High-dose intravenous vitamin C is not associated with an increase of pro-oxidative biomarkers.

OBJECTIVE: High-dose vitamin C therapy might mediate beneficial clinical effects by counteracting reactive oxygen species. However, concerns are raised whether this approach might provoke diametrical (ie pro-oxidative) effects. The objective was to determine ascorbyl free radical (AFR) concentrations and potential variables of pro-oxidative damage. DESIGN: Crossover study; six healthy males received daily infusions of 750 or 7500 mg vitamin C for six consecutive days. Fasting concentrations of vitamin C and AFR were determined daily. On day 1, concentrations of vitamin C and AFR were measured at 0.25, 0.5, 1, 2, 4 and 8 h post infusion. Plasma concentrations of thiobarbituric acid-reactive substances (TBARS), tocopherol and urine concentrations of 8-oxoguanosine were determined on days 1 and 6. RESULTS: Kinetic studies on day 1 showed that concentrations of vitamin C and AFR displayed parallel dose- and time-dependent kinetics and elimination was highly efficient. Vitamin C and AFR fasting concentrations on days 2-6 were slightly above the baseline, suggesting new, stable steady states. TBARS decreased in both groups, whereas tocopherol and 8-oxoguanosine concentrations remained unchanged. CONCLUSION: Kinetics of AFR largely depend on plasma vitamin C concentrations and AFR is eliminated efficiently. Our data do not support induction of pro-oxidative effects in healthy volunteers given intravenous high-dose vitamin C. SPONSORSHIP: Pascoe Pharmazeutische Präparate GmbH, Giessen, Germany.

Electron spin resonance and fluorescence studies of the bound-state conformation of a model protein substrate to the chaperone SecB.

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.

Nucleotide-binding sites in the functional unit of sarcoplasmic reticulum Ca2+-ATPase as studied by photoaffinity spin-labeled 2-N3-SL-ATP.

2-N3-SL-ATP [2-azido-2',3'-O-(1-oxyl-2,2,5,5-tetramethyl-3-carbonyl-pyrroline) adenosine triphosphate], a photoaffinity spin-labeled derivative of ATP with a nitroxide moiety attached to the ribose ring and an azido group attached to C2 of the adenine ring, was used to study the nucleotide-binding site stoichiometry of sarcoplasmic reticulum (SR) Ca2+-ATPase. The label was shown to bind at the catalytic site of the enzyme, even though the rate of hydrolysis was poor. A maximal binding ratio of 1 mol/mol of ATPase was found. The ESR spectra showed signals from spin-spin interactions between two radicals corresponding to a distance of about 15 A between labels bound to adjacent sites on the enzyme. This indicates that the minimal functional unit of the Ca2+-ATPase is a dimer with the nucleotide-binding sites in close proximity.

Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma.

Cigarette smoke is widely believed to increase free radical concentrations causing subsequent oxidative processes that lead to DNA damage and hence, to several diseases including lung cancer and atherosclerosis. Vitamin C is a reducing agent that can terminate free-radical-driven oxidation by being converted to a resonance-stabilized free radical. To investigate whether short-term supplementation with the antioxidants vitamin C and E decreases free-radical-driven oxidation and thus decreases DNA damage in smokers, we determined the frequency of micronuclei in lymphocytes in 24 subjects and monitored the electron paramagnetic resonance signal of ascorbate free radical formation in plasma. Further parameters comprised sister-chromatid exchanges and thiobarbituric acid-reactive substances. Twelve smokers and twelve non-smokers took 1000 mg ascorbic acid daily for 7 days and then 1000 mg ascorbic acid and 335.5 mg RRR-alpha-tocopherol daily for the next 7 days. Baseline concentrations of both vitamins C and E were lower and baseline numbers of micronuclei were higher (p < 0.0001) in smokers than in non-smokers. After 7 days of vitamins C and E, DNA damage as monitored by the number of micronulei was decreased in both, smokers and non-smokers, but it was more decreased in smokers as indicated by fewer micronuclei in peripheral lymphocytes (p < 0.05). Concomitantly, the plasma concentrations of vitamin C (p < 0.001) as well as the ascorbate free radical (p < 0.05) were increased. The corresponding values in non-smokers, however, did not change. Our findings show that increased ascorbate free radical formation in plasma after short-term supplementation with vitamins C and E can decrease the number of micronuclei in blood lymphocytes and thus DNA damage in smokers.

