Testosterone regulates epidermal growth factor levels in the thyroid gland of hypothyroid mice. The effects of testosterone (TP) and thyroxine (T4) on the level of epidermal growth factor (mEGF) in the thyroid were compared in a hypothyroid mouse model. Groups of five adult female BALB/c mice were given a "severe" hypothyroid regimen consisting of an iodine deficient diet together with oral and s.c propylthiouracil (PTU). Sialoadenectomy or sham operation was performed after 18 days on the hypothyroid regimen. The mice convalesced on normal diet for 5 days and beginning from day 23 received either T4, 1 ug/g or 2 ug/g, s.c daily or TP, 0.3 mg or 0.75 mg, i.m. every third day until day 33, while continuing the hypothyroid regimen. Control mice received normal diet and vehicles for the various injections. The mice were killed on day 33 and thyroidal EGF levels determined by radioimmunoassay. The mean+S.E. levels of mEGF in the thyroid were 10.12 +/- 1.75 ng/mg protein (control), 3.82 +/- 0.67 ng/mg (hypothyroid; p < 0.01), 3.07 +/- 1.52 (T4, 1 ug/g; p < 0.02), 2.59 +/- 0.46 ng/mg (T4, 2 ug/g; p < 0.01), 8.58 +/- 2.48 (TP, 0.3 mg), and 9.65 +/- 1.86 (TP, 0.75 mg). Thus thyroidal mEGF levels decreased significantly in all groups except those subsequently treated with testosterone; T4 was ineffective in reversing the tissue depletion of mEGF in this model. These results show that mEGF levels in the thyroid could be depleted by hypothyroidism and may also be androgen responsive.


Vasoactive intestinal peptide enhances thyroidal iodide uptake during dietary iodine deficiency. The presence of vasoactive intestinal peptide and neuropeptide Y in thyroid nerves and their effects on thyroid blood flow are well known. However, the effects of these two neuropeptides on the various processes involved in thyroid hormone biosynthesis and release have not been fully explored. We have now tested these two peptides for effects on an early step in thyroid hormone biosynthesis, namely iodide uptake, a process which is comprised of trapping and organification. In these experiments, we have used anesthetized adult male rats pretreated with thyroxine or fed a low iodine diet to increase thyroidal sensitivity. Vasoactive intestinal peptide significantly increased iodide uptake in rats fed an iodine deficient diet but not in those fed a normal iodine diet. This effect disappeared if animals were pretreated with propylthiouracil. Neuropeptide Y did not alter iodide uptake in rats on either the low or the high iodine diet, regardless of the presence or absence of propylthiouracil. The effect of vasoactive intestinal peptide on iodide uptake could be due to its influence on the organification of iodine, or on thyroid blood flow, or on both processes.


Detection of albumin binding abnormalities in sera of patients with familial dysalbuminaemic hyperthyroxinaemia using isoelectric focusing. Definitive diagnosis of familial dysalbuminaemic hyperthyroxinaemia (FDH) requires finding a high concentration of [125I]T4 bound to albumin. We used isoelectric focusing (IEF) in agarose gels to study the sera of three members of a family with FDH and compared the distribution of [125I]T4 obtained by IEF with that obtained by conventional agarose gel electrophoresis (AGE). Both IEF and AGE confirmed the diagnoses of FDH. In case #1, the % [125I]T4 bound to albumin was 15.2 and 20.0, with IEF and AGE, respectively, in case #2 23.6 and 26.5, and in case #3, 22.1 and 23.0, compared to normal controls of 5.9 and 7.4, respectively. IEF has not previously been used to diagnose FDH, to our knowledge. IEF has the advantage of eliminating TBG interference with albumin migration, which can potentially complicate the diagnosis of FDH when the AGE method is used. To date, reverse flow electrophoresis, a more cumbersome method with poorer resolution than IEF, has been utilized to eliminate TBG interference. IEF, in agarose gels, is a relatively simple and accurate method to diagnose FDH, and avoids artifacts which may be encountered with AGE.


A point mutation of low-density-lipoprotein receptor causing rapid degradation of the receptor. The exons of the low-density-lipoprotein-(LDL)-receptor gene from a Japanese patient with homozygous familial hypercholesterolemia were amplified by the polymerase chain reaction (PCR), and their nucleotide sequences were determined. A point mutation from G to C was found in exon 9, which was expected to change Asp at position 412 to His. This amino acid change occurred within the epidermal-growth-factor-precursor homology domain of the LDL receptor, slightly impairing the processing from the precursor to the mature form and causing rapid degradation of the mature form in the fibroblasts of the patient. The mutant LDL-receptor gene transfected into COS-1 cells expressed a LDL-receptor protein with the same properties as the protein expressed in the fibroblasts of the patient; impaired processing and rapid degradation of the synthesized receptor protein. The mutation was identified in family members of the patient by dot-blot hybridization of PCR-amplified DNA with the mutant oligonucleotide. The family members carrying the mutant gene showed higher serum cholesterol levels than the others. However, their cholesterol levels were also greatly influenced by the apolipoprotein-E phenotype.


Investigation of the mechanism of non-turnover-dependent inactivation of purified human 5-lipoxygenase. Inactivation by H2O2 and inhibition by metal ions. Human 5-lipoxygenase is a non-heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non-turnover-dependent inactivation of purified recombinant human 5-lipoxygenase for the following reasons: catalase protects against oxygen-dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 microM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton-type reaction of the ferrous enzyme with H2O2, resulting in the formation of an activated oxygen species. Purification of 5-lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 mumol 5(S)-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5-lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half-maximal product formation being obtained at 0.9 microM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5-Lipoxygenase is strongly inhibited by low concentrations (< or = 10 microM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2 = 2 min) in the presence of excess EDTA. These observations on the mechanism of non-turnover-dependent inactivation of 5-lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.


Nuclear-magnetic-resonance analysis of the capsular antigen of Actinobacillus pleuropneumoniae serotype 9. Its identity with the capsular antigen of Escherichia coli K62 (K2ab), Neisseria meningitidis serogroup H and Pasteurella haemolytica serotype T15. The specific capsular antigen of Actinobacillus pleuropneumoniae serotype 9 was characterized by one-dimensional and two-dimensional high-field nuclear-magnetic-resonance methods, and by chemical analyses, as a teichoic-acid-type polymer of a repeating unit having the structure [formula: see text] The basic polymer structure is identical to capsular antigens of Neisseria meningitidis group H, Escherichia coli K62 (K2ab) and Pasteurella haemolytica serotype T15.


The 70-kilodalton heat-shock proteins of the SSA subfamily negatively modulate heat-shock-induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae. In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress-related metabolite that accumulates upon exposure of cells to heat shock or a variety of non-heat inducers of the stress response. Here, we describe the influence of mutations in individual heat-shock-protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70-kDa heat-shock proteins (hsp70) overproduced trehalose during heat shock at 37 degrees C or 40 degrees C and showed abnormally slow degradation of trehalose upon temperature decrease from 40 degrees C to 27 degrees C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27 degrees C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat-stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heat-stressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a delta hsp104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat-shock proteins, in particular hsp70, appear to be involved in recovery from the heat-stressed state rather than in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27 degrees C after heat shock at 40 degrees C. Trehalase activity is known to be under positive control by cAMP-dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein-kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.


Amino acid and DNA sequences of an extracellular basic protease of Dichelobacter nodosus show that it is a member of the subtilisin family of proteases. A DNA fragment encoding an extracellular basic protease (pI approximately 9.5) from Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative agent of ovine footrot, has been cloned and expressed in Escherichia coli and sequenced. E. coli harbouring a plasmid with a 3-kb DNA fragment containing the D. nodosus basic-protease gene exhibited proteolytic activity when tested on skim-milk plates. The sequence of the native basic protease isolated from D. nodosus was also determined by direct amino acid sequencing. Comparison of the deduced sequence of the primary translation product (603 residues) and that of the native protease (344 residues) indicates that the protease is synthesized as a precursor molecule, containing a signal peptide (21 residues), a 111 amino acid pro-peptide and a 127 residue C-terminal extension which is subsequently processed to the mature active form. Comparison of the D. nodosus basic protease sequence with that of other serine proteases showed that it is related to the subtilisin family of proteases with strong conservation of sequence identity around the catalytic site residues. A remarkable similarity in structure was found to the serine protease of Xanthomonas campestris, a plant pathogen, with respect to the length of the precursor segments, conservation of disulfide bridges and approximately 50% sequence identity of the mature proteases.


Control of the metabolic flux in a system with high enzyme concentrations and moiety-conserved cycles. The sum of the flux control coefficients can drop significantly below unity. In a number of metabolic pathways enzyme concentrations are comparable to those of substrates. Recently it has been shown that many statements of the 'classical' metabolic control theory are violated if such a system contains a moiety-conserved cycle. For arbitrary pathways we have found: (a) the equation connecting coefficients CEiJ (obtained by varying the Ei concentration) and CviJ (obtained by varying the kicat), and (b) modified summation equations. The sum of the enzyme control coefficients (equal to unity under the 'classical' theory) appears always to be below unity in the systems considered. The relationships revealed were illustrated by a numerical example where the sum of coefficients CEiJ reached negative values. A method for experimental measurements of the above coefficients is proposed.


Protection by glutathione and other thiol compounds against the loss of protein thiols and tocopherol homologs during microsomal lipid peroxidation. Microsomes from rat liver were used to investigate the mechanisms by which thiol compounds protect cellular membranes against damage from oxidants. Glutathione (GSH), dihydrolipoate and dithioerythritol, but not cysteine, ameliorated the loss of thiol groups of microsomal proteins attacked by Fe/ADP/NADPH or Fe/ADP/ascorbate prooxidant systems. The protection by GSH, but not dihydrolipoate or dithioerythritol, appeared to be enzymic since it was lost after microsomes were heated or treated with trypsin. The blocking of microsomal protein thiols with N-ethylmaleimide also diminished the protective effect of GSH. Lipid peroxidation, as assessed by chemiluminescence and vitamin-E loss, was inhibited in parallel with the protection of protein thiols. In microsomes lacking vitamin E, the protection of protein thiols by exogenous thiols was diminished. However, the GSH-dependent protection of vitamin E showed no preference for alpha-tocopherol over other tocopherol homologs. It is suggested that a GSH-dependent enzyme maintains protein thiols in the face of oxidative damage during microsomal peroxidation. A maintenance of protein thiols might not only protect important metabolic functions, but may also afford an antioxidant capacity to membranes, and account for one facet of the GSH-dependent inhibition of lipid peroxidation.


Interleukin-1 beta and transforming growth factor-beta 2 enhance cytosolic high-molecular-mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells. Interleukin-1 beta induces gene expression and secretion of group-II phospholipase A2 and release of prostaglandin E2 from rat mesangial cells. The interleukin-1 beta-induced synthesis of group-II phospholipase A2 is prevented by transforming growth factor-beta 2, whereas transforming growth factor-beta 2 potentiated the interleukin-1 beta-evoked prostaglandin E2 production. Transforming growth factor-beta 2 itself did not induce synthesis of group-II phospholipase A2, although it stimulated prostaglandin E2 formation. Here we describe the effect of interleukin-1 beta and transforming growth factor-beta 2 on a cytosolic phospholipase A2 activity and prostaglandin E2 formation in rat mesangial cells. Based on the resistance to dithiothreitol and migration profiles on a Mono-Q anion-exchange column and a Superose 12 gel-filtration column, the cytosolic phospholipase A2 activity was assigned to a high-molecular-mass phospholipase A2. Measured with 1-stearoyl-2-[1-14C]arachidonoylglycero-phosphocholine as substrate, both interleukin-1 beta and transforming growth factor-beta 2 enhanced the high-molecular-mass phospholipase A2 activity. The stimulation of rat mesangial cells with interleukin-1 beta and transforming growth factor-beta 2 was time- and dose-dependent with maximal cytosolic phospholipase A2 activities at 10 nM and at 10 ng/ml respectively, after 24 h of stimulation. Under these conditions, interleukin-1 beta and transforming growth factor-beta 2 enhanced the cytosolic phospholipase A2 activity 2.2 +/- 0.6-fold and 2.5 +/- 0.6-fold, respectively. These results strongly suggest that an enhanced cytosolic high-molecular-mass phospholipase A2 activity is involved in the formation of prostaglandin E2 mediated by transforming growth factor-beta 2. Whether interleukin-1 beta induced group-II phospholipase A2 and/or interleukin-1 beta-enhanced cytosolic phospholipase A2 activity is involved in prostaglandin E2 formation in rat mesangial cells is discussed.


Isolation and functional analysis of histidine-tagged elongation factor Tu. The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.


Synthetic peptides as antagonists of the anaphylatoxin C3a. Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.


Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy poly(rC) and poly(dC). Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.


Alpha-crystallin exists in a non-spherical form. A study on the rotational properties of native and reconstituted alpha-crystallin. Native alpha-crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient-electric-birefringence measurements and ultraviolet linear-dichroism spectroscopy. These techniques were also performed on 6-M-urea-dissociated and reconstituted alpha-crystallin. Transient-electric-birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non-spherical occurrences of alpha-crystallin in more detail. Although not distinguishable with size-exclusion chromatography, we could identify at least two different classes of both native and reconstituted alpha-crystallin, from which at least one consists of non-spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the alpha-crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three-layer model for alpha-crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711-724].


Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding. Thermal denaturation of two homologous proteins, low-M(r) cysteine-proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65 degrees C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half-denaturation at 50.2 degrees C and 90.8 degrees C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively. Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three-dimensional structure has been determined. The calculated enthalpy, heat-capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.


Inhibition of chloroplast ATPase by the K+ channel blocker alpha-dendrotoxin. Possible structural and functional similarities between the channel part, CF0, of chloroplast ATPase (CF0CF1) and ion channels permeable to monovalent cations were investigated using high-affinity toxins mainly targeted against voltage-sensitive K+ channels. In particular, the effect of the K(+)-channel blocker alpha-dendrotoxin and the crude scorpion venom of Leiurus quinquestriatus hebraeus (LQ venom) on ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes was characterised. Alpha-dendrotoxin (K(i) approximately 5.05 microM) and the LQ venom (K(i) approximately 1.55 micrograms/ml) specifically inhibited ATP synthesis in thylakoid membranes and in CF0CF1-containing liposomes. Our results show that alpha-dendrotoxin and peptides of the LQ venom with an apparent molecular mass of about 4.0 kDa, probably isoforms of charybdotoxin, specifically bind to CF0CF1. This binding was reversible and induced a high leak conductance for H+ through CF0. The Ca(2+)-dependent ATPase activity of the isolated soluble part of CF0CF1 (CF1) was completely inhibited by 1 microM alpha-dendrotoxin, while the crude LQ venom, at concentrations up to 10 micrograms/ml, had no affect on ATPase activity. The concentration dependence of the inhibition by alpha-dendrotoxin indicates that approximately 2 mol alpha-dendrotoxin bind/mol CF0CF1 and 1 mol alpha-dendrotoxin/mol CF1. Known inhibitors of H(+)-flow-through CF0 acted in the presence of alpha-dendrotoxin synergistically. Dicyclohexylcarbodiimide and venturicidin, in contrast to their known effect of blocking H(+)-flow-through CF0, increased the leak conductance through CF0 in the presence of alpha-dendrotoxin drastically. This uncoupling effect indicates that their normal mode of blocking is a secondary effect. Binding of the inhibitors to their respective sites apparently does not affect the proton pathway in CF0, but induces a conformation which closes the channel part for H+. Protein sequence comparison between the known binding site of charybdotoxin in the shaker K+ channel from Drosophila [MacKinnon, R. & Heginbotham, L. (1990) Neuron 5, 767-771] and the choroplast ATPase showed that subunit III reveals a significant similarity (64%) in parts of its sequence (Gln28-Leu53) to the helix 5 and helix 6 (S5-S6) linker region (Ala413-Cys462; the charybdotoxin-binding site) of the shaker K+ channel. According to secondary-structure predictions, the homologous sequences in subunit III and the shaker K+ channel represent putative hydrophilic loops connecting two transmembrane alpha-helices. Apparently the shaker K+ channel and subunit III share significant topological features in these hydrophilic loops which may be part of the respective channel entrance.


Kinetics of ATP synthesis catalyzed by the H(+)-ATPase from chloroplasts (CF0F1) reconstituted into liposomes and coreconstituted with bacteriorhodopsin. The H(+)-ATPase from chloroplasts (CF0F1) was isolated, purified and reconstituted into liposomes from phosphatidylcholine/phosphatidic acid. A transmembrane pH difference, delta pH, and a transmembrane electric potential difference, delta psi, were generated by an acid/base transition. The rate of ATP synthesis was measured at constant delta pH and constant delta psi as a function of temperature between 5 degrees C and 45 degrees C. The activation energy was 55 kJ mol-1. CF0F1 was coreconstituted with bacteriorhodopsin at a molar ratio of approximately 1:170 in the same type of liposomes. Illumination of the proteoliposomes leads to proton transport into the vesicles generating a constant delta pH = 1.8. The dependence of the rate of ATP synthesis on ADP concentration was measured with CF0F1 in the oxidized state, E(ox), and in the reduced state, E(red). The results can be described by Michaelis-Menten kinetics with the following parameters: Vmax = 0.5 s-1, Km = 8 microM for E(ox) and Vmax = 2.0 s-1, Km = 8 microM for E(red).


Contribution of de-novo and salvage synthesis to the uracil nucleotide pool in mouse tissues and tumors in vivo. The relative contribution of de-novo and salvage synthesis to tissue pyrimidine nucleotide pools is an important parameter in the rational design of anti-pyrimidine therapies, but has not been measured in vivo. We have measured the contribution of de-novo synthesis to the total acid-soluble uracil nucleotide pool in mouse tissues by analysis of the incorporation of label after intra-peritoneal infusion of L-[15N]alanine. The contribution of salvage synthesis was measured by the incorporation of radiolabel after intravenous infusion of [14C]uridine. The results show that de-novo synthesis makes the larger contribution to the intestine uracil nucleotide pool, salvage synthesis makes the larger contribution to the kidney pool, and de-novo and salvage synthesis make roughly equal contributions to the liver pool. In tumors studied (L1210, P388, B16, Nettesheim), the contribution of de-novo synthesis was at least five times the contribution of salvage synthesis. The measurements were repeated 24 hours after a 400-mg/kg dose of N-phosphonacetyl-L-aspartic acid. De-novo synthesis was substantially inhibited in all tissues and tumors after this treatment, although significant residual activity was observed in the intestine and L1210 cells. Nettesheim carcinoma was the only tumor or tissue to show a significant increase in salvage synthesis after N-phosphonacetyl-L-aspartic acid treatment.


Effect of 2-mercaptoethanol on glutathione levels, cystine uptake and insulin secretion in insulin-secreting cells. The role of glutathione (GSH) in the differentiated state of insulin-secreting cells was studied using 2-mercaptoethanol as a means of varying intracellular GSH levels. 2-Mercaptoethanol (50 microM) caused a marked increase of GSH in two rat insulinoma cell lines, RINm5F and INS-1, the latter being dependent on the presence of 2-mercaptoethanol for survival in tissue culture. The effect of 2-mercaptoethanol on GSH was shared by other thiol compounds. Since in other cell types 2-mercaptoethanol is thought to act on cystine transport, thereby increasing the supply of cysteine for GSH synthesis, we have studied [35S]cystine-uptake in INS-1 cells. At equimolar concentrations to cystine, 2-mercaptoethanol caused stimulation of [35S]cystine-uptake. The effect persisted in the absence of extracellular Na+, probably suggesting the involvement of the Xc- carrier system. INS-1 cells with a high GSH level, cultured 48 h with 2-mercaptoethanol, displayed a lower cystine uptake than control cells with a low GSH content. The effect of variations of the GSH levels on short-term insulin release was studied. No alteration of glyceraldehyde-induced or KCl-induced insulin release in RINm5F cells was detected. In contrast, both in islets and in INS-1 cells, a high GSH level was associated with a slightly lower insulin release. In INS-1 cells the effect was more marked at low glucose concentrations, resulting in an improved stimulation of insulin secretion. On the other hand, in islets, a decrease in the incremental insulin release evoked by glucose was seen. As in other cell types, oxidized glutathione (GSSG) was less than 5% of total GSH, and in INS-1 cells no change in the GSH/GSSG ratio was detected during glucose-induced or 3-isobutyl-1-methylxanthine-induced insulin release. In conclusion, 2-mercaptoethanol-dependent INS-1 cells, as well as RINm5F cells and islets of Langerhans, display a low capacity in maintaining intracellular levels of GSH in tissue culture without extracellular thiol supplementation; 2-mercaptoethanol possibly acts by promoting cyst(e)ine transport; changes in GSH levels caused a moderate effect on the differentiated function of insulin-secreting cells.


Characterization of a nitric-oxide-catalysed ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase. Auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GraPDH) has recently been demonstrated to be dramatically stimulated in the presence of nitric oxide. In order to obtain insight into the sequence of events leading to ADP-ribosylation of GraPDH, we studied the target amino acid, the nucleotide cofactor requirement, pH dependency and the stoichiometry of the reaction. Basal as well as stimulated ADP-ribose transfer is inhibited by the SH-group alkylating reagent, N-ethylmaleimide. Furthermore, the radiolabel of auto-[32P]ADP-ribosylated GraPDH is removed by treatment with HgCl2, suggesting an ADP-ribose-cysteine bond. Several indirect and direct mechanistic considerations point to NAD+ as the only cofactor for the ADP-ribosylation reaction, excluding the possibility of a reaction sequence involving a NAD-glycohydrolase(s) followed by nonenzymatic ADP-ribose transfer to GraPDH. Optimal ADP-ribosylations were carried out at alkaline pH values using 10 microM free NAD+ as the sole nucleotide cofactor. Bovine serum albumin with an S-nitrosylated SH group can serve as a model of ADP-ribose transfer from NAD+ and suggests that the nitric-oxide-modified SH group (S-nitrosylated SH group) is a prerequisite for the reaction.


The use of alternative substrates in the characterization of actin-methylating and carnosine-methylating enzymes. Actin isolated from nearly every eukaryotic species contains approximately 1 mol 3-methylhistidine/mol protein. His73 in actin has been shown, by protein sequencing, to be the site of methylation. The methylation occurs enzymically and post-translationally. A rabbit skeletal muscle myofibrillary fraction has previously been shown to contain a histidine methyltransferase activity that is actin specific. Detailed study of this enzyme has been hampered by lack of a suitable substrate assay. Naturally occurring actins are poor substrates for the enzyme, presumably due to prexistent methylation at His73. In this study, two potential alternative substrates have been investigated. These are a chicken beta-actin expressed in Escherichia coli as a fusion protein with 80 amino acids of an influenza protein, NS1, and a synthetic peptide, Tyr-Pro-Ile-Glu-His-Gly-Ile-Ile-Thr, corresponding to residues 69-77 of actin. Both substrates were covalently methylated at histidine residues in the presence of S-adenosylmethionine and partially purified enzyme fractions from rabbit muscle. In methylation experiments employing the fusion actin in the form of inclusion bodies, 3-methylhistidine is the major product, as is the case when soluble muscle or non-muscle actin is used. However, for the synthetic peptide, the methylated product primarily contained 1-methylhistidine and only a small amount of the isomeric 3-methylhistidine. Further investigations revealed that the peptide was recognized by carnosine N-methyltransferase, another histidine methyltransferase found in muscle tissue. Carnosine N-methyltransferase appears to copurify with the actin-methylating enzyme in preliminary fractionation experiments. Separation of the two methyltransferase activities is described.


The human rhabdomyosarcoma cell line A204 lays down a highly insoluble matrix composed mainly of alpha 1 type-XI and alpha 2 type-V collagen chains. The biosynthesis of collagen by the A204 cell line was examined using polyclonal antibodies raised against collagen type V and type XI. The study of the pepsin-digested collagen showed that it is composed mainly of alpha 1(XI) and alpha 2(V) collagen chains in an apparent 2:1 ratio, suggesting the formation of heterotypic molecules [alpha 1(XI)]2 alpha 2(V). The existence of this chain stoichiometry was further demonstrated by immunoprecipitation of the molecule with an antibody recognizing alpha 2(V) but not alpha 1(XI) collagen chains. Electron microscopy analyses of 24-h cultures showed that this matrix is composed of thin fibrils, that can be decorated with immunogold-labelled anti-(type-V collagen) IgG, but not with anti-(type-XI collagen) IgG. The collagen matrix laid down by A204 cells is highly insoluble. In the presence of beta-aminopropionitrile, an inhibitor of lysyl oxidase, only a small proportion of intact collagen could be extracted without proteolytic treatment. Immunoblotting of intact medium collagen from cultures performed in the presence of beta-aminopropionitrile showed four distinct bands with each antibody. The migration of the bands, stained with anti-(type-V collagen) IgG, had apparent molecular masses of 127, 149, 161 and 198 kDa (compared to globular standards) while the bands stained with anti-(type-XI collagen) IgG had apparent masses of 145, 182, 207 and 225 kDa. These data indicate that type-V and type-XI collagen chains can assemble in heterotypic isoforms. In this system, the synthesized isoforms are able to aggregate into a highly cohesive matrix and they undergo a proteolytic processing closely similar to that of other fibrillar collagens.


The Soret magnetic circular dichroism of ferric high-spin myoglobins. A probe for the distal histidine residue. To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.


Purification of assembly-competent tubulin from Saccharomyces cerevisiae. We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.


Nuclear substrates of protein kinase C.

Nuclear substrates of protein kinase C. Starting from the finding that, for neuronal cells, the nuclear-membrane-associated protein kinase C (PKC) is the so-called 'membrane inserted', constitutively active form, we attempted to identify substrates of this nuclear PKC. For this purpose, nuclear membranes and other subcellular fractions were prepared from bovine brain, and in-vitro phosphorylation was performed. Several nuclear membrane proteins were found, the phosphorylation of which was inhibited by specific PKC inhibitors and effectively catalyzed by added PKC. Combining the methods of two-dimensional gel electrophoresis, in-situ digestion, reverse-phase HPLC and microsequencing, two of these nuclear PKC substrates were identified; the known PKC substrate Lamin B2, which serves as a control of the approach and the nucleolar protein B23. Our data suggest, that, for B23, Ser225 is a site of phosphorylation by PKC.


The effect of metal binding on the structure of annexin V and implications for membrane binding. The structure of annexin V, crystallised in the presence of two calcium or barium ions for each protein molecule, was solved by molecular replacement to 0.24 nm resolution. The two metal ions are found in domains I and IV, i.e. on the same side of the channel that lies in the centre of the molecule. The structures of the barium and calcium form are extremely close, the only differences localised in the metal-binding sites that lie on the surface of the molecule. The occupancies of the metal ions, however, are lower for barium than for calcium, expressing the lower affinity of the protein for the former. The packing of the annexin molecules in the crystal asymmetric unit may represent a model for the calcium driven association of membrane-bound annexins that leads to membrane fusion.


Purification and characterization of rat brain cytosolic 3,5,3'-triiodo-L-thyronine-binding protein. Evidence for binding activity dependent on NADPH, NADP and thioredoxin. A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3-Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a Ka of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 microM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the Ka was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.


Coordination and internal exchange of two DNA molecules in a RecA filament in the presence of hydrolysing ATP. Information on ATP-RecA-DNA structure from linear dichroism spectroscopy. Solution structure of complexes between DNA and recombinase RecA from Escherchia coli, in the presence of the physiological cofactor ATP, is probed by flow linear dichroism (LD) spectroscopy. A problem of ADP accumulation which promotes dissociation of DNA-RecA is circumvented by using an ATP-regenerating system. The LD features indicate that the local structure of the complex is very similar to that found in the presence of the non-hydrolysable analog of ATP, adenosine-5'-O-[gamma-thio]triphosphate (ATP[gamma S]); the DNA bases are oriented with their planes preferentially perpendicular to the long axis of the filament, while the indole chromophores of the two tryptophan residues of RecA are rather parallel to this reference direction. A much smaller overall amplitude of the LD spectrum, compared to ATP[gamma S], is interpreted as a result of fast dissociation of RecA due to hydrolysis of ATP, producing transiently naked DNA regions which act like flexible joints, diminishing the macroscopic orientation of the RecA filaments. However, the ATP hydrolysis is not found to prevent simultaneous accommodation of two non-complementary DNA molecules in the RecA complex, as judged from the LD behaviour upon successive addition of two different polynucleotides or modified DNA strands. A notable difference from corresponding complexes formed with ATP[gamma S] is that, in the presence of ATP hydrolysis, the order in which the two DNA molecules have been added is insignificant as judged from virtually identical resulting structures; this observation indicates that exchange of DNA occurs between the two DNA accommodation sites within the RecA filament.


Improvement in the heterogeneous N-termini and the defective N-glycosylation of human interleukin-6 by genetic engineering. Recombinant human interleukin-6 (IL-6), expressed in Chinese hamster ovary cells, has heterogeneous N-termini of Ala1 and Val3, as does naturally occurring IL-6. This heterogeneity is thought to be caused by difficulty in cleavage of the signal sequence. To obtain homogeneous IL-6, Pro at -1 was exchanged for Ala by site-directed mutagenesis. Alternatively, the signal sequence was replaced with that of human granulocyte-colony-stimulating factor. In both cases, the IL-6 designed to start with Ala1 was still heterogeneous, while the IL-6 designed to start with Val3 showed a homogeneous N-terminus. It is suggested that the heterogeneity of the N-terminus is caused not only by the signal sequence, but also by the succeeding sequences of the mature protein. Only a portion of recombinant human IL-6 is N-glycosylated. Asn46, being exchanged for Gln by site-directed mutagenesis, was confirmed to be partially N-glycosylated. The defective N-glycosylation was assumed to be caused by interference or tension from a disulfide bond near the N-glycosylation site. To verify this hypothesis, the Cys45 and Cys51 forming the disulfide bond were exchanged for Ser. The N-glycosylated species became predominant upon this substitution, suggesting that formation of the disulfide bond is a cause of the defective N-glycosylation.


Aminopeptidase P from human leukocytes.

Aminopeptidase P from human leukocytes. Cytosolic aminopeptidase P was obtained in highly purified form from human leukocytes by a four-step procedure. Buffy coats were the starting material. A M(r) of 140,000 was obtained by size-exclusion HPLC for the native enzyme. As shown by SDS/PAGE under reducing and denaturing conditions, the enzyme consisted of likely identical subunits with M(r) of 71,000. Purified aminopeptidase P cleaved off, specifically and efficiently, the N-terminal residues from peptides with N-terminal Xaa-Pro sequences. The penultimate proline was not replaceable by hydroxyproline, alanine and glycine in di-, tri- and tetrapeptides. Polyproline was not hydrolyzed. Dipeptides were cleaved (Arg-Pro, Phe-Pro > Trp-Pro > Pro-Pro) although slower than longer peptides. Cleavage was observed of several biologically active peptides; C-terminal fragment (residues 201-206) of C-reactive protein, oxytocin fragment Tyr-Pro-Leu-Gly, morphiceptin, peptide Gly-Pro-Arg-Pro (inhibitor of fibrin polymerization) and kentsin. In addition, cleavage of a protein, interleukin-6, was also demonstrated. Aminopeptidase P was maximally activated by Mn2+, and to a lesser extent by Co2+. The activity was optimal at pH 8. Ni2+, Zn2+ and especially Cd2+ caused marked inhibition. EDTA, 1,10-phenantroline and dithiothreitol were also inhibitory. Carbobenzoxy-phenylalanine, as well as several N-carbobenzoxy-proline-containing peptides, caused partial inhibition. The observed resistance of Gly-Pro, Pro-Gly, Pro-Phe and Pro-Ile to hydrolysis by the purified enzyme strongly indicates absence of known proline-specific dipeptidases in the aminopeptidase-P preparation.


Factorial invariance issues in the study of adult personality: an example using Levenson's locus of control scale. Confirmatory factor analysis was used to examine factorial invariance across young, middle-aged, and elderly age groups, using Levenson's multidimensional locus of control (LoC) scale, which measures beliefs in Internal Control (I), Control by Powerful Others (P), and Chinese (C). Data were obtained from 563 individuals ranging in age from 25 years to 75 years of age who resided in Southeastern Louisiana. Results indicated that Levenson's 3-factor conceptualization of control was not a valid representation of the samples' responses. A model that specified the elimination of 17 unreliable items and the formation of both an internal and an external control factor that was based on the seven remaining I and P items provided an adequate fit to the data for the three age groups, though when additional constraints were specified, factorial invariance was not demonstrated. The 7-item I and P factor model generated a pattern of relationships with other measures which was similar to the pattern found if all items of the I and P scales were used.


An adult developmental study of the encoding of spatial location. The present study was designed to test the hypothesis that there is a differential deficit in the ability to encode contextual information with increasing age. Young, middle-aged, and elderly adults were shown target words in various quadrants of a computer screen (contexts) and were told to either (a) remember the words and their locations, (b) remember the words, or (c) tell whether the words referred to something that was alive or not. Following presentation of the words, subjects were given a recognition test for the words and were asked to identify the quadrant in which each word had been presented. If older adults have a contextual encoding deficit, than an interaction between age and instruction condition would be expected in memory for quadrants. Older adults would be expected to perform better relative to younger adults when the locations were target information (intentionally learned) than when they were contextual (not intentionally learned). Since such an interaction was not obtained, the results provide no support for the hypothesis that the elderly have an encoding deficit that is specific to contextual information.


Self-judged comprehension in adults: effects of age and skill. We used an error detection paradigm to examine the effects of skill on young (M = 23.74) and old (M = 68.87) adults' self-judged comprehension of texts. Skill was defined as the ability to evaluate comprehension by detecting text problems. Multiple regression results indicated that problematic texts produced lower comprehension judgments for older readers, regardless of level of skill, than for younger readers. Only more skilled younger readers tended to lower their comprehension judgments for problematic text. Younger adults who were less skilled at evaluating their comprehension assessed their comprehension as superior to their more skilled peers. Less skilled older adults appear to know, better than less skilled younger adults, when they do not comprehend a text. Research is needed to examine older adults' perceived self-efficacy and use of regulation strategies to resolve comprehension failures.


Age-related differences in adults' macrospatial cognitive processes. Young and elderly adults' performance was compared on the Landmark Selection Task, designed to assess perceptual selection, and the Scrambled Route Task, designed to assess temporospatial integration. Age-related performance decrements were found on both tasks. Subjects' scores on psychometric tests hypothesized as involving some of the same processes as these experimental tasks yielded positive correlations to measures of task performance. Unexpectedly, self-estimates of wayfinding and distance estimation skills were negatively correlated to experimental task performance for elderly adults. Results were discussed in the context of declines in the effectiveness of selective attention, which is considered critical to perceptual selection, and in the proficiency of working memory, which is considered central to temporospatial integration.


Variation in external context and adult age differences in action memory. Adult age differences were determined for the memory and retention of the content of actions. Variation in the physical context from the time of performance of the actions to the time of testing for their memory had no effect for either young or elderly adult subjects on the recall or the recognition of the actions either shortly after acquisition or 24 hours later. Both young and elderly subjects showed significant forgetting of actions over a 24-hour retention interval as measured by recall.


Continuously distributed random variables in factorial designs. Age as a variable in lifespan research usually is sampled as several age blocks which, in turn, are combined with additional variables in a factorial design. Sampling a continuous variable in discrete blocks increases the difficulty in obtaining adequate sampling, reduces power, and prevents a fine grain analysis of age x treatment interactions. Age can be sampled as a continuously distributed variable, factorially combined with treatment groups, and analyzed as an analysis of variance by the use of regression analysis and comparison of multiple R2 coefficients. The advantages of such a sampling strategy include both practical sampling advantages as well as statistical advantages when compared with the usual sampling approach.


Adult age differences in the storage of information in working memory. The performance of 97 young and 91 old persons were compared to determine if a deficiency in working memory resources for processing, storage, or allocation could be detected. Persons simultaneously performed a storage and one of two processing tasks while instructed to allocate resources to processing, storage, or both tasks. The storage task involved remembering the names of one, three, or five persons. Processing tasks involved solving addition problems presented on flashcards or answering common knowledge questions. Results showed increased age differences on the storage task as demands for resources increased but no differences on processing tasks. Individuals seemed unable to allocate resources as instructed. A comparison of young-old and old-old groups showed the same results as those obtained comparing young and old groups and support the hypothesis of a deficiency of storage, but not processing, resources in working memory for old, especially old-old, adults.


In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up-regulation of major histocompatibility complex class I molecules by short synthetic peptides. Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC-I). Externally added peptides can sensitize target cells by binding directly to MHC-I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC-I interactions can also be reflected as up-regulation of MHC-I in vitro on certain cells. We have compared the capacity of Db, Kb- and Ld-binding peptides, which are slightly different in length, to up-regulate MHC-I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9-mer Db-binding peptide by adding cystein to the amino terminus and lysine to the amino- or carboxy terminus and studied the effects on MHC-I up-regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen-binding groove of Db. These small modifications of the optimal 9-mer peptide strongly influenced their functions but still there was a correlation between MHC-I up-regulation and CTL responses. Up-regulation of MHC-I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen-presenting cells in vivo.


Immunomodulation of streptococcal cell wall-induced arthritis. Identification of inflammatory cells and regulatory T cell subsets by mercuric chloride and in vivo CD8 depletion. Streptococcal cell wall (SCW)-induced arthritis is a chronic, erosive polyarthritis which can be induced in susceptible Lewis rats by one intraperitoneal injection of a sterile, aqueous suspension of SCW. The chronic phase of the disease is dependent on T cells. Mercuric chloride is an immunomodulating agent, causing autoimmunity in BN rats, but an OX8+ cell-mediated immunosuppression in Lewis rats. Therefore, we investigated the effect of mercuric chloride, whether or not combined with in vivo OX8 depletion, on SCW-induced arthritis in Lewis rats. We show that (a) depletion of OX8+ cells leads to a more chronic arthritis with a more rapid onset, (b) treatment with mercuric chloride induces a rapidly developing disease which is not chronic, and (c) treatment with mercuric chloride and OX8+ cell depletion induces an arthritis with a very rapid onset and enhanced chronicity. Together with histological data this suggests an important role for OX8+ T cells in controlling both the acute and chronic phase of the disease. In addition, mercuric chloride seems to induce an early activation of T cells resulting in an enhanced onset of disease, which is controlled later by enhanced activation of OX8+ cells.


Age differences in sensitivity, response bias, and reaction time on a visual discrimination task. Twelve young adults (age 21-25 years) and twelve older adults (age 57-65 years) performed a visual discrimination task (parallel lines) in which the following factors were manipulated: the difference in the length of the lines (3% and 9%), signal exposure duration (150, 750, and 2000 ms), and presentation modality (simultaneous and successive). The effect of repeating the task over three blocks was also controlled. Reaction time, sensitivity (d'), the decision criterion (beta), and the level of confidence the subjects had in their responses were measured and analyzed. The reaction time of the older subjects was longer than for younger ones. The difference remained constant across conditions. Signal detection analysis indicated that the ability to discriminate, as measured by d', was the same, on the whole, in the two age groups, but the sensitivity of the older was more affected by signal exposure duration than that of their younger counterparts. Contrary to what might have been expected, the older subjects used a more risky decision criterion than the younger subjects (preferring false alarms to omissions) and did not have significantly less confidence in their responses. Task repetition led to reduced reaction times and to a slightly higher level of confidence, but no age-related effects were found. The findings suggest that, in addition to a perceptual compensatory component, the longer reaction times of the older subjects include a decision-making component not necessary for their level of discrimination accuracy. The outcome of a lower criterion in the older group supports other findings according to which a more conservative decision strategy is not a general, systematic feature of the aged.


Age cohort differences in the ability to perform closure on degraded figures. Young (17-26) and old (60-80) men and women performed a perceptual closure task for degraded line drawings under three conditions of prior picture knowledge (exact, similar, no prior knowledge) and two conditions of perceptual noise (contour or detail drawings) resulting in six levels of task difficulty. Young and old subjects took equal advantage prior knowledge conditions, however, old subjects required a greater percentage of picture and more time to make closure under all conditions when compared to young subjects. To test the perceptual slowing hypothesis, old subject performance was regressed on that of young across three levels of task difficulty. The slope of these regression equations supported the notion of a perceptual slowing hypothesis to explain age cohort differences.


Interferon-alpha prevents endotoxin-induced mortality in mice. Endotoxins, the lipopolysaccharide (LPS) moieties on the bacterial cell wall, cause many of the pathological features of Gram-negative septicemia. Tumor necrosis factor (TNF), primarily a product of monocyte/macrophages, has been shown to mediate many of the pathophysiological effects of endotoxin. Kupffer cells, the largest macrophage population in the body, release TNF when stimulated by LPS in vitro. A recombinant human hybrid interferon-alpha A/D (rIFN-alpha) markedly inhibited this LPS-elicited TNF production by Kupffer cells. The effects of rIFN-alpha were further tested in C57BL/6 mice receiving a lethal dose (400 micrograms/mouse) of LPS. All LPS-treated mice died within 2 days. Pretreatment with rIFN-alpha 1 h before LPS challenge improved the survival at 3 days to 22% (5/23, p < 0.04). In contrast, rIFN-alpha was more effective when administered 20 min after LPS injection, increasing the survival rate to 81% (13/16, p < 0.0001). TNF mRNA expression in the liver and spleen 50 min after LPS challenge, and plasma TNF 1.5 h after LPS were also reduced by either pretreatment or post-treatment with rIFN-alpha. Subsequently, experiments were carried out to test the efficacy of delayed rIFN-alpha treatment. A significant protective effect was still apparent when rIFN-alpha was administered 6, 10 and even 14 h (81%, 62% and 28% survival, respectively) after LPS challenge when serum TNF levels had already returned to near baseline. These experimental results suggest that rIFN-alpha might have a therapeutic potential for the prevention and treatment of the deleterious effects associated with endotoxemia besides mechanisms initially blocking TNF production.


Functional role of alpha 2/beta 1 and alpha 4/beta 1 integrins in leukocyte intercellular adhesion induced through the common beta 1 subunit. Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the alpha 4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein beta 1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-beta 1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common beta 1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-beta 1 mAb. Induction of cell aggregation by anti-beta 1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to alpha 4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA alpha 4/beta 1 heterodimer, intercellular adhesion induced through the VLA-beta 1 chain could be selectively inhibited by other anti-beta 1 mAb as well as by anti-alpha 4 mAb. Interestingly, anti-beta 1 mAb which induced strong aggregation of VLA-alpha 2- or VLA-alpha 4-transfected K562 cells, had minimal effect on the alpha 2- alpha 4- alpha 5+ K562 cell line. Furthermore, the beta 1-mediated induction of cell aggregation on alpha 2-K562- and alpha 4-K562-transfected cells was blocked by preincubation with either anti-alpha 2 or anti-alpha 4 mAb, respectively, as well as by other anti-beta 1 mAb. Interestingly, parental K562 cells were able to interact with both alpha 2- and alpha 4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of alpha 2/beta 1 and alpha 4/beta 1 heterodimers in intercellular interactions and underline the pivotal role of the common beta 1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.


Binding of T cell receptor to major histocompatibility complex class II-peptide complexes at the single-cell level results in the induction of antigen unresponsiveness (anergy). Using dispersion cultures performed in semi-solid medium we demonstrate here that the interaction of T cell antigen receptor molecules with Ia-peptide complexes on the same cell surface results in T cell activation, without the production of lymphokines. This recognition of antigen at the single-cell level, induced a state of anergy which was not due to a decrease of surface T cell antigen receptor/CD3 complexes. The induction of anergy by peptide could not be prevented by the addition of various co-stimulatory signals, including antibodies to CD28, or CD2, high doses of interleukin-2 or phorbol ester.


Aging, narrative organization, presentation mode, and referent choice strategies. We examined age differences in referent choice strategies when narrative organization and presentation mode taxed working memory. Readers could use the following cues to choose referents for an ambiguous pronoun: (a) thematic cue (main/minor character), (b) foreground cue (character in main/subordinate clause), and (c) recency of mention of character. Subjects read short narratives with a critical sentence that mentioned the main character in the main clause of the critical sentence (congruent condition) or in the subordinate clause of this sentence (incongruent condition). They chose the main or minor character as referent for a pronoun in the next (target) sentence. Experiment 1 examined how older and young readers use these cues with the Memory narrative presentation (target sentence presented on a separate page from the rest of the narrative). Young readers used similar strategies as those in Morrow (1985), where narrative presentation did not tax memory. Thus, the Memory presentation had little influence on their strategies. However, older readers were less consistent and tended to split in choosing the two characters. In Experiment 2, where narrative presentation did not tax working memory, older readers used similar strategies to the young readers in Experiment 1. Experiment 3 found that elders reading narratives with the Memory presentation recalled the nonpreferred character name after the narrative, suggesting that both characters were accessible from working memory. The study suggests that the Memory presentation mode in Experiment 1 interfered with elders' consistent use of narrative cues in choosing referents, especially when these cues conflict.


Vaccination or tolerance to prevent diabetes. Experiments with transgenic mice expressing the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) under the control of the rat insulin promoter (RIP) have demonstrated that potentially self-reactive T cells that normally ignore self peptides may nevertheless be induced by self peptides or "cross-reactive" foreign (e.g. viral) peptides that arise in the host in an immunogenic form; once activated these potentially self-reactive T cells may cause autoaggressive diseases (e.g. diabetes). The possibility of vaccinating against such T cell-mediated immunopathological diseases was evaluated in the RIP-GP transgenic mouse line Bln. Any attempt to vaccinate with the self antigen itself (e.g. recombinant vaccinia virus expressing LCMV-GP) failed to protect mice from disease. However, immunization with a recombinant vaccinia virus expressing LCMV-nucleoprotein (vacc-NP) as a non-GP LCMV vaccine was able to modulate the immune response and prevented autoaggressive disease in a MHC-dependent fashion. In contrast, tolerance induction neonatally or, more generally applicable, by lethal irradiation and reconstitution with neo-self antigen-expressing bone marrow cells always resulted in prevention of virally induced diabetes in this model situation.


Immunoglobulin kappa light chain germ-line transcripts in human precursor B lymphocytes. B lymphoblastoid cell lines (BLCL), established from bone marrow and peripheral blood mononuclear cells from two severe combined immunodeficiency (SCID) patients, manifested a complete absence of genomic rearrangements of the immunoglobulin (Ig) heavy (H) and light (L) chain loci. The BLCL contained germ-line transcripts of the Ig kappa region locus of approximately 1.2 kilobase (kb). By cDNA cloning and sequence analysis the transcripts were shown to consist of a C kappa segment, a J kappa 1 gene segment, 160 base pairs (bp) of J kappa 1 5' intervening sequence, containing the heptamer/nonamer recombination recognition sequences and at the 5' end a 523-bp segment designated human kappa zero, The first 206 bp of this 5' segment were homologous to the reported murine kappa zero region. Genomic restriction mapping and DNA sequence analysis demonstrated that the human kappa zero segment is located approximately 4 kb upstream of J kappa 1. The kappa zero segment contains a putative promoter region with an OCT2 binding site, and has a splice donor site to accomplish splicing to an acceptor site 160 bp upstream of J kappa 1. Expression of the kappa zero gene segment was found in BLCL derived from normal fetal bone marrow, in which both Ig kappa loci were in the germ-line configuration. These findings indicate that the described transcripts are not only present in SCID, but also in normal developing pre-B lymphocytes. The expression of germ-line Ig kappa L chain transcripts may be associated with the locus becoming accessible to gene rearrangement.


Modulation of oral tolerance to ovalbumin by cholera toxin and its B subunit. Oral administration to mice of ovalbumin (OVA), if given together with cholera toxin (CT) or its B subunit (CTB) prevented the hyporesponsiveness to OVA subsequently injected parenterally. Oral immunization with CT plus OVA or OVA plus CTB in fact primed the immune system, inducing a stronger response to a subsequent parenteral injection of OVA with complete Freund's adjuvant than in mice prefed only with OVA or with saline. Oral CT plus OVA also induced good serum IgG1 and IgA anti-OVA responses, with slightly (not significant) decreased IgG2a and IgG2b responses. Our in vivo findings agree well with earlier in vitro data from others, including CT inhibition of the Th1 CD4+ T cell subset and with CT effect on B cells (induction of LPS-stimulated IgM+ B cells to undergo increased switch differentiation to IgG1- and IgA-secreting cells).


Induction of primary anti-viral cytotoxic T cells by in vitro stimulation with short synthetic peptide and interleukin-7. The present study investigated whether a short synthetic peptide NPP, with a modified sequence (147-158 R156-) derived from influenza A virus nucleoprotein with high affinity for Kd major histocompatibility complex class I molecules, could induce primary influenza virus-specific cytotoxic T (Tc) cells in vitro. Naive BALB/c (H-2d) splenocytes did not respond to the stimulation with only NPP with the generation of effector Tc cells specific for influenza A virus-infected target cells in vitro. However, they were able to do so if cultured with NPP in the presence of IL-7. IL-7 activity in this system differed significantly from IL-2 activity in the specificity of the effect. The use of exogenous IL-2, instead of IL-7, with NPP resulted in the induction of lytic cells that lysed both influenza virus-infected and uninfected syngeneic target cells. These results suggest that IL-7 is a potent regulatory cytokine in the antigen-specific activation of resting naive Tc cell precursors and may provide the necessary conditions for the induction of human primary Tc cells in vitro.


Object recognition by component features: are there age differences. This study extended aspects of Biederman's (1987) recognition-by-components (RBC) theory to the analysis of age differences in the recognition of incomplete visually-presented objects. RBC theory predicts that objects are recognizable or recoverable under conditions of fragmentation if a sufficient amount of essential structural information remains available. Objects are rendered nonrecoverable by the omission or obstruction of essential structural features at vertices and areas of concavity. Fifteen young adults and 15 older adults participated in a study of the effects of amount (25%, 45%, 65%) and type of fragmentation (recoverable, nonrecoverable) on object naming. Age-related declines in recognizing incomplete objects were associated with the amount of fragmentation, but type of fragmentation did not affect the performance of older adults. For the young adults, accuracy of performance was affected by both amount and type of fragmentation, consistent with Biederman's RBC theory. These results were interpreted as suggesting that age-related declines in perceptual closure performance have to do with non-structural factors such as the ability to inferentially augment degraded or missing visual information.


Peritoneal exudate lymphocyte and mixed lymphocyte culture hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin. We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.


Cocaine and dopamine differentially protect [3H]mazindol binding sites from alkylation by N-ethylmaleimide. The binding of cocaine, d-amphetamine and dopamine to the site on the dopamine transporter labeled by [3H]mazindol was investigated in rat striatal membranes. N-Ethylmaleimide inhibited about 95% of the specific binding of 5 nM [3H]mazindol in a concentration-dependent manner. The effect of 10 mM N-ethylmaleimide was completely prevented by cocaine (EC50 of 3 microM), but neither 300 microM dopamine nor d-amphetamine afforded any significant protection. On the other hand, high concentrations of cocaine, d-amphetamine and dopamine provided similar protection against inhibition by 0.1 mM N-ethylmaleimide. Taken together these data support the hypothesis that a significant portion of the cocaine binding domain on the transporter is distinct from that of either dopamine or amphetamine. This distinction may be sufficient to allow properly designed drugs to prevent cocaine binding without inhibiting DA transport.


Evidence for mutually exclusive binding of cocaine, BTCP, GBR 12935, and dopamine to the dopamine transporter. The present study addressed the possibility that there are distinct but allosterically interacting populations of binding sites for dopamine/cocaine and BTCP/GBR (N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine/1-(2-diphenylmethox y) - ethyl]-4-(3-phenylpropyl)piperazine) (selective dopamine uptake blockers) on the dopamine transporter in the rat striatum. Dopamine uptake sites were labeled in vitro with the cocaine analog [3H]CFT (2 beta-carbomethoxy-3 beta-(4-fluorophenyl)-tropane), and the inhibition of binding by CFT or cocaine was measured. A graphic method was adopted for studying shifts in inhibitory potency resulting from the addition of a second compound. Under the conditions used, the co-presence of dopamine, GBR 12935, or BTCP decreased the inhibitory potency of CFT or cocaine to the extent predicted by a model in which all compounds bind to the same site or the binding of all compounds is mutually exclusive. No evidence for negative allosteric interactions between CFT and BTCP was found in experiments comparing inhibition of [3H]CFT binding by BTCP at a low and high concentration of [3H]CFT.


The positive inotropic calcium sensitizer EMD 53998 antagonizes phosphate action on cross-bridges in cardiac skinned fibers. The diazinone derivative EMD 53998 sensitizes skinned myocardial fibers to Ca2+ and enhances maximal calcium-activated force (pCa = 4.5) by approximately 100%; the EC50 is 10 microM in the absence and about 30 microM in the presence of added inorganic phosphate (10 mM). Although concentrations of added phosphate as low as 0.5 mM inhibit force, at high concentrations of EMD 53998 (> or = 50 microM), phosphate only inhibits at concentrations exceeding 20 mM. These data suggest that the effects of EMD 53998 and phosphate are mutually antagonistic. Importantly, both EMD 53998 and phosphate had similar effects on force generation in troponin I-depleted (Ca(2+)-independent) skinned fibers, thus demonstrating that these compounds are likely to affect cross-bridges directly and not via the Ca(2+)-regulatory system.


High basal expression of zif268 in cortex is dependent on intact noradrenergic system. The transcription factor Zif268 displays high basal levels of expression in cortex that appear to be dependent on physiological synaptic activity. We report that selective lesions of the noradrenergic system induced by DSP4 markedly suppress basal zif268 mRNA levels in cortex. Accordingly, the noradrenergic system which projects extensively to the cortex and is tonically active may play a key role in maintaining normal patterns of gene expression in target neurons.


Antisense oligonucleotide eliminates in vivo expression of c-fos in mammalian brain. Immediate-early genes such as c-fos and NGFI-A are rapidly and transiently expressed in the striatum following amphetamine administration in vivo. Here we show that direct infusion of an antisense oligodeoxynucleotide to c-fos into striatum will reduce amphetamine-induced production of Fos-like immunoreactivity without affecting NGFI-A expression. These results suggest that it is possible to use antisense technology to study the role of immediate-early genes in specific sites in the brain in vivo.


Immunological and neurobiochemical alterations induced by repeated oral exposure of phenol in mice. Phenol, a major metabolite of benzene, is a potentially immunotoxic and neurotoxic substance of environmental significance. Male CD-1 mice were continuously exposed to 0, 4.7, 19.5, and 95.2 mg phenol/l in drinking water for 4 weeks. Various immune functions were evaluated and levels of selected neurotransmitters and metabolites measured in discrete brain regions. The doses of phenol did not produce any overt clinical signs of toxicity; peripheral red blood cell counts and hematocrits decreased. A dose of 95.2 mg/l suppressed the stimulation of cultured splenic lymphocytes by lipopolysaccharide, pokeweed mitogen, and phytohemagglutinin and the response in mixed lymphocyte cultures. The two high doses suppressed antibody production response to the T cell-dependent antigen (sheep erythrocytes), as determined by plaque-forming cells, and serum antibody levels. Mice treated with phenol had lower levels of neurotransmitters in several brain regions. In the hypothalamus, a major norepinephrine-containing compartment, the concentrations of norepinephrine significantly decreased by 29 and 40% in groups dosed with 19.5 and 95.2 mg/l, while dopamine concentrations decreased in the corpus striatum by 21, 26, and 35% at 4.7, 19.5 and 95.2 mg/l, respectively. Phenol also decreased 5-hydroxytryptamine in the hypothalamus, medulla oblongata, midbrain and corpus striatum. Levels of monoamine metabolites decreased in the hypothalamus (5-hydroxyindoleacetic acid), midbrain (vanillylmandelic acid), corpus striatum (vanillylmandelic acid and dihydroxyphenylacetic acid), cortex (vanillylmandelic acid), and cerebellum (dihydroxyphenylacetic acid).


Effects of cholesterol and oxysterols on gap junctional communication between human smooth muscle cells. Intercellular communication is considered to play an essential role in maintaining and controlling cell growth, cell differentiation and homeostasis. Cell-cell communication can be regulated by factors that influence gap junctional function. In this study it was demonstrated that cholesterol and oxidized cholesterol have the potential to modulate gap junctional communication between human smooth muscle cells in an opposite way. Cholesterol supplementation to human smooth muscle cells resulted in an increase of gap junctional communication up to 130% with regard to the control values. However, autooxidized cholesterol inhibited gap junctional communication more than 40%. Testing of several pure cholesterol oxidation derivates on gap junctional communication demonstrated that all of them were capable to inhibit intercellular communication in the order 25-hydroxycholesterol greater than cholestan-3 beta,5 alpha,6 beta-triol greater than 7-ketocholesterol greater than cholesterol 5,6 alpha-epoxide. The cell-cell communication-inhibiting potency of these oxysterols is in accordance with their atherogenic potency. This implies that cholesterol oxidation products, instead of pure cholesterol, can be promoting factors in the atherogenesis by influencing gap junctional communication between arterial smooth muscle cells, the target cells of atherosclerotic lesions.


Eosinophil peroxidase and 125I- ion binding in guinea-pig trachea. The present study further characterizes ascorbic acid-dependent 125I- ion binding in guinea-pig trachea. Binding of 125I- ion in the presence of ascorbic acid was detected at the epithelial/submucosal interface, apparently involving individual cells containing peroxidase enzyme. A similar binding pattern was observed when hydrogen peroxide was substituted for ascorbic acid. Binding could be inhibited by the addition of the H2O2 degrading enzyme catalase or the peroxidase inhibitor thiourea. Results demonstrated that this binding was dependent upon the addition of, or endogenous production of hydrogen peroxide and its metabolism via airway eosinophil peroxidase. The relevance of this anomalous iodine binding phenomenon to radioiodinated ligand binding studies is discussed.


Pharmacological aspects of arthritis induced by a muramyl dipeptide analogue in rats. Fourteen consecutive daily subcutaneous injections of 4 mg/kg of the muramyl dipeptide analogue MDP-Lys(L18) into rats caused arthritis characterized by swelling of the tarsal joint, increases in lymphocytes and monocytes in the peripheral blood, and elevated serum immunoglobulin G (IgG). The present study was performed to evaluate the effects of indomethacin, phenylbutazone, dexamethasone, D-penicillamine, aurothioglucose, cyclophosphamide and cyclosporin A on this arthritis. Administration of indomethacin, phenylbutazone or dexamethasone inhibited the development of the tarsal joint swelling, suggesting that prostaglandins may be involved in the pathogenesis of the arthritis. Cyclophosphamide reduced the arthritis, together with decreases in the lymphocyte count and the serum IgG level. Cyclosporin A worsened the arthritis in a dose-dependent manner and increased the neutrophil count without raising the serum IgG level, but inhibited the induction of adjuvant arthritis in rats with Mycobacterium bacilli. MDP-Lys(L18) may therefore induce arthritis differing in mechanism from adjuvant arthritis.


Inhalation of carbon monoxide does not accelerate arteriosclerosis in cockerels. The effects of chronic exposure to moderate levels of carbon monoxide (CO) upon the augmentation of arteriosclerotic plaque development were investigated in a series of in vivo studies. Cockerels were exposed to carefully regulated CO levels in dynamic exposure chambers. The plaque volume percentage in the aortic walls of experimental and control animals was determined by point-counting. Chronic CO inhalation, at levels up to 200 ppm, did not stimulate arteriosclerotic plaque development (at 200 ppm CO, carboxyhemoglobin (COHb) levels 10 min after exposures ended were 11-12%). When administered concomitantly with cholesterol feeding, CO did not augment plaque development. When administered after either carcinogen-associated or diet-promoted plaque size increases had occurred, CO elicited no further plaque size increases. Thus, in this animal model, daily exposures to moderately high CO levels were without discernable effect upon arteriosclerotic plaque development, although high COHb levels were attained.


The nitromethylene heterocycle 1-(pyridin-3-yl-methyl)-2-nitromethylene-imidazolidine distinguishes mammalian from insect nicotinic receptor subtypes. Effects of the insecticidal compound 1-(pyridin-3-yl-methyl)-2-nitromethylene-imidazolidine (PMNI) on different subtypes of the nicotinic acetylcholine receptor were studied in voltage-clamped locust thoracic ganglion neurons, mouse BC3H1 muscle cells and mouse N1E-115 neuroblastoma cells. In locust neurons 10 microM PMNI induced agonistic effects and subsequent complete block of 1 mM acetylcholine-induced inward currents. The same concentration of PMNI produced no significant agonistic and antagonistic effects on the endplate type nicotinic receptor in BC3H1 cells. Neuronal type nicotinic receptor operated ion currents in N1E-115 cells were blocked by 10 microM PMNI to 50% of the control value, while agonistic effects were not observed. The differential action of PMNI designates this compound as a potential new tool to distinguish between subtypes of the nicotinic acetylcholine receptor.


Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450IA2. V79 Chinese hamster cells were genetically engineered for stable expression of human P450IA2. Full length cDNA, encoding human P450IA2, was inserted into an SV40 early promoter containing eukaryotic expression vector and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to the neomycin derivative G418) into V79 Chinese hamster cells. The recombinant expression vector was introduced into two different V79 sublines, one expressing an endogenous acetyltransferase (V79-NH), the other not (V79-MZ). The presence of human cytochrome CYP1A2 cDNA in the G418 resistant V79 cell clones was confirmed by Southern blotting. The transcription of the cDNA into mRNA was detected by Northern blotting and the translation into an authentic cytochrome P450IA2 protein was shown by Western blotting. The enzymatic activity in these cells was determined by the cytochrome P450IA2-dependent methoxy-, ethoxy-, benzoxy-, and pentoxyresorufin dealkylation activity.


Mechanisms of thrombocytopenia in patients with lymphoproliferative diseases. Different factors are involved in the development of thrombocytopenia in patients with lymphoproliferative disorders. Significant correlation was detected between the number of megakaryocytes in bone marrow and platelet count (r = 0.485, p = 0.002, n = 37) and significant difference between the number of megakaryocyte in patients with normal platelet count (> 200,000/microliters) and patients with marked thrombocytopenia (platelet count < 100,000/microliters). All patients in the latter group (n = 15) had a relatively low number of megakaryocytes. Low but significant reverse correlation was found between the level of platelet-associated IgG (PA-IgG) and platelet count (r = -0.249, p = 0.024, n = 82) and significant difference between the mean levels of PA-IgG in the groups of patients with platelet count > 200,000/microliters and < 100,000/microliters. PA-IgG were increased in 46% of patients in the total group and in 65% of patients with platelet count < 100,000/microliters. The correlation between platelet count and PA-IgG was about 2 times higher in splenectomized (r = -0.549, p = 0.005, n = 24) than nonsplenectomized patients. All splenectomized patients with platelet count < 100,000/microliters (n = 8) had a significant increase in PA-IgG. Serum antibodies were detected in only 7% of tested patients. This group was characterized by severe thrombocytopenia (in 6 of 10 patients--platelet count < 50,000/microliters) and a high incidence of haemorrhages (in 5 of 10 patients). Thus the depression of platelet production was suggested to be the basic cause of thrombocytopenia in lymphoproliferative disorders. Involvement of immune mechanisms was revealed in a large number of patients and correlated with a deeper and more complicated thrombocytopenia.


Reduced erythropoietin response to anaemia in elderly patients with normocytic anaemia. We studied the effect of age on the relationship between haemoglobin and serum erythropoietin (EPO) levels in anaemic patients. 568 patients over 70 years of age were compared with 137 patients under 70 and a reference group of 144 patients of all ages with proven iron deficiency. EPO was measured using a radioimmunoassay. We found that elderly patients with a normocytic anaemia (N = 375) had a statistically lower EPO response than younger patients with normocytic anaemia (N = 61) (p < 0.05) or patients of all ages with iron-deficiency anaemia (p < 0.05). There was no difference between the sexes. Elderly patients with microcytic or macrocytic anaemia had a normal EPO response as compared to the "gold standard" of iron deficiency. These findings suggest that a proportion of elderly patients with normocytic anaemia has an impaired EPO response.


Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets. There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.


Electrokinetic behaviour and surface sialic acid status of blood platelets in essential thrombocythaemia (ET). We have previously reported, for platelets from patients with idiopathic thrombocytopenic purpura, that there was in increased electrophoretic mobility that was related to their increased sialic acid content. Heterogeneity in the circulating platelet pool of patients with essential thrombocythaemia (platelet counts 600-1000 x 10(9)/l) has also been investigated by preparative continuous flow electrophoresis (CFE). The surface charge-dependent separation profiles of all the patients' platelets showed marked anodal shifts compared with the profiles of platelets from age- and sex-matched control subjects separated under identical conditions. The increase above normal in the net electronegativity of the patients' platelets could not be accounted for by differences in surface neuraminidase-labile or total platelet sialic acid content.


Results in hairy-cell leukemia patients treated with alpha-interferon: predictive prognostic factors. Fourty-four evaluable patients with hairy cell leukemia (HCL) were treated with human lymphoblastoid alpha-interferon (alpha-IFN), at a dose of 3 x 10(6) Units a day for 12-18 months while 18 of them continued to receive a three times per week schedule at the same dose as maintenance treatment. Eighteen percent of patients achieved complete response, 64% partial response, and 18% minor response with a median duration of 37.5, 22.9 and 3.5 months respectively. Twenty patients (45%), all partial or minor responders, subsequently had progression of the disease. The progression occurred more frequently in patients who presented at diagnosis with a hairy-cell index value > 0.50 than in those who presented with a hairy-cell index < 0.50: 14/26 (54%) versus 2/11 (18%) respectively. In addition, the progression rate was more evident in "non-maintained" than in "maintained" patients: 16/26 (61.5%) versus 4/18 (22%). Restarting alpha-IFN treatment in 16 of the 20 progressed patients proved effective only in 9 of them. From these findings it appears that a low hairy-cell index at diagnosis correlates favorably with a good hematological response. Furthermore, continuous therapy with alpha-IFN seems very useful in reducing the progression of the disease, in particular in patients with a very high hairy-cell index at diagnosis.


Treatment of myelodysplastic syndromes with human granulocytic-macrophage colony stimulating factor (GM-CSF) or GM-CSF combined with low-dose cytosine arabinoside. In a phase II study, 21 patients with MDS (RAEB, RAEBt, CMML and RA and RAS with severe cytopenia) were randomized to be treated with 3 courses of GM-CSF (3 micrograms/kg/day s.c.) alone (11 patients) or in combination with AraC (20 mg/m2/d s.c.) (10 patients) for 14-d periods, interrupted by 14-d rest periods. Eight patients discontinued the treatment. In the GM-CSF group a marked increase in WBC and neutrophil counts during each course of treatment administration were seen in most patients. Platelet counts decreased in 14 of 24 courses of treatment in the GM-CSF plus AraC group but in none of the GM-CSF group. Although the changes in the circulating blood cells were transient and the counts tended to return to the pretreatment levels during the rest periods, some more durable effects were seen. In 3/6 patients of the GM-CSF group who completed the designed treatment, both WBC and neutrophils remained elevated above the pretreatment levels throughout the 3-month period of treatment, while in one of them thrombocytopenia improved considerably. In the GM-CSF plus AraC group, 4 out of the 7 patients who completed the treatment showed an improvement of neutropenia as well as anaemia. In these 4 patients the BM percentage of blasts was also decreased. In conclusion, the results of this study indicate that GM-CSF given intermittently improves leukopenia in some patients with MDS. In addition, the administration of GM-CSF seems to prevent granulocytopenia of concurrent AraC treatment and may be of benefit in the treatment of these diseases.


Identification of nuclear factors which interact with the 5' flanking region of the EF-1 alpha O gene in Xenopus laevis. The EF-1 alpha O gene of Xenopus laevis is a stage-specific gene, being transcribed in oogonia and oocytes, but not in postmeiotic germ cells and terminally differentiated cells. We found that two trans-acting factors from oocyte nuclear extract are able to interact with a DNA sequence in the 5'-upstream region of the EF-1 alpha O gene. Methylation interference experiments suggested that the two factors recognised the same DNA element. Gel retardation assays indicated that part of the protein binding site could be confined to a 21 bp sequence, located between -51 and -72, relative to the cap site. Interestingly, this region shares great homology to a negative regulatory segment in the promoter of the TFIIIA gene, another developmentally regulated gene.


Interferon-mediated intracellular signalling. Modulation of different phospholipase activities in Burkitt lymphoma cells. The effect of interferon-alpha on Daudi lymphoma cells either sensitive or resistant to the action of this cytokine has been analysed in terms of phospholipase C (PLC) and D (PLD) activities. Results have shown a combined modulation of PIP2-specific phospholipase C and phospholipase D. In particular, a decreased activity of PIP2-specific PLC has been found, concomitant to a PLD-mediated phosphatidylcholine hydrolysis, suggesting that the intracellular signalling activated by interferon in Daudi cells involves a phospholipase D/phosphohydrolase pathway.


Xenopus laevis oocyte G alpha subunits mRNAs. Detection and quantitation during oogenesis and early embryogenesis by competitive reverse PCR. The expression of mRNAs coding for different Xenopus laevis oocyte G alpha subunits was analyzed by the PCR technique. Using the nucleotide sequences of five previously cloned cDNAs for oocyte G alpha subunits [FEBS Lett. 244, 188-192, 1989; FEBS Lett. 268, 27-31, 1990] and the highly sensitive reverse PCR reaction we found that G alpha o, G alpha i-1, G alpha i-3 and G alpha s species are present in oocyte stage VI, G alpha o mRNA being the most abundant transcript. G alpha o mRNA was further quantitated through oogenesis, unfertilized eggs and early embryogenesis stages by a competitive PCR reaction using an 'in vitro' deleted G alpha o mRNA as the internal standard. Using this approach we found that Xenopus G alpha o mRNA levels were constant during oogenesis and unfertilized eggs at a concentration of 3.5 pg of mRNA/stage (5 x 10(5) molecules) and diminish gradually during early embryogenesis, reaching a level of 0.3 pg in the gastrula stage. These findings show that oocyte G alpha o, and perhaps the rest of the alpha subunits, are expressed as maternal mRNAs and could play an important role in signal transduction at the beginning of oocyte cell differentiation.