant kidney oxygen consumption: hypermetabolism or hyperbole? On the basis of observations in surgically created remnant kidneys of rat and dog, a novel hypothesis for progressive injury in the setting of reduced renal mass has been put forth. Both rat and dog remnant kidneys exhibit significant hypertrophy that is accompanied by an increased rate of oxygen consumption (QO2) per remaining nephron but not per gram of tissue. This putative "hypermetabolism" is seen in the face of progressive scarring of the tubulointerstitial compartment of the remnant kidney, and interventions that reduce QO2 in these models have been associated with reduced tissue injury in previous studies. The proposed pathway by which an increase in QO2 leads to cellular damage is via the production of oxygen-reactive species or free radicals. In this article, the available data upon which this "hypermetabolism" hypothesis is based are reviewed and the constructs within which these data have been analyzed are examined. From these considerations, a set of questions not yet answered that may serve to direct more fruitful query into this intriguing problem


Determination of glomerular size-selectivity in the normal rat with Ficoll. Diffusion studies in vitro indicate that Ficoll behaves more like an ideal spherical molecule than does dextran, suggesting that Ficoll would be a better probe of glomerular pore size than the commonly used dextran. To examine the differences between these macromolecules in vivo, the fractional clearances of tritiated Ficoll and dextran were measured over a wide range of molecular sizes (Stokes-Einstein radius, rs, from 19 to 65 A) in normal euvolemic Munich-Wistar rats. Whole-kidney and single-nephron hemodynamic conditions were characterized through a combination of clearance and micropuncture measurements. The fractional clearance, or sieving coefficient (theta), for dextran significantly exceeded that of Ficoll at all molecular sizes examined, theta for dextran being approximately 10 times that for Ficoll for rs greater than 30 A. Thus, the results with Ficoll imply a more size-restrictive barrier than do the results with dextran. The values of theta for Ficoll approximated previously reported values for uncharged globular proteins. Although theta for Ficoll at rs = 35 A was much smaller than the corresponding value for dextran, it was still approximately 30 times greater than typical values of the filtrate-to-plasma concentration ratio reported for serum albumin (a polyanion) in the rat, in agreement with the concept that glomerular charge-selectivity normally plays an important role in the prevention of albuminuria. Three membrane-pore models were compared in their ability to represent the dextran and Ficoll sieving data. A lognormal pore-size distribution in parallel with a nonselective "shunt" pathway was found to be more effective than either an isoporous membrane with a shunt or a purely lognormal distribution. On the basis of these laboratory results and computations, Ficoll may be preferred over dextran in future studies of glomerular size-selectivity.


Phospholipase A2-induced changes in AMPA receptor: an autoradiographic study. The expression of long-term potentiation and learning of a classical conditioning task increase [3H]-AMPA binding in hippocampus. Phospholipase A2 (PLA2) has been proposed to underly these changes, as PLA2 treatment of membrane preparations increases the affinity of AMPA receptors for agonists. We demonstrate here that preincubation of thin (10 microns) frozen rat brain sections with exogenous PLA2 and calcium at physiological temperature changes the binding properties of AMPA receptors. Quantitative autoradiography reveals that PLA2-treatment produces a differential increase in [3H]-AMPA binding across brain regions. The same treatment also decreases the binding of an antagonist ([3H]-CNQX) throughout the brain. We propose that PLA2 treatment results in a modification of the AMPA receptors which is regionally specific, probably due to different AMPA receptor subunit compositions.


Effect of NO synthase inhibition on NMDA- and ischaemia-induced hippocampal lesions. We assessed the effect of NG-Nitro-L-arginine methylester (L-NAME), an inhibitor of nitric oxide synthase, on the hippocampal lesions induced either by the focal injection of N-methyl-D-aspartate (NMDA) or (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (s-AMPA) or by 10 min of severe forebrain ischaemia (4-vessel occlusion), in the rat. We find that L-NAME, 20 or 40 mg kg-1, selectively decreases NMDA-induced CA1 lesions while it has no effect on AMPA toxicity. L-NAME, 20 mg kg-1, does not decrease hippocampal damage induced by ischaemia. These results suggest that NO contributes to NMDA toxicity and support data indicating that NMDA receptor antagonists fail to protect against hippocampal CA1 lesions in the 4-vessel occlusion model.


Follicular dendritic cells in the alternative antigen transport pathway: microenvironment, cellular events, age and retrovirus related alterations. Follicular dendritic cells (FDC) are located in lymphoid follicles of secondary lymphoid tissues and play a pivotal role in the initiation and maintenance of the secondary antibody response. FDC are an integral part of the microenvironment of the follicle and function as members of the 'alternative antigen transport pathway.' This pathway consisting of the antigen transport cell-FDC-ICCOSOME-B cell axis, leads to the formation of germinal centers where antibody-forming cell and memory B cell development are initiated through interaction of FDC-retained antigen, B cells and T helper cells. Evidence suggests that these interactions are down-regulated through antibody feedback, or as needed, reactivated with the utilization of FDC-retained antigen for the maintenance of antibody levels. Age- and retrovirus-related FDC defects seriously compromise the capacity of this pathway to maintain immunity.


Analysis of cost-effectiveness of different strategies for hepatocellular carcinoma screening in hepatitis B virus carriers. A mathematical model was used to calculate the efficacy of screening to detect hepatocellular carcinoma at a resectable stage in hepatitis B virus carriers. Data relating to tumour incidence, efficacy of screening tests and tumour growth times were obtained from a literature review. Various tests were costed according to charges currently prevailing at the authors' institution. The cost per early tumour detected is inversely proportional to tumour incidence. It is relatively low for populations with high incidences of hepatocellular carcinoma for example, male carriers over the age of 30. Both the costs and the proportions of early tumour detected increase with increasing frequency of screening. However, the use of ultrasonography at 10 monthly intervals or both ultrasonography and alpha-fetoprotein estimation at yearly intervals will detect 90% of tumours early at a cost of S$20,000 (US$11,800) per early tumour detected. The results would be significantly altered if tumour growth times were markedly different from those reported in the literature.


Treatment of diffuse non-Hodgkin's lymphoma with combined chemotherapy using methotrexate, adriamycin, cyclophosphamide, vincristine, prednisolone and bleomycin (MACOP-B). Forty-nine previously untreated adult patients with diffuse non-Hodgkin's lymphoma were treated with MACOP-B (methotrexate, adriamycin, cyclophosphamide, vincristine, prednisolone and bleomycin) between December 1986 and December 1990. Forty patients (82%) achieved a complete response (CR), three (6%) a partial response (PR), while four (8%) had either no response or progression of disease, one (2%) patient ceased MACOP-B therapy and received other chemotherapy because of sustained neutropenia, and one patient (2%) died of sepsis during therapy. The factors that adversely affected the CR rate were by stage IV, the presence of B symptoms, the presence of a large mass (greater than 5 cm), and low serum total protein level. The 4-year survival for all 49 patients was 70% and the 4-year disease-free survival (DFS) for the 40 CR patients was 77%. Relapses were higher in patients whose initial serum lactic dehydrogenase (LDH) level was higher than 660 IU/1 (DSF 89% vs. 49%). Toxicity was substantial but acceptable, with neutropenia and mucositis proving to be the most frequent severe side-effects. These preliminary results confirmed the effectiveness of MACOP-B therapy for diffuse non-Hodgkin's lymphoma.


Hypercholesterolaemia in patients with hepatocellular carcinoma. Ninety-one (11.4%) subjects with hypercholesterolaemia (serum cholesterol level more than 250 mg/dL) of 792 Chinese patients with hepatocellular carcinoma (HCC) were studied in Taiwan. All 91 patients had large tumours greater than 7 cm in diameter and a tumour volume greater than 50%; 56 (61%) of these patients manifested tumour involvement in both lobes of the liver. The HCC patients with hypercholesterolaemia had significantly higher mean serum levels of albumin, triglyceride and alpha-fetoprotein (AFP) compared with age-sex-tumour volume matched HCC patients without hypercholesterolaemia. The associated incidence of hypoglycaemia in hypercholesterolaemic HCC patients was significantly higher than in HCC patients without hypercholesterolaemia (15/90 vs 4/90; P = 0.01). There was no significant difference in the survival analysis between HCC patients with and without hypercholesterolaemia. Eight and 11 of hypercholesterolaemic HCC patients had their tumours surgically resected and received transcatheter hepatic arterial chemoembolization (TAE), respectively. Serum cholesterol levels fell to the normal range after treatment and rose to abnormal levels again when tumours recurred after surgery or progressively enlarged after TAE. The change in pattern of serum cholesterol was parallel to the change in serum AFP. Serum cholesterol levels may serve as another marker in identifying tumour recurrence and the presence of a viable tumour mass in hypercholesterolaemic HCC patients who have received surgical resection or TAE.


Exposure of platelet fibrinogen receptors by a monoclonal antibody to the GPIIb-IIIa complex, PMA4. We found that a monoclonal antibody to the glycoprotein (GP) IIb-IIIa complex, PMA4, induces fibrinogen binding to platelets, and we examined the mechanism involved. Affinity chromatography and crossed immunoelectrophoresis showed that PMA4 recognized an epitope on the GPIIb-IIIa complex-specific domain. The binding of 125I-fibrinogen to platelets was induced by PMA4 in a concentration-dependent manner and was blocked by EDTA, RGDS peptides and an anti-GPIIb-IIIa monoclonal antibody, PMA1. Binding of the divalent antibody to the GPIIb-IIIa complex was necessary to induce fibrinogen binding and subsequent platelet aggregation, since Fab fragments, unlike PMA4 IgG and F(ab')2 fragments, did not induce fibrinogen binding or aggregation. The PMA4 IgG induced fibrinogen binding, serotonin secretion, and Ca2+ mobilization, whereas F(ab')2 induced fibrinogen binding only. In addition, F(ab')2-induced fibrinogen binding was not abolished in the presence of aspirin, H-7, a protein kinase C inhibitor, PGE1 or dibutyryl cyclic AMP. These results demonstrate that the binding of PMA4 divalent molecules to the GPIIb-IIIa complex can expose platelet fibrinogen receptors in the absence of the stimulatory effects of intracellular mediators on platelets. Thus, we conclude that the fibrinogen receptors on the GPIIb-IIIa complex can be exposed by direct action of the antibody on the complex molecules.


Plasma disappearance of homologous and heterologous pancreatic enzymes in dogs. Homologous and heterologous (human) pure pancreatic juice was infused into dogs to compare the behaviour of pancreatic enzymes in the circulation. This was achieved by estimating the magnitude of elevation and speed of disappearance of pancreatic enzymes in plasma. The half-lives of the plasma enzymes did not differ significantly between dogs and humans. However the half-lives of other enzymes (longest for amylase and shortest for trypsinogen) did differ. The magnitude of elevation expressed as a ratio of peak (5 min) to basal level was greatest for trypsinogen (28 times) and least for amylase (1.4 times). However, 289% and 410% of the initial lipase enzyme activity was recovered 5 min after infusion of dog and human pure pancreatic juice, respectively (44% when measured by immunoreactive human lipase activity). The excess recovery requires re-evaluation of diagnostic value of turbidimetric activity of plasma pancreatic lipase.


Relationship between fatty liver, alanine aminotransferase, HBsAg and hepatitis C virus. A community health survey of 923 residents aged 30 years or more was performed in Putai Township of Taiwan. To elucidate the relationships between hepatitis C virus (HCV) and surrogate tests for non-A, non-B hepatitis in hyperendemic areas of hepatitis B virus (HBV) serum levels of alanine aminotransferase (ALT), triglycerides, cholesterol, hepatitis B surface antigen (HBsAg) and antibody to HCV (anti-HCV) were examined. Glucose tolerance tests and the history of diabetes treatment were used to define the diabetes status. Fatty liver was diagnosed by sonography. The prevalence of anti-HCV was 2.6% (95% confidence interval, 1.6-3.6%). Elevated ALT and fatty liver were significantly associated with anti-HCV in univariate analysis. Anti-HCV was not an associated factor for fatty liver after adjusting for serum triglycerides and cholesterol, sex, body mass index and diabetes status through multiple logistic regression. However elevated ALT was still associated with anti-HCV after adjusting for serum triglycerides, sex, body mass index, HBsAg and age through multiple linear regression. The anti-HCV prevalence was similar between HBsAg-positive and negative subjects. Aggregation of HCV infection was found among spouses. It was concluded that elevated ALT and intimate contact with HCV carriers might be associated factors for HCV infection, and that HBV infection and fatty liver were not related to HCV infection in Taiwan.


Hepatitis C as the cause of chronic non-A, non-B hepatitis: high sensitivity of simultaneous measurement of core and non-structural antibodies. First generation serologic tests (ELISA-1) for hepatitis C virus infection measure antibodies directed against a short non-structural segment of the virus (anti-c100-3). A major disadvantage of this test is that it lacks sensitivity in the identification of hepatitis C virus among patients at risk of infection. Thus, only 70-90% of chronic non-A, non-B cases are ELISA-1 positive. The present study set out to determine whether antibodies directed against the core region would be a more sensitive indicator of hepatitis C virus infection in patients with chronic non-A, non-B hepatitis. Sera were studied from 97 patients with raised serum alanine aminotransferase levels for more than 6 months in whom other causes of abnormal alanine aminotransferase were excluded. Using ELISA-1, 85 sera (87%) were anti-c100-3 positive. Sera were then tested for presence of antibody directed against Po, a core peptide of a Japanese strain of hepatitis C virus, using an ELISA method. Eighty-eight sera (91%) were anti-Po positive. Among the 12 anti-c100-3 negative patients, six were anti-Po positive. A second generation ELISA for anti-HCV (ELISA-2) incorporates a different antibody to the core region (c22-3) in addition to an expanded non-structural region, c200, which consists of c100-3 plus c33c. With these tests, all sera but one were positive, including 11 of 12 ELISA-1 negative and eight of nine anti-Po negative sera.(ABSTRACT TRUNCATED AT 250 WORDS)


Late postprandial pancreatic secretion periods in conscious dogs. Effect of vagotomy. It is well established that, in the dog, the exocrine pancreatic secretion in response to food intake is a two-phased mechanism with a first phase during 0-4 h period and a second one during 8-12 h period. In the present study we have investigated the role played by the vagus nerve in the genesis of this late pancreatic hypersecretion (second phase) in dogs with truncal vagotomy and pyloroplasty. Truncal vagotomy totally suppressed the first phase of the pancreatic secretion; it did not abolish the second postprandial phase but it increased its latency by delay of 4 hours. In fact, during the 12-18 h period a pancreatic hypersecretory response was evidenced after vagotomy which appeared to be statistically significant as compared to basal values (P less than 0.001). Our results indicate that the vagus nerve does not play a role in the genesis of the late hypersecretory second phase.


In vitro effects of divalent metal ions and metabolic modulators on purified glutamine synthetase from brain of teleostean fish. The purified brain glutamine synthetase of a teleostean fish, Clarias batrachus has been examined under the influence of divalent metal ions activation and in vitro studies using different amino acids and metabolic modulators. All observations of the enzyme activity are based on transferase reaction at optimum temperature and pH. The enzyme exhibits an absolute requirement for Mn2+ (most potent) Mg2+ and Co2+. The activity is markedly inhibited by leucine, asparagine, isoleucine, carbamyl phosphate, uridine monophosphate and glutamate and activated by alpha-ketoglutarate. The significance of these results has been discussed in brief with emphasis on its involvement in teleostean physiology and metabolism.


Effect of food deprivation and refeeding on rat organ temperatures. Small thermocouple sensors were surgically implanted in the liver, kidney, hind leg muscle, interscapular brown adipose tissue, small and large intestines, dorso-lumbar internal side of the skin, periovaric adipose tissue and the lower aorta of Wistar rats. The aortic temperature was taken as core temperature. The sensors allowed continuous long-term monitoring of the temperatures of these organs. A period of 18 hours of food deprivation resulted in an overall decrease of mean core and organ temperature, brown adipose tissue temperature dropping to values lower than those of the aorta in the fed state. Liver, kidney and small intestine maintained higher temperatures than the aorta both in fed and starved states. Refeeding overshot the core temperature with increases in most organs versus both the fed and food-deprived situations. The results are concordant with an active role of brown adipose tissue in dietary induced thermogenesis. Three days of food deprivation did not alter the basic circadian rhythm of core temperatures in the rat kept at 22 degrees C, whereas it did modulate both nightly maximum and diurnal minimum temperatures to much lower settings than either in the fed or refed situations. The rat adapts to starvation by decreasing core and organ temperatures and widening the amplitude of the daily temperature cycle.


[Detailed analysis of human pancreatic secretions collected by retrograde catheterization. Parallel or non-parallel excretion of digestive enzymes?]. Pancreatic juices were collected by selective reverse catherism of the chief pancreatic duct in two patients, one free from pancreatic disease and the other having a pancreas cancer. They were analysed in detail especially in order to get information on the mechanism of enzyme excretion. The variations of the digestive enzyme activities (amylase, lipase, trypsin, chymotrypsin, carboxypeptidase A and B) were not superimposable among them or with the fluctuations of the protein concentration in the pancreatic juice samples. These results agree with a non-parallel enzyme-excretion mechanism by the pancreas. However deep electrophoresis analyses of pancreatic juice samples showed that the ratio of each digestive enzyme concentration remained almost constant in the same patient. This observation disagrees with the above conclusion and suggests that the data obtained by using classical methods for estimating digestive enzyme activities have to be considered prudently. By another way, two main significant differences were reported by analysing the ionic composition of the pancreatic juice samples following their origin. The pancreatic juice samples of the patient having a pancreas cancer had a lower and more variable Na+ concentration than those coming from the patient who was free from pancreas disease. They had a HCO3- concentration which was almost constant, contrary to what was observed for the pancreatic juice secreted by the other patient.


On the latency of blood phenoloxidase of painted locust Poeciloceros pictus. The blood phenoloxidase of Poeciloceros pictus exists as proenzyme. The amidase activity present in the haemocyte is responsible for the activation of haemocyte phenoloxidase. There is an amidase inhibitor in the plasma. Plasma phenoloxidase could be activated by various fatty acids, biological compounds like lipopolysaccharide zymosan and detergents. Electrophoretic studies suggest that activation of haemocyte phenoloxidase involves limited proteolysis. It is suggested that the natural activator of plasma phenoloxidase may be fatty acid and it does not undergo proteolysis during activation. The biological compounds like zymosan or LPS are easily recognised by the factor(s) from the membranes of haemocytes.