Phosphatidylcholine activation of human heart (R)-3-hydroxybutyrate dehydrogenase mutants lacking active center sulfhydryls: site-directed mutagenesis of a new recombinant fusion protein.

(R)-3-Hydroxybutyrate dehydrogenase (BDH) is a lipid-requiring mitochondrial enzyme with a specific requirement of phosphatidylcholine (PC) for function. A plasmid has been constructed to express human heart (HH) BDH in Escherichia coli as a hexahistidine-tagged fusion protein (HH-Histag-BDH). A rapid two-step affinity purification yields active HH-Histag-BDH (and six mutants) with high specific activity ( approximately 130 micromol of NAD(+) reduced.min(-1).mg(-1)). HH-Histag-BDH has no activity in the absence of phospholipid and exhibits a specific requirement of PC for function. The HH-Histag-BDH-PC complex (and HH-BDH derived therefrom by enterokinase cleavage) has apparent Michaelis constants (K(m) values) for NAD(+), NADH, (R)-3-hydroxybutyrate (HOB), and acetoacetate (AcAc) similar to those for bovine heart or rat liver BDH. A computed structural model of HH-BDH predicts the two active center sulfhydryls to be C69 (near the adenosine moiety of NAD) and C242. With both sulfhydryls derivatized, BDH has minimal activity, but site-directed mutagenesis of C69 and/or C242 now shows that neither of these cysteines is required for PC activation or catalysis (the double mutant, C69A/C242A, is highly active with essentially normal kinetic parameters). Six cysteine mutants each have an increased K(m)(NADH) (2-6-fold) but an unchanged K(m)(NAD)+. The C242S and C69A/C242S enzymes (but not the analogous C242A mutants nor the C69A or C69S mutants) exhibit approximately 10-fold increases in K(m)(HOB) and K(m)(AcAc), reflecting an altered substrate binding site. Thus, although C242 (in the C-terminal lipid binding domain of BDH) is close to the active site, it appears to be in a hydrophobic environment and only indirectly defines the substrate binding site at the catalytic center of BDH.

A thermodynamic coupling mechanism for the disaggregation of a model peptide substrate by chaperone secB.

Molecular chaperones prevent protein aggregation in vivo and in vitro. In a few cases, multichaperone systems are capable of dissociating aggregated state(s) of substrate proteins, although little is known of the mechanism of the process. SecB is a cytosolic chaperone, which forms part of the precursor protein translocation machinery in Escherichia coli. We have investigated the interaction of the B-chain of insulin with chaperone SecB by light scattering, pyrene excimer fluorescence, and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B-chain of insulin. We show that SecB is capable of dissociating soluble B-chain aggregates as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B-chain aggregate by SecB has been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B-chains, rather it binds the small population of free B-chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate toward the individual B-chains. Thus SecB can rescue aggregated, partially folded/misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B-chain to chaperone SecB. The data suggests that the B-chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate.

Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes.

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6. The specific caspase-3 inhibitor, Ac-Asp-Glu-Val-Asp-aldehyde, inhibited caspase-3 activation and attenuated SDZ IMM 125-induced apoptosis and lactate dehydrogenase leakage. After 20 h of SDZ IMM 125 incubation, the parameters of apoptosis were further increased. Decreased mitochondrial membrane potential (measured by rhodamine 123 uptake) and cytochrome c release went in parallel with ultrastructural mitochondrial changes, and might be regarded as early events that trigger the apoptotic cascade. Transmission electron microscopy showed cytoplasmic blebbing after 4 h of SDZ IMM 125 incubation. As observed by transmission electron microscopy, treatment with SDZ IMM 125 resulted in an increase in the number of necrotic cells after 20 h, but not after 4 h. Our findings suggest that in rat hepatocyte cultures, SDZ IMM 125 is a specific inducer of apoptosis after short-term incubation, and this overlaps with necrosis after longer treatment periods. It is very likely that the necrosis occurring later is the result of the early apoptotic events.

Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures.

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms. Rat hepatocytes were cultured for 4 or 20 h with CsA at concentrations of 0, 10, 25, and 50 microM. Chromatin condensation and fragmentation, DNA fragmentation (TUNEL), membrane phosphatidylserine distribution (Annexin V), caspase-1, -3, and -6 activity, mitochondrial membrane potential (Rhodamine 123), and cytochrome c release into the cytosol were investigated. Four hours after CsA treatment, chromatin condensation and fragmentation and the number of TUNEL- and Annexin V-positive cells increased dose-dependently without any observable enzyme leakage, which indicated the integrity of the outer cell membrane. After 20 h of CsA incubation apoptosis parameters were further increased and were accompanied by the increased activity of the cysteine protease, caspase-3 (CPP 32), and slightly increased caspase-6 (Mch 2), but not caspase-1 (ICE). The caspase-3 inhibitor, Ac-DEVD-CHO, inhibited caspase-3 activation and attenuated CsA-induced apoptosis and LDH leakage. The caspase-6 inhibitor, Ac-VEID-CHO, only marginally inhibited CsA-induced apoptosis. Decreased mitochondrial membrane potential and cytochrome c release went in parallel with ultrastructural mitochondrial changes and might be regarded as early events that trigger the apoptosis cascade. Transmission electron microscopy confirmed an increase in the number of necrotic cells after 20 h, but not after 4 h, compared with controls.

Synthesis and application of novel bifunctional spin labels.

The synthesis of new bifunctional spin-labeled cross-linking reagents is described. Covalent attachment to papain was achieved via a thiol-specific thiosulfonate residue and, for the second anchor point, via a nonspecific photoreactive azido function. The thiosulfonate formed a reversible disulfide linkage, which could be cleaved again reductively by dithiothreitol. The spin label, a pyrroline-1-oxyl radical, was highly immobilized after attachment to papain by both functional groups and showed little if any relative motion with respect to the protein.

EPR kinetic studies of the LDL oxidation process driven by free radicals.

Oxidation is widely assumed to play a causal role in the pathogenesis of atherosclerosis. Oxidative modification of low density lipoprotein (LDL) can be followed by analyzing the lag phase of the conjugated diene formation at 234 nm in LDL exposed to Cu2+. This procedure is restricted to isolated LDL fractions. To make this assay applicable to different biological systems, the present paper introduces a method to determine the time course of lipid peroxidation by measuring the EPR signal intensity and thereby the concentration of the radicals formed. Stable radical spin adducts were generated using the spin trap PBN (N-tert.-butyl-alpha-phenylnitrone) and were detected by EPR spectroscopy. Comparing the specific formation of radicals and the generation of conjugated dienes as measured by UV absorbance revealed analogous lag, propagation and decomposition phases.

Hepatocellular effects of cyclosporine A and its derivative SDZ IMM 125 in vitro.

The novel immunosuppressive drug O-hydroxyethyl-D(Ser)8-cyclosporine (SDZ IMM 125) and cyclosporine A (CyA) were compared in different in vitro models with respect to hepatocellular side effects. SDZ IMM 125 was less lipophilic than CyA and also decreased liposomal membrane anisotropy less. Furthermore, SDZ IMM 125 increased Na+ and Ca++ permeability across the liposomal membranes significantly more than CyA. The uptake of CyA and SDZ IMM 125 into freshly isolated rat hepatocytes was neither saturable, Na+ dependent or temperature sensitive, nor could it be inhibited vice versa, indicating passive diffusion. The diffusion coefficient of CyA was about two times higher than that of SDZ IMM 125, reflecting its higher lipophilicity. In primary hepatocyte monolayers the cellular concentrations of CyA were about two times higher than that of SDZ IMM 125. As an indicator of cholestasis the saturable uptake of cholyltaurine into isolated cells was found to be apparently competitively inhibited to the same extent by both compounds. In isolated perfused rat livers SDZ IMM 125 caused a significantly greater decrease in bile flow than did CyA. Release of lactate dehydrogenase from hepatocyte primary cultures and from isolated perfused livers were determined as parameter of cell damage. In both systems the cytotoxicity of SDZ IMM 125 was significantly higher than that of CyA. The data suggest that SDZ IMM 125 causes greater cholestatic and cytotoxic effects than CyA at equimolar cellular exposure.

Cyclosporine A-induced oxidative stress in rat hepatocytes.

In man the immunosuppressive drug Cyclosporine A (CsA) has been used successfully in organ transplantation and in the treatment of autoimmune disorders. The drug, however, causes side effects which occur mainly in the kidney but also in the liver. The mechanisms leading to the hepatic side effects are not yet fully understood. Because reactive oxygen production is a common mechanism of drug toxicity, the goal of this study was to evaluate whether CsA induces oxidative stress in rat liver cells. In primary rat hepatocyte 20-h cultures, CsA caused a concentration-dependent increase of free reactive oxygen species, thiobarbituric acid reactive substances, loss of protein thiols and decrease of molar ratios of glutathione and glutathione disulfide in the range of 0 to 50 microM CsA. The weakening or enforcement of the cellular glutathione state by the glutathione synthesis inhibitor buthionine sulfoximine or the glutathione disulfide -reducing agent dithiothreitol either increased or inhibited the CsA cytotoxicity, as determined by lactate dehydrogenase release. CsA also decreased the level of endogenous antioxidant ascorbic acid and increased its oxidation product dehydroascorbic acid. Supplementation of the cell cultures with ascorbic acid significantly reduced the CsA toxicity. The antioxidant DL-alpha-tocopherol-polyethylene-glycol-1000-succinate partly decreased CsA-mediated reactive oxygen species formation, totally decreased thiobarbituric acid reactive substances formation, prevented the loss of protein-bound sulfhydryl groups and in addition totally inhibited the CsA cytotoxicity. The present data provide good evidence that oxidative stress is part of the mechanism by which CsA causes toxicity in rat liver cells.

Nucleotide binding to the heat-shock protein DnaK as studied by ESR spectroscopy.

We employed ESR spectroscopy using spin-labeled adenine nucleotides to investigate nucleotide binding to the 70-kDa heat shock protein, DnaK, from Escherichia coli. Binding stoichiometries of 1 mol/ mol for both ATP and ADP to previously nucleotide-depleted protein in the presence of Mg2+ were determined directly and under equilibrium binding conditions. Of the spin-labeled adenine nucleotides available to us, only the derivatives with the spin label attached to the C8 position of the adenine moiety, 8-SL-AdoP3 and 8-SL-AdoP2 [8-(2,2,6,6-tetramethyl-piperidin-4-yl -1-oxyl-)amino-adenosine-5'-triphosphate or diphosphate], were bound sufficiently tightly by the heat-shock protein, resulting in ESR spectra typical for immobilized radicals. In the absence of Mg2+, only approximately 0.5 mol were bound. Subsequent addition of Mg2+, however, led to the previously observed maximum binding of 1 mol/mol. Both 8-SL-AdoP3 and 8-SL-AdoP2 were fully exchangeable upon addition of excess ATP or ADP suggesting that the analogs bound directly to the nucleotide binding sites within the protein. 8-SL-AdoP2 release was also observed in the presence of the co-chaperone GrpE, indicating that the spin-labeled analogs of adenine nucleotides function like the natural nucleotide-substrates of the heat-shock protein. Small differences in the ESR spectra of 8-SL-AdoP3 and 8-SL-AdoP2 in complex with DnaK were observed.

Specific interaction of (R)-3-hydroxybutyrate dehydrogenase with membrane phosphatidylcholine as studied by ESR spectroscopy in oriented phospholipid multibilayers: coenzyme binding enhances the interaction with phosphatidylcholine.

The interaction of phospholipid with (R)-3-hydroxybutyrate dehydrogenase, a phosphatidylcholine-requiring membrane enzyme, has been studied using ESR spectroscopy of spin-labeled lipids, both as ordered multibilayers and in lipid vesicle suspensions (liposomes). Partially oriented phospholipid multibilayers were prepared from lipid vesicles composed of a 1:1 mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Vesicles containing (R)-3-hydroxybutyrate dehydrogenase yielded active preparations of the enzyme in such multibilayers. With increasing protein/lipid ratio, the order of the multibilayers was disrupted as monitored by ESR spectroscopy with a spin-labeled analogue of PC, 5-doxyl-PC (5 mol %, 10% of total PC) as a probe. The outer peak separation of 5-doxyl-PC varied with the lipid/protein ratio. The lower the ratio, the larger was the separation, with higher activity enzyme being more effective in exerting this effect. When 5-doxylstearic acid was substituted for 5-doxyl-PC or when the enzyme was inactive, the 2A(zz) value stayed practically constant at its lower limit (about 54 G). Multilayers composed of 81% PE, 11% diphosphatidylglycerol (DPG), and 8% 5-doxyl-PC (no unlabeled PC present) gave similar results. With this lipid mixture, the maximal 2A(zz) value (about 61 G) was reached at lower protein/lipid ratios, although the enzymic activity of (R)-3-hydroxybutyrate dehydrogenase is reduced to 40% in this system. The outer peak separation also depended on the presence of the coenzyme, NAD+, and 2-methylmalonate. The latter enhances binding of NAD+ about 100-fold by forming a ternary complex. With this ternary complex, the 2A(zz) values were increased unless the maximal values had been reached already in the absence of coenzyme. In all these experiments only a single ESR spectral component was observed. Similar results were obtained for the enzyme in liposomes, although the effect was less pronounced apparently due to the higher mobility of the probe. It is concluded that PC is motionally restricted by (R)-3-hydroxybutyrate dehydrogenase and yet is in rapid exchange with the bulk lipid on the ESR time scale. PC is required for formation of tight and functional complexes with NAD [Rudy et al. (1989) Biochemistry 28, 5354-5366], and such complexes strengthen the interaction of the enzyme with PC.

EPR spectroscopy of 5-DOXYL-stearic acid bound to the mitochondrial uncoupling protein reveals its competitive displacement by alkylsulfonates in the channel and allosteric displacement by ATP.

Competition of fatty acids (FA) and alkylsulfonates with 5-DOXYL-stearic acid (5-SASL) binding to isolated mitochondrial uncoupling protein (UcP) is demonstrated using EPR spectroscopy. A distinct peak of the bound 5-SASL (h+1I) decreased with increasing concentration of competitors. Since alkylsulfonates are UcP substrates, it suggests that the FA binding site is located in the anion channel. Moreover, with increasing ATP the h+1I peak decreased and was smoothed with the 'micellar' peak into a single wider peak. A pH of 8.5 reversed this effect. It could reflect an allosteric release of 5-SASL from the ATP binding site which mimics the ATP gating mechanism.

Effects of magnesium ions on the relative conformation of nucleotide binding sites of F1-ATPases as studied by electron spin resonance spectroscopy.

Cations like Mg2+ play an important role in the catalytic mechanism of F1-ATPases. In this study we applied ESR spectroscopy and used the ATP analog 2-azido-2',3'-(2,2,5,5-tetramethyl-3-pyrroline-1-oxyl-3-carboxylic acid ester)ATP (2-N3-SL-ATP) to investigate the effects of Mg2+ ions on the structure of the nucleotide binding sites of F1-ATPases from beef heart mitochondria (MF1) and from the thermophilic bacterium PS3 (TF1). The results demonstrated that Mg2+ ions not only influenced the binding of the nucleotide analogs to F1 but also altered the structure and geometry of the nucleotide binding sites. We observed that the dipolar interactions that are indicative of the close proximity of enzyme-bound 2-N3-SL-ANP (Vogel, P.D., Nett, J.H., Sauer, H.E., Schmadel, K., Cross, R.L., and Trommer, W.E. (1992) J. Biol. Chem. 267, 11982-11986) were only detectable in MF1-ATPase when the enzyme was preincubated with Mg2+ ions. In the absence of Mg2+, the enzyme exhibited ESR spectra indicative of spin label bound in at least two different environments (binding sites) with no dipolar interactions visible. TF1-ATPase did not exhibit clear dipolar interactions in the presence or absence of Mg2+. The ESR spectra of TF1 in the absence of Mg2+ indicated two different environments of the spin labels. Subsequent addition of Mg2+, however, led to exactly the same spectra as if the enzyme was incubated with the ions, indicating a rearrangement of the nucleotide binding sites. In summary, clear differences in the structures of the nucleotide binding sites of MF1 and TF1 in the presence or absence of Mg2+ were observed. Conformational differences between F1-bound spin-labeled nucleotides were also observed between TF1- and MF1-ATPases.

Labeling of the mitochondrial membrane D-3-hydroxybutyrate dehydrogenase (BDH) with new bifunctional phospholipid analogues.

D-3-Hydroxybutyrate dehydrogenase (BDH), an inner mitochondrial protein, is a well-known phospholipid dependent enzyme. It is a primary dehydrogenase of the oxidative phosphorylation system and is involved in the redox balance of the NAD+/NADH pool. The preparation of fluorescent phospholipids and newly synthesized bifunctional phospholipid analogues (fluorescent and photoactivatable) allowed us to study the structural requirement for lipid activation of the purified enzyme. This paper reports the chemical synthesis protocols to prepare these new phospholipids and their characterization. Illumination experiments of complexes between bifunctional phospholipids and BDH which lead to a cross-linked polypeptide indicate that both the polar head and the hydrophobic moiety of phospholipids interact with BDH. The bifunctional phospholipids were also tested on other lipid-binding proteins, i.e., horse cytochrome c and bovine serum albumin, and demonstrated the promising potential of this new type of photoactivatable molecules which can be followed merely by fluorescence without radioactive labeling.

Antigen-specific therapy of experimental myasthenia gravis with acetylcholine receptor-gelonin conjugates in vivo.

Rats suffering from experimental autoimmune myasthenia gravis (EAMG) induced by previous immunization with foreign acetylcholine receptor (AChR) were treated with AChR-toxin conjugates using the plant toxin gelonin. This led to a marked improvement of clinical symptoms as well as a significant increase in functional AChR compared to untreated rats with EAMG as determined 6 to 10 weeks later. No therapeutic effect was observed after treatment with gelonin or AChR alone. The immune response to irrelevant control antigens was not altered by this treatment.

Analysis of lymphocyte subsets in patients with aplastic anemia before and during immunosuppressive therapy.

To define the contribution of T-lymphocyte subsets in the development of aplastic anemia (AA), T-cell subpopulations including alpha beta T cells, gamma delta T cells, and delta TCS1-positive gamma delta T cells, were analyzed by cytophotometry in the peripheral blood (PB) and bone marrow (BM) of patients with AA before and after 6 weeks of therapy with anti-lymphocyte globulin (ALG), methylprednisolone, and cyclosporin A (CSA). In nine patients with AA a significant decrease of PB- and BM-derived T cells was observed after 6 weeks of therapy as compared with normal controls. At diagnosis, the CD4/CD8 ratio in PB and BM of the patients did not differ from the ratio in the control population; however, a reversed ratio (< 1) was present in PB as well as in BM after weeks of therapy. Interestingly, lymphocytes expressing the gamma delta T-cell receptor (TCR tau delta) were significantly decreased both before (PB 1.2 +/- 0.1%; BM 0.8 +/- 0.1%) and after 6 weeks of therapy (PB 0.7 +/- 0.1%; BM 0.7 +/- 0.1%) as compared with healthy controls (PB 2.4 +/- 0.2%; BM 2.3 +/- 0.2%). However, the proportion of the gamma delta-T-cell subpopulation expressing the delta TCS1 phenotype was markedly increased before (PB 42 +/- 3.5%; BM 31 +/- 3%) and especially after 42 days of therapy (PB 77 +/- 12%; BM 45 +/- 2%) as compared with that in normal subjects (PB 19 +/- 2%; BM 9.7 +/- 0.8%). At present, follow-up is under evaluation to correlate these findings with hematological response. The pathophysiological significance of the observed alterations within the T-cell subsets and especially the gamma delta T-cell populations will require further functional analyses, in particular since delta TCS1-positive gamma delta T cells exhibit autoimmunological capacity.

ATP binding to bovine serum albumin.

Specific binding of ATP to bovine serum albumin (BSA) is demonstrated employing ATP derivatives spin-labeled at either N6 or C8 of adenine ring or at the ribose moiety. Based on a 1:1 stoichiometry binding constants are in the 50-100 microM range. Binding is largely competitive with ATP or stearic acid. A small fraction of the labeled nucleotides could not be liberated by these ligands. Binding of AMP is in the millimolar range, only